WO2023089159A1

WO2023089159A1 - New strategy targeting stroma/tumor cell crosstalk to treat a cancer

Info

Publication number: WO2023089159A1
Application number: PCT/EP2022/082555
Authority: WO
Inventors: Richard TOMASINI; Jérémy NIGRI; Sarah TUBIANA
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique; Université D'aix Marseille; Institut Jean Paoli & Irene Calmettes
Priority date: 2021-11-22
Filing date: 2022-11-21
Publication date: 2023-05-25

Abstract

In this study, the Inventors report that CD9 is a key component of PC-associated CAFs-derived ANXA6+-EVs. They determined that CD9 is expressed by PC-associated CAFs in vitro as well as in vivo. Targeting CD9 impaired CAFs-derived ANXA6+-EVs uptake by pancreatic cancer cells, which consequently decreases their migratory abilities. Signaling pathway arrays highlighted p38/MAPK as activated in pancreatic cancer cells following CAFs-derived ANXA6+/CD9+-EVs uptake. The use of CD9 blocking antibody, p38 siRNA or chemical inhibitors impaired pancreatic cancer cells abilities following incubation with CAFs-derived ANXA6+/CD9+-EVs. Finally, they revealed CD9 expression as an independent poor-prognosis marker in human PC samples. Collectively their data highlight the key role of CD9 in CAFs-derived ANXA6+-EVs internalization by pancreatic cancer cells and the consequent, and mandatory, activation of p38/MAPK pathway to foster their migratory abilities. Measuring the oncogenic CAFs-derived ANXA6+/CD9+-EVs then limiting their action on pancreatic cancer cells abilities might be a promising option for PC stratification and treatment.

Description

NEW STRATEGY TARGETING STROMA/TUMOR CELL CROSSTALK TO

TREAT A CANCER

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology.

BACKGROUND OF THE INVENTION:

Intercellular communication is at the basis of cellular and tissue homeostasis maintenance insuring adapted and efficient responses to environmental modulation of cells. While intercellular communications are classically described through cell-cell contacts and soluble factors such as cytokines, hormones and metabolites among others, secretion of extracellular vesicles (EVs) consists of the most complex mode of cellular crosstalk [1], EVs are membrane vesicles having the same topology than cell of origin and contain a broad array of biological active components such as genetic material, proteins, or metabolites [2], Following EVs uptake, numerous cellular abilities were shown to be drastically modified in a physiological or pathological situation, thus leading to the development of therapeutic applications for EVs, especially in the context of cancer [3,4],

Indeed, the role of EVs-producing/accepting cells and their consequent impact in carcinogenesis then tumor evolution have been extensively investigated in several cancers. Cancer cell EVs were shown to influence immune cell activation [5], stroma-assisted tumor growth [6], chemotherapy resistance [7], miRNA biogenesis [8] or metastatic organotropism [9,10], while tumor stroma EVs were described, among other biological impacts, as regulating therapy resistance pathways [11], cancer cell metabolism [12], or invasive behavior [13], Results from those approaches highlighted EVs as critical mediators of intercellular communication between tumor cells and stromal cells in local and distant environment with a consequent impact on patient’s fate and survival.

With a 5-year overall survival rate at only ~7% together with an increased incidence [14], pancreatic ductal adenocarcinoma (PC) figures as the solid cancer with the worst prognosis. In trying to understand why survival statistics hardly changed in the last 20 years, the main reasons include a late diagnosis, a lack of biomarkers, and the prevalence of therapeutic resistance. However, one has to consider the specific cellular context of those tumors. Indeed PC consists of 80% of non-tumor cells, mainly Cancer-Associated Fibroblasts (CAFs), immune and nerve cells composing the intra-tumoral microenvironment (TME) or stroma compartment [15], TME, and consequently its dialogues with tumor cells, has been clearly shown as impacting PC development as well as therapeutic resistance to treatment by the scientific community, who so far, failed to transform those stroma-related knowledges into clinical tools. Recently, several groups reported improvements in the stroma-based knowledge with potential implications in patient care and prognosis, through classical molecular biology discoveries [16,17] or the use of stromal subtyping [18,19] as an important stratification tool for future therapies.

Regarding TME-composing cells, immune cells were demonstrated to have both anti- and pro-tumor actions under dependence of multiples factors and recent studies highlighted such variable abilities for CAFs [20], Those findings suggest that general depletion of CAFs is not a relevant option and could led to effects opposite to the intended but rather open stromalbased therapies necessitating to target only pro-tumourigenic CAFs or their specific crosstalk with tumor cells. In this last field of research, stroma-derived EVs and specifically CAFs- derived EVs are of large interest. Considered as pheno-copying part of the originating cells, CAFs-derived EVs detection could drastically improve the potential of CAFs subtyping in PC stratification. Besides, studies revealing the impact of CAFs-derived EVs on PC tumor cell aggressiveness [21,22], metabolism [12], and chemotherapy resistance [23] improved the biological relevance of targeting their production by CAFs or their uptake by PC tumor cells.

Following such approaches, deciphering molecular mechanisms driving CAFs-derived EVs production, cargo, release then uptake is of crucial importance. We previously reported that, within hostile areas of PC, CAFs-derived EVs promote pancreatic cancer cells aggressiveness [22], Such effect was dependent on the presence of a protein complex involving ANXA6, LRP1, and TSP1 in CAF-derived EVs. While the biological outcome of those specific EVs was clearly demonstrated, the driving pathway activated following EVs uptake by tumor recipient cells as well as the molecular mechanism of EVs internalization remain unclear.

SUMMARY OF THE INVENTION:

The present invention relates to a method for predicting the survival time of a patient suffering from a cancer with preponderant stroma, comprising i) determining the expression level of CD9 in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value. DETAILED DESCRIPTION OF THE INVENTION:

Taking into account those conceptual gaps and the potential translational impact of such knowledge, in the current study, the Inventors used i-TRAQ-quantitative proteomic analysis to identify the specific protein composition of CAFs-derived ANXA6⁺-EVs and CAFs-derived ANXA6'-EVs. They found the tetraspanin CD9 as one of the most deregulated proteins, then further revealed CD9 as involved in CAFs-derived ANXA6⁺-EVs uptake. Moreover, they found that p38/MAPK pathway was induced following CAFs-derived ANXA6⁺/CD9⁺-EVs uptake in pancreatic tumor cells and mandatory for the consequent improved migration.

Collectively, our findings suggest that CD9 is a crucial member of CAFs-derived ANXA6⁺-EVs involved in controlling their uptake by recipient pancreatic cancer cells, which induces p38/MAPK pathway activation then a consequent improvement of tumor cell aggressiveness. Analysis of publicly available human expression database confirmed CD9 expression as an independent poor-prognosis factor in PC patients. These results highlight a novel function of tetraspanin CD9 in CAFs-derived EVs and their consequent impact on PC tumor cells abilities, reinforcing the need of targeting stromal/tumor cells crosstalk.

Methods for predicting the survival time of a patient and the aggressiveness of a cancer

A first aspect of the invention relates to a method for predicting the survival time of a patient suffering from a cancer with preponderant stroma, comprising i) determining the expression level of CD9 in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

In some embodiments, the invention relates to a method for predicting the survival time of a patient suffering from a cancer with preponderant stroma, comprising i) isolating extracellular vesicles from a sample obtained from the patient ii) determining the expression level of CD9 in extracellular vesicles from the sample iii) providing a good prognosis when the expression level determined at step ii) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step ii) is higher than its predetermined reference value. A second aspect of the invention relates to a method for predicting the aggressiveness of a cancer with preponderant stroma, comprising i) determining the expression level of CD9 in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

In some embodiments, the invention relates to a method for predicting the aggressiveness of a cancer with preponderant stroma, comprising i) isolating extracellular vesicles from a sample obtained from the patient ii) determining the expression level of CD9 in extracellular vesicles from the sample iii) providing a good prognosis when the expression level determined at step ii) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step ii) is higher than its predetermined reference value.

In some embodiments, the extracellular vesicles are derived from tumor stromal cells. In some embodiments, the extracellular vesicles are Cancer-Associated Fibroblast (CAF)-derived extracellular vesicles. In some embodiments, the extracellular vesicles are Cancer-Associated Fibroblast (CAF)-derived ANXA6⁺ extracellular vesicles.

According to these particular embodiments, the sample can be blood, peripheral-blood, serum, plasma, stromal cells, tumoral circulating cells or a tumor sample. In some embodiment, the sample contains extracellular vesicles derived from tumor stromal cells. In some embodiment, the sample contains cancer-associated fibroblast (CAF)-derived extracellular vesicles. In some embodiments, the extracellular vesicles are Cancer-Associated Fibroblast (CAF)-derived ANXA6⁺ extracellular vesicles.

As used herein, the term “subject” or “patient” denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with a cancer with a preponderant stroma, in particular a pancreatic cancer, a cholangiocarcinoma, a squirrhus of breast, Hodgkin Lymphoma, colorectal cancer or prostate cancer. In some embodiment, the cancer with a preponderant stroma is a metastatic cancer. In some embodiment, the cancer is a metastatic pancreatic cancer. As used herein, the term “CD9” denotes a tetraspanin membrane protein considered as one of the main exosome surface markers. Tetraspanins are cell surface glycoproteins with four transmembrane domains that form multimeric complexes with other cell surface proteins. For the gene sequence, its Entrez Gene reference number is: 928; for the protein, the UniProt reference number is: P21926 and NCBI references are: NP_001317241.1 or NP_001760.1; for the mRNA, the GenBank reference is: NM_001769.

The present invention focus on and targets the expression of CD9 in extracellular vesicles derived from the tumor stromal cells, more precisely, derived from cancer-associated fibroblasts.

As used herein, the term “stroma” denotes the intra-tumoral microenvironment. Tumors are composed of neoplastic cells and non-neoplastic cells in various ratio depending tumors type and grade. The total amount of non-neoplastic cells composed the stroma, which are mainly cancer-associated fibroblasts (CAFs) and immune cells. As demonstrated in the present invention, CD9 is involved in stroma/tumor cell crosstalk mediated by extracellular vesicles derived from stroma, improving tumor cells abilities, such as tumor cell survival and cancer aggressiveness by enhancing migratory abilities.

As used herein, the term “cancer with preponderant stroma” or “cancer with abundant stroma” or “stroma-rich tumor” denotes a cancer where tumor/stroma ratio is superior to 0.5. As example, the abundance of stroma may be assessed with a histological section analyzed under microscope. The abundance of stroma is also strongly correlated with the consistency of tumor formation. When the stroma is abundant, the whole tumor or the stroma itself adopt a rigid structure, sometimes retracted. Since stroma is mainly composed of CAFs, a cancer with preponderant stroma is associated with an enriched proportion of CAF.

In some embodiments, the cancer with preponderant stroma is a pancreatic cancer, a cholangiocarcinoma, a squirrhus of breast, Hodgkin Lymphoma, colorectal cancer or prostate cancer. In some embodiments, the cancer with preponderant stroma is a metastatic cancer.

As used herein, the term “extracellular vesicles” denotes lipid bilayer-delimited particles formed inside cells and released from cells. Extracellular vesicles are implicated in cell-cell communication as means for intercellular communication and transmission of macromolecules (such as nucleic acids, proteins, amino acids, lipids among others) between a producing cell and a receiving cell. Thus, extracellular vesicles may contain disease-associated cargo, released into the intracellular medium after fusion with the targeted cell plasma membrane.

As used herein, “extracellular vesicles derived from tumor stromal cells” denotes extracellular vesicles secreted by any tumor stromal cell, in particular cancer-associated fibroblasts (CAF) or macrophages. In order to isolate extracellular vesicles derived from stromal cells or macrophages, conditioned media (EVs free) from stromal cells/macrophages co-culture can be processed through serial ultracentrifugation step combined with exclusion columns in order to recover specifically the extracellular vesicles in the range of 50 to 150 nm. In order to isolate cancer associated fibroblast-derived extracellular vesicles, conditioned media from CAFs cultured in EVs-free medium can be processed through serial ultracentrifugation step combined with exclusion columns to recover specifically extracellular vesicles from the range of 50 to 150nm.

As used herein, the term “CAF-derived ANXA6⁺ extracellular vesicles” denotes extracellular vesicles secreted by cancer-associated fibroblasts (CAF) and lined by Annexin 6 (ANXA6). In order to isolate CAF-derived ANXA6⁺ extracellular vesicles, CAFs are cocultured with macrophages under hypoxia and lipid deprivation. Those culture conditions drive ANXA6 and its protein multicomplexe to become carge of extracellular vesicles. Exclusion column and serial ultracentrifugation lead to recovery of extracellular vesicles.

From these isolated extracellular vesicles, CD9 may be quantified by western-blot using specific CD9 antibodies. The quantity of CD9 may be compared to other extracellular vesicles markers such as CD81, ALIX, TSG101.

As used herein, the term “survival time” denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as cancer with preponderant stroma (according to the invention). The survival time rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment. As used herein and according to the invention, the term “survival time” can regroup the term “Overall survival (OS)”. As used herein, the term “OS” denotes the time from diagnosis of a disease such as cancer with preponderant stroma (according to the invention) until death from any cause. The overall survival rate is often stated as a two-year survival rate, which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.

As used herein, the term “aggressiveness of a cancer” reflects the capacity of a cancer to lead to the formation of metastasis by improving cancer cell dissemination, cancer cell migration and invasion abilities, modifying their adhesive capacities and favoring pre- metastatic and metastatic niche formation. Metastasis represents the growth of cancer cell in a secondary site/organ following cancer cell dissemination from a primary site/organ. In vitro, invasion and migration abilities can be monitored using a Boyden Chamber or using spheroid cell cultures embedded in matrix, in 3D. The skilled person well-know how to detect metastasis in vivo, as example with an echography, a radiology or a scanner.

Measuring the expression level of CD9 can be done by measuring the gene expression level of CD9 or by measuring the level of the protein CD9 and can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence or mRNA) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthal ene-1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol -reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrromethene boron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912). In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOT® (obtained, for example, from Life Technologies (Quantum Dot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the band gap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif.). Additional labels include, for example, radioisotopes (such as ³H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+.

Detectable labels that can be used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase. Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH). Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pinkel et al., Proc. Natl. Acad. Sci. 83:2934- 2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am.l. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929. Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP (dinitrophenol), and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore. In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153. It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can be labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti- DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 nm). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can be envisioned, all of which are suitable in the context of the disclosed probes and assays. Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate). The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microspheresized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

According to the invention, the level of CD9 proteins may also be measured and can be performed by a variety of techniques well known in the art. For measuring the expression level of CD9, techniques like ELISA (see below) or ELLA allowing to measure the level of the soluble proteins are particularly suitable. In the present application, the “level of protein” or the “protein level expression” or the “protein concentration” means the quantity or concentration of said protein. In another embodiment, the “level of protein” means the level of CD9 protein fragments. In still another embodiment, the “level of protein” means the quantitative measurement of CD9 protein expression relative to an internal control. Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample. Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresismass spectroscopy technique (CE-MS), etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Methods of the invention may comprise a step consisting in comparing the proteins and fragments concentration in circulating extravesicles with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the protein CD9. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the protein CD9. Thus, in a particular embodiment, fragments of CD9 protein may also be measured.

Predetermined reference values used for comparison of the expression levels may comprise “cut-off’ or “threshold” values that may be determined as described herein. Each reference (“cut-off’) value for CD9 level may be predetermined by carrying out a method comprising the steps of: a) providing a collection of samples from patients suffering of a cancer and/or samples of the corresponding uninvolved tissues as described in the invention; b) determining the level of CD9 for each sample contained in the collection provided at step a); c) ranking the tumor tissue samples according to said level d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level, e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient; f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve; g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets h) selecting as reference value for the level, the value of level for which the p value is the smallest.

For example the expression level of CD9 may be assessed for 100 cancer samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the highest expression level and sample 100 has the lowest expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels. In routine work, the reference value (cut-off value) may be used in the present method to discriminate cancer samples and therefore the corresponding patients.

Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art. The man skilled in the art also understands that the same technique of assessment of the expression level of a protein should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a protein of a patient subjected to the method of the invention.

A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of CD9 in the sample obtained from the patient. The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively, the kit of the invention may comprise amplification primers that may be pre-labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

CD9 as a biomarker for differential diagnostic between pancreatic cancer and pancreatitis

A third aspect of the invention relates to a method to perform the differential diagnostic between pancreatic cancer and pancreatitis, comprising a step of determining the expression level of CD9 in a sample obtained from the patient. Pancreatic ductal adenocarcinoma, referred here as pancreatic cancer, is a tumor in the pancreas which occurs when pancreatic cells integrate genomic mutations in oncogenes or tumor suppressor genes. Pancreatitis is an inflammation of the pancreas which may be acute or chronic. Pancreatic cancer and pancreatitis have similar symptoms such as abdominal pain, back pain, bloating, nausea, weight loss, onset of diabetes and depression. Actual biomarkers do not provide satisfying results in such case since they provide essentially non-discriminating results. Imaging data and cytology results after biopsy can provide sufficient information to make a diagnosis. Thus, non-invasive diagnostic of pancreatic cancer is quite difficult, even more in patients also suffering from chronic pancreatitis. These two pathologies are closely related since pancreatitis is a risk factor for pancreatic cancer. Accordingly, their differential diagnosis is of capital interest.

According to the invention, such differential diagnostic between pancreatic cancer and pancreatitis may be performed by comparing the expression level of CD9 between tumor samples and inflammatory tissues. In order to provide such differential diagnostic, EVs could be extracted and CD9 measured in these EVs by drawing blood from patients suffering from pancreatitis or pancreatic cancer. The main advantage of this method is its fully discriminating and non-invasive approach.

CD9 inhibitor

In a fourth aspect, the present invention relates to a CD9 inhibitor for use in the treatment of a cancer with preponderant stroma in a subject in need thereof.

In some embodiment, the invention relates to a CD9 inhibitor for use in the treatment of a metastatic cancer with preponderant stroma in a subject in need thereof.

In some embodiments, the cancer with preponderant stroma is a pancreatic cancer, a cholangiocarcinoma, a squirrhus of breast, Hodgkin Lymphoma, colorectal cancer or prostate cancer.

As used herein, the term “CD9 inhibitor” denotes a molecule or compound which can inhibit the interactions of the protein, or a molecule or compound which destabilizes the proteins. The term “CD9 inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein. In the context of the invention, using a CD9 inhibitor impair tumor stromal cells-derived extracellular vesicles uptake by cancer cells. Since CD9 is mandatory to activate p38/MAPK pathway in the context of tumor/stroma cell crosstalk, the use of a CD9 inhibitor is particularly relevant in the treatment of primary and metastatic cancers.

In one embodiment, the inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In one embodiment, the inhibitor according to the invention (CD9 inhibitor) is an antibody. Antibodies directed against CD9 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against CD9 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-CD9 single chain antibodies. Compounds useful in practicing the present invention also include anti-CD9 antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to CD9. Humanized anti-CD9 antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of CD9 are selected. As example, an antibody directed against CD9 may be AT 14-012 (Villaudy et al., 2020) or ALB6 antibody (Boucheix et al., 1983).

In another embodiment, the antibody according to the invention is a single domain antibody directed against CD9. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B- cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then, for this invention, neutralizing aptamers of CD9 are selected.

In one embodiment, the compound according to the invention is a polypeptide. In a particular embodiment the polypeptide is an antagonist of CD9 and is capable to prevent the function of CD9. Particularly, the polypeptide can be a mutated CD9 protein or a similar protein without the function of CD9. In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell. The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moi eties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In another embodiment, the CD9 inhibitor according to the invention is an inhibitor of CD9 gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of CD9 expression for use in the present invention. CD9 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that CD9 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of CD9 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of CD9 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of CD9 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-m ethyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing CD9. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non- cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles are provided in Kriegler, 1990 and in Murry, 1991. Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno- associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In another embodiment, the invention relates to a method for treating a pancreatic cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CD9 inhibitor.

In order to test the functionality of a putative CD9 inhibitor a test is necessary. For that purpose, to identify CD9 inhibitors, ANXA6⁺CD9⁺EVs can be prepared from cancer-derived CAFs and incubated with cancer cells. The migratory ability of cancer cells in presence or not of the inhibitor can be monitored, as well as the EVs uptake by cancer cells or p38 MAPK activation or CD9 level in EVs. Preferentially, the migratory ability of cancer cells is monitored by EVs uptake by cancer cells.

Kit of part

A fifth aspect of the present invention relates to i) a CD9 inhibitor, and ii) at least one anti-cancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a cancer with preponderant stroma.

In some embodiment, the present invention relates to i) an antibody directed against CD9, and ii) at least one anti-cancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a cancer with preponderant stroma.

In some embodiment, the at least one anti-cancer agent is gemcitabine and/or FOLFIRINOX and/or a p38MAPK inhibitor.

As used herein, the term “simultaneous use” denotes the use of a CD9 inhibitor and at least one anti-cancer agent occurring at the same time.

As used herein, the term “separate use” denotes the use of a CD9 inhibitor and at least one anti-cancer agent not occurring at the same time.

As used herein, the term “sequential use” denotes the use of a CD9 inhibitor and at least one anti-cancer agent occurring by following an order. Therapeutic composition

A sixth aspect of the invention relates to a therapeutic composition comprising a CD9 inhibitor for use in the treatment of a cancer with preponderant stroma in a subject in need thereof.

In some embodiments, the invention relates to a therapeutic composition comprising a CD9 inhibitor and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof.

In some embodiment, the invention relates to a therapeutic composition comprising a CD9 inhibitor and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof, wherein the at least one anti-cancer agent is gemcitabine.

In some embodiment, the invention relates to a therapeutic composition comprising a CD9 inhibitor and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof, wherein the at least one anti-cancer agent is FOLFIRINOX.

In some embodiment, the invention relates to a therapeutic composition comprising a CD9 inhibitor and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof, wherein the at least one anti-cancer agent is a p38MAPK-inhibitor.

In some embodiment, the invention relates to a therapeutic composition comprising an antibody directed against CD9 and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof.

In some embodiment, the invention relates to a therapeutic composition comprising an antibody directed against CD9 and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof, wherein the anti-cancer agent is gemcitabine.

In some embodiment, the invention relates to a therapeutic composition comprising an antibody directed against CD9 and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof, wherein the anti-cancer agent is FOLFIRINOX.

In some embodiment, the invention relates to a therapeutic composition comprising an antibody directed against CD9 and at least one anti-cancer agent for use in the treatment of a cancer with preponderant stroma in a subject in need thereof, wherein the anti-cancer agent is a p38MAPK inhibitor.

As used herein, “gemcitabine” denotes a nucleoside analog in which the hydrogen atoms on the 2’ carbon of deoxycytidine are replaced by fluorine atoms. Gemcitabine is a chemotherapy medication used to treat cancers, in particular, as a first line therapy for the palliative treatment of locally advanced or metastatic cancer.

As used herein, “FOLFIRINOX” denotes a combination chemotherapy regimen consisting of oxaliplatin, irinotecan, fluorouracil and leucoverin (FOLFIRINOX). FOLFIRINOX is a chemotherapy medication used to treat cancers, in particular for the palliative treatment of locally advanced or metastatic cancer.

In some embodiments, the cancer is a metastatic cancer.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent.

Thus, the present invention also relates to a kit comprising an inhibitor of the expression according to the invention and a further therapeutic active agent.

For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

Anti-cancer agents may be Folfirinox and/or Gemcitabine and/or a p38MAPK inhibitor. Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L- asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. As example, a p38MAPK inhibitor may be SB203580, SB202190, SB202474.

In some embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.

Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies. Other additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can be an opioid or nonopioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent. Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7- Hl). Typically, the checkpoint blockade cancer immunotherapy agent is an antibody. In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. CD9 expression is increased in PC, according to the molecular subtypes, and associated with shorter disease-free survival. (A) Box plot of CD9 mRNA expression (log?) in PC primary samples (N=793) and normal pancreatic samples (N=158). For each box plot, median and ranges are indicated. The p-value is for the Student t-test. ***p < 0.001. (B- D) Box plot of CD9 mRNA expression (log?) in PC primary samples according to the molecular subtypes defined by Bailey (B), Collisson (C) and Moffitt (D). For each box plot, median and ranges are indicated. The p-value is for the ANOVA test in (b) and (c), and Student t-test for (d). ***p < 0.001, **p < 0.01, N.S not significant. (E) Kaplan-Meier DFS curves in patients with PC according to the CD9-based classification (“CD9-high” and “CD9-low”). The -value is for the log-rank test.

Figure 2. CD9 mRNA expression in PC compared to healthy pancreas (A) Relative cd9 mRNA expression in pdxl-cre/Kras^G12D/Ink4A^fl/flmice at 4 and 9 weeks expressed as fold change to healthy control (Kras^G12D/Ink4A^fl/fl) of similar age. *p < 0.05. (B) Relative cd9 mRNA expression, analyzed by RT-qPCR, in pancreata from mice with chronic pancreatitis (n=5) or PC (n=6). Data were normalized to TATA-Box Binding Protein. Results are presented as mean ± SD of triplicates *p < 0.05.

Figure 3. CD9 expression in PC is mainly driven by CAFs. Determination of CD9 mRNA level, through RT-qPCR analyses, in primary human PC derived-CAFs cultured in control or physiopathologic culture condition. Data were normalized to TATA-Box Binding Protein. Results are presented as mean ± SD of triplicates using 3 different pCAFs ***p < 0.001.

Figure 4. CD9 blockage limits EVs uptake and PC cancer cell migration. (A) Graphical scheme describing the experimental workflow in which EVs, derived from either NHF/iCAFs or CAFs cultured in physiopathological condition, are stained with PKH26 as a fluorescent dye then incubated with PC cancer cells. Percentage of PC cancer cells uptaking EVs NHF (top panel) or iCAF (bottom panel). (B) CD9 blocking antibody decrease NHF EVs uptake of pancreatic cancer cells. Results are presented as mean ± SD of triplicates *p < 0.05, **/?<0.01. (C) CD9 blocking antibody decrease iCAF EVs uptake of pancreatic cancer cells. Results are presented as mean ± SD of triplicates **/?<0.01. (D) Percentage of pancreatic cancer cells with internalization of CAFs PKH26⁺-EVs without treatment (Ctrl), under incubation of CD9 blocking antibody at I g, with EIPA treatment (EIP A) or with EIPA treatment + incubation with CD9 blocking antibody at Ipg. Results are presented as mean ± SD of triplicates *p < 0.05, **p < 0.01. (E) CD9 blocking antibody decrease pancreatic cancer cells migration induced by iCAF EVs. Results are presented as mean ± SD of triplicates **/?<0.01, ***/?<0.001.

Figure 5. p38MAPK pathway is induced in pancreatic cancer cells following CAFs- derived ANXA6⁺/CD9⁺-EVs uptake then is mandatory for improved migration. (A) Analysis of signaling pathway activated in PANC-1 following stroma-derived EVs incubation. Relative quantification of phospho-p38MAPK dot blots from each conditions (B) Quantification of phospho-p38MAPK and p38MAPK protein level in PANC-1 cells pre-treated with the corresponding chemical drugs (SB203580, SB202190 and SB202474) or mock control (DMSO) than incubated with stroma-derived EVs. P-Tubulin served as a loading control. (C) Migration ability measurement of PANC-1 cells treated as in (B). (D) Quantification ofp38MAPK protein level in PANC-1 cells transfected with control (siCtr) or p38 siRNA. (E) Migration ability of PANC-1 cells treated as in (D).

Figure 6. mRNA level of EMT markers. Relative Snail, Snai2, Twist, N-Cadherin and E-Cadherin mRNA expression in pdxl-cre/Kras^G12D/Ink4A^fl/fl mice at 4 and 9 weeks expressed as fold change to healthy control (Kras^G12D/Ink4A^fl/fl) of similar age. *p < 0.05.

EXAMPLE:

Material & Methods

Isolation of extracellular vesicles (EVs)

For co-culture experiments, 600 000 CAFs (infected with shRNA Ctrl or shRNA ANXA6 as in previous study[22]) were first plated in 6 wells and the day after 300 000 Raw 264.7 were plated in culture insert of 0.4 pm (Millipore, MCHT12H48). Cells were then washed twice 24h later in PBS IX, before adding specific media (EVs free): DMEM F12, 1% serum or DMEM F12, 1% delipidated serum (Lonza, DE14-840E) and hypoxia was introduced at 1% (vol/vol) O2 and 5% (vol/vol) CO2 in nitrogen atmosphere in a subchamber system (Biospherix). Cell-conditioned media were collected 48h later. EVs were isolated from these media by three sequential centrifugation steps at 4 °C: 10 min at 500 x g, to remove cells; then 30 min at 10,000 x , to remove cell debris and large extracellular vesicles; and 3 h at 100,000 x g, to pellet EVs, followed by one wash (suspension in PBS/centrifugation at 100,000 x g), to remove soluble serum and secreted proteins. The high speed pellet was then resuspended in 100 pL of PBS and if necessary lyzed in lysis buffer 5X (300 mM Tris Hcl pH 6.8, 10% SDS, 50% glycerol, 500 mMDTT, 0.01% bromophenol blue and 5% of P-mercaptoethanol) to be analyzed by western blot. For comparative analyses, the HSP was collected from equivalent amounts of culture medium, conditioned by equivalent amounts of cells. The corresponding cell layers were washed and lysed as described below.

Protein extraction and in-gel trypsin digest

EVs were resuspended in 40 pL of HEPES (50 mM, pH 7.5)/NaCl 150 mM lysis buffer supplemented by 1 mM EDTA, 1 mM EGTA, 10 %, glycerol, 1 % triton, 25 mM NaF, 1 % SDS, PMSF 1 :200 (v:v), Na3VO4 1 mM and SDS 1% and protein extracted. Samples were reduced with 5 mM Tris-(2-carboxyethyl) phosphine (SIGMA-ALDRICH, Saint-Quentin Fallavier, France) at 60 °C for 60 min, before their alkylation with 55 mM iodoacetamide (SIGMA-ALDRICH, Saint-Quentin Fallavier, France) at room temperature (in dark) for 30 min. Proteins from each sample were separated on a NuPAGE 4-12 % gel (Life Technologies) and stained with Imperial Blue (Pierce, Rockford, IL). The entire lane was cut into pieces and followed by in-gel trypsin digestion. Briefly, gel pieces were washed three times for 15 min in destain solution containing 50 mM ammonium bicarbonate and 50% methanol (v/v). The spots were then dried in a Speedvac concentrator (Thermo Fisher Scientific) for 5 min, which was followed by overnight in-gel digestion in 100 pL of 50 mM ammonium bicarbonate buffer containing 6 ng/pL sequencing grade modified porcine trypsin (Promega, le Madison, WI). The digestion mixture was extracted with 50% ACN and 5% formic acid (v/v). Speed-vac-dried peptide extracts were then resuspended in 15 pL of 3% ACN/0.1% formic acid (v/v) prior to LC-MS/MS analysis.

Mass spectrometry analysis

Three biological samples were prepared for this study, each sample were further analyzed thrice by LC-MSMS. Briefly, EVs protein extract were stacked on NuPAGE 4-12% Bis-Tris acrylamide gels (Life Technologies) in a single band, stained with Imperial Blue (Pierce, Rockford, IL) and cut from the gel. Gels pieces were submitted to an in-gel trypsin digestion as previously described [24], Peptides mixtures were extracted, dried down with speedvacuum and reconstituted with 0.1% TFA in 4% acetonitrile (ACN) before analyze by LC-MS/MS using an LTQ Velos Orbitrap Mass Spectrometer (Thermo Electron, Bremen, Germany) online with a an Ultimate 3000RSLCnano chromatography system (Thermo Fisher Scientific, Sunnyvale, CA). For each run, 10% on the Peptide mixture was separated on a PepMap RSLC Cl 8 column. First peptides were concentrated and purified on a pre-column (C18 PepMaplOO, 2 cm * 100 pm I.D, 100 A pore size, 5 pm particle size) in solvent A (0.1% formic acid in water). In the second step, peptides were separated on a reverse phase LC EASY- Spray Cl 8 column (PepMap RSLC Cl 8, 15 cm x 75 pm I.D, 100 A pore size, 2 pm particle size) at 300 nL/min flow rate. After column equilibration using 4% of solvent B (20% water - 80% acetonitrile - 0.1% formic acid), peptides were eluted from the analytical column by a two steps linear gradient (4-22% acetonitrile/H2O; 0.1 % formic acid for 110 min and 22-32% acetonitrile/H2O; 0.1 % formic acid for 10 min). For peptide ionization in the EASY-Spray nanosource, spray voltage was set at 1.9 kV and the capillary temperature at 275 °C. The LTQ velos Orbitrap was used in data dependent mode to switch consistently between MS and MS/MS. MS spectra were acquired in the Orbitrap in the range of m/z 300-1700 at a FWHM resolution of 30 000 measured at 400 m/z. For internal mass calibration the 445.120025 ions was used as lock mass. The more abundant precursor ions were selected and collision-induced dissociation fragmentation was performed in the ion trap on the 10 most intense precursor ions measured to have maximum sensitivity and yield a maximum amount of MS/MS data. The signal threshold for an MS/MS event was set to 500 counts. Charge state screening was enabled to exclude precursors with 0 and 1 charge states. Dynamic exclusion was enabled with a repeat count of 1, exclusion list size 500 and exclusion duration of 30 s.

Protein identification and quantification

Relative intensity-based label-free quantification (LFQ) was processed using the MaxLFQ algorithm from the freely available MaxQuant computational proteomics platform, version 1.5.2.8 [25], The acquired raw LC Orbitrap MS data were first processed using the integrated Andromeda search engine. Spectra were searched against the Human database (UniProt Proteome reference, date 201508; 20197 entries), the Mouse database (date 201508; 16717 entries) and a set of 177 more abundant proteins from bovine serum used for cells culture. The following parameters were used for searches: (z) trypsin allowing cleavage before proline; (ii) two missed cleavages were allowed; (zz) monoisotopic precursor tolerance of 20 ppm in the first search used for recalibration, followed by 4.5 ppm for the main search and 0.5 Da for fragment ions from MS/MS ; (zzz) cysteine carbamidomethylation (+57.02146) as a fixed modification and methionine oxidation (+15.99491) and N-terminal acetylation (+42.0106) as variable modifications; (iv) a maximum of five modifications per peptide allowed; and (v) minimum peptide length was 7 amino acids and a maximum mass of 4,600 Da. The match between runs option was enabled to transfer identifications across different LC-MS/MS replicates based on their masses and retention time within a match time window of 0.7 min and using an alignment time window of 20min. The quantification was performed using a minimum ratio count of 1 (unique+razor) and the second peptide option to allow identification of two cofragmented co-eluting peptides with similar masses. The false discovery rate (FDR) at the peptide and protein levels were set to 1% and determined by searching a reverse database. For protein grouping, all proteins that cannot be distinguished based on their identified peptides were assembled into a single entry according to the MaxQuant rules. The statistical analysis was done with Perseus program [26] (version 1.5.1.6) from the MaxQuant environment (www.maxquant.org). The LFQ normalised intensities were uploaded from the proteinGroups.txt file. First, proteins marked as contaminant, reverse hits, and “only identified by site” were discarded. Protein LFQ normalized intensities were base 2 logarithmized to obtain a normal distribution. Quantifiable proteins were defined as those detected in at least 70% of the samples in one or more condition. Missing values were replaced using data imputation by randomly selecting from a normal distribution centred on the lower edge of the intensity values that simulates signals of low abundant proteins using default parameters (a downshift of 1.8 standard deviation and a width of 0.3 of the original distribution). To determine whether a given detected protein was specifically differential a two-sample t-test were done using permutation based FDR-controlled at 0.05 and employing 250 permutations. The p value was adjusted using a scaling factor sO with a value of 0.2.

Bioinformatics analysis

Gene ontology (GO) enrichment analyses were performed using DAVID (Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/) [27,28],

Co-immunoprecipitation

Tissue lysates (3 mg), cell lysates (250 pg) were cleared using protein G-sepharose beads (Invitrogen) for 45 min at 4°C. The cleared lysates were then incubated with either CD9 and ANXA6 or Tag-HA as non-relevant antibody (1 pg) overnight at 4°C, followed by 45 min incubation with 20 pl of protein G-sepharose beads. After washing the beads five times in cold lysis buffer, the complexes were dissolved in Laemmli sample buffer and boiled for 10 min. Eluates were analyzed by western blot as described below. Real-time-quantitative PCR analysis (RT-qPCR)

RT-qPCR experiments were carried out as described in detail previously [22],

Human and mouse samples

Fixed and freshly frozen tissue samples of PC were obtained from patients who underwent surgery at the department of Digestive Surgery, North Hospital (Marseille, France) between 2009 and 2010. Three patients underwent pancreaticoduodenectomy and one a left pancreatectomy. No distant metastasis was revealed at initial diagnosis. Histological examination confirmed diagnosis of PC in all cases. Tumor staging was performed according to the International Union Against Cancer TNM System (the 6th edition). Prior to surgery, all patients had signed an informed consent form that had been approved by the local ethics committee (agreement reference of CRO2 tissue collection: DC-2013-1857). Pdxl-Cre;LSL- Kras^G12D;Ink4a/Arf^fl/fl and Pdxl-Cre;LSL-Kras^G12D;p53^R172H mice were obtained by crossing the following strains: Pdxl-Cre;LSL-Kras^G12D and Inkda/Arf^{1 11} or p53^R172H mice kindly provided by Dr. D. Melton (Harvard Stem Cell Institute, Cambridge, MA), Dr. R. Depinho (Dana-Farber Cancer Institute, Boston) and Dr. T Jacks (David H. Koch Institute for Integrative Cancer Research, Cambridge, MA), respectively. PC-bearing 8-12 wk-old mice were euthanized with their mating control littermates. For induced-chronic pancreatitis in mice, 7- week-old KI mice were daily injected with cserulein (250 pg/kg, #C9026, Sigma-Aldrich) for 12 days by intraperitoneal route. Pancreatic samples (healthy, PC or chronic pancreatitis) were fixed in 4% (wt/vol) formaldehyde for immunochemistry or frozen in cold isopentane for protein extraction. All animal care and experimental procedures were performed in agreement with the Animal Ethics Committee of Marseille under reference 16868-2018092614457139.

Immunochemistry

A total of 5 pm formalin-fixed, paraffin-embedded human or mouse sections were deparaffinized in xylene and rehydrated through a graded ethanol series. An antigen retrieval step (Dako) was performed before quenching endogenous peroxidase activity [3% (vol/vol) H2O2], Tissue sections were then incubated with CD9 as primary antibody, and immunoreactivities were visualized using the Vectastain ABC kit (PK-4001, Vector Laboratories) or Streptavidin-HRP (Dako, P0397) according to the manufacturers’ protocol. Peroxidase activity was revealed using the liquid diaminobenzidine substrate chromogen system (Dako, K3468). Counter staining with Mayer hematoxylin was followed by a bluing step in 0.1% sodium bicarbonate buffer, before final deshydration, clearance, and mounting of the sections.

Immunofluorescence

A total of 5 pm formalin-fixed, paraffin-embedded human or mouse sections were deparaffinized in xylene and rehydrated a through graded ethanol series. An antigen retrieval step (10 mM sodium citrate, 0.05% Tween 20, 95 °C) was then performed before tissue sections were pre-incubated in blocking solution [3% (wt/vol) BSA/10% (vol/vol) goat serum] for Ih. Tissue sections were incubated in a mixture of two primary antibodies against CD9 with pan- cytokeratin (PKRT, 1 :50), cytokeratin 19 (CK19, 1 : 100), alpha smooth muscle actin (aSMA, 1 :200), or platelet derived growth factor receptor beta (PDGFR0, 1 : 100) in blocking solution overnight at 4°C. After washing in PBS, slides were incubated with a mixture of two secondary antibodies in blocking solution (Alexa Fluor 568-conjugated or Alexa Fluor 488-conjugated antibody, 1 :500, Molecular Probes). Stained tissue sections were mounted using Prolong Gold Antifade reagent with DAPI (Life Technologies) before being sequentially scanned at a 20 x magnification under a fluorescent microscope (Nikon Eclipse 90i) equipped with a CCD camera (Nikon DS-1QM).

Extracellular vesicles labeling and uptake by tumor cells

EVs were labeled with PKH26 (Sigma-Aldrich), according to the manufacturer’s protocol, with some modifications. Briefly, EVs-containing pellets were resuspended in 1 mL Diluent C. Separately, 1 mL Diluent C was mixed with 2 L of PKH26. The EVs suspension was mixed with the stain solution and incubated for 4 min. The labeling reaction was stopped by adding an equal volume of 1% BSA. Labeled EVs were ultracentrifuged at 100,000 xg for 70 minutes, washed with PBS, and ultracentrifuged again. In parallel, 70 000 tumor cells are plated on glass for immunofluorescence analysis or in 12 wells for flow cytometry analysis. After 24h, the cells were washed twice in PBS IX, before adding specific media (EVs free): DMEM Fl 2, 1% serum or DMEM Fl 2, 1% delipidated serum (Lonza, DE14-840E), and hypoxia was introduced at 1% (vol/vol) O2 and 5% (vol/vol) CO2 in nitrogen atmosphere in a subchamber system (Biospherix) during 24h. Then, cells were exposed to EVs in the indicated concentrations and timing. For immunofluorescence, glass coverslip were fixed with 4% PF A for 10 min, washed in PBS, mounted using Prolong Gold Antifade reagent with DAPI (Life Technologies) and observed with a Zeiss Meta confocal microscope (LSM 510 META, Zeiss, France) with a UV laser and x 40 objectives. For flow cytometry, trypsinized cells were resuspended in lx PBS and EVs uptake was analyzed in IxlO⁴ cells using a MACSQuant VYB instrument (Milteny Biotec). For blocking experiments, anti-CD9 antibody was incubated with EVs for 2 hrs before addition to PANC-1 cells.

Migration assay

PANC-1 migration was studied using Boyden chambers. Culture inserts of 0.8 pm (BD Falcon, BD353097) with a porous membrane at the bottom (8 p pores) were coated with a mix made of 1% gelatin and 10 pg/ml fibronectin, before being seeded with PANC-1 from coculture after 24h of treatment (50 000 cells per insert) and placed into the wells containing the co-culture media. Migration was performed during 4h. After cleaning and briefly staining inserts with Coomassie blue, migration was assessed by counting the number of colored cells in 10 high power fields with the EVOS™microscope (Ozyme, Magnification 20x).

Western blot

Proteins were resolved by SDS-PAGE, transferred to nitrocellulose filters, blocked for Ih at room temperature in Tris-buffered saline / 5% non-fat dry milk / 0.1% Tween20, and blotted overnight with primary antibodies in blocking solution (p-Akt 1 : 100, Akt 1 : 100, p-p38 1 :200, p38 1 :2000, p-Erk5 1 :100, Erk5 1 : 100, p-Erkl/2 1 : 100, Erkl/2 1 : 100, p-JNK 1:200, JNK 1 :500, p-Tubulin 1 : 1000, Snail 1 : 1000, Twist 1 :200, N-Cadherin 1 :500, E-Cadherin 1 :500). After extensive washings in TBS / 0.1% Tween20, filters were incubated for Ih at room temperature with a HRP conjugated secondary antibody at 1 :5000 before being revealed with an enhanced chemiluminescence substrate (Millipore). Acquisition was performed with a Fusion FX7 imager (Vilber-Lourmat, France) and measurements of band intensities were determined using ImageJ software (National Institutes of Health). Experiments using the PathScan® Intracellular Signaling Array Kit (Chemiluminescent Readout, #7323) from Cell Signaling were done according to the manufacturer’s recommendations.

Chemicals and other reagents p38 MAPK inhibitor (SB203580), p38 MAPKa and p inhibitor (SB202190), and SB 202474 (a structural analog of SB 202190 and SB 203580 that is used as a negative control in studies of p38 inhibition) from Sigma-Aldrich were resuspended in DMSO, used as mock control, and used at lOpM as final concentration. Signal Silence® p38 MAPK siRNA (6564S, Cell Signaling Technology, Danvers, USA) were transfected at lOpM in Panc-1 cells using Oligofectamine (Life Technology). Statistical analysis

All experiments were done at least three times. One representative experiment is shown. EVs uptake and migration assays are displayed as one representative experiment of three independent experiments, mean ± s.e.m. RT-qPCR are displayed as mean ± SD of triplicates of three independent experiments. Statistical analysis was performed with Mann-Whitney U test to detect significant differences between two experimental groups. -values less than 0.05 were considered to be statistically significant, and data are presented as the median ± interquartile range or mean + SEM.

Gene expression analysis in human clinical samples

We gathered clinicopathological and gene expression data of clinical pancreatic samples from 15 publicly available data sets (data not shown). Data were collected from the National Center for Biotechnology Information (NCBI)/Genbank GEO, ArrayExpress, EGA, and TCGA databases and had been generated using DNA microarrays (Affymetrix, Agilent) and RNA-seq (Illumina). The pooled data set contained 951 samples, including 793 primary PC samples, and 158 normal pancreatic samples. The study was approved by our institutional board. A total of 756 primary PC samples were informative for disease-free survival (DFS). Data analysis required pre-analytic processing as previously published [29], CD9 expression in primary tumors was measured as discrete value by using the median expression level as cut-off and defined two tumor classes thereafter designated “CD9-high” and “CD9-low”. The molecular subtype of tumors was determined by applying to each sample in each data set separately the multigene classifiers reported by Bailey [30-32], Correlations between CD9-based tumor classes and clinicopathological features were analysed using the t-test or the Fisher’s exact test when appropriate. Disease-free survival was calculated from the date of diagnosis to the date of relapse or death from PC. Follow-up was measured from the date of diagnosis to the date of last news for event-free patients. Survivals, calculated using the Kaplan-Meier method, were compared with the log-rank test. Univariate and multivariate DFS analyses were done using Cox regression analysis (Wald test). Variables tested in univariate analyses included patients’ age at diagnosis (>60 vs <60 years), sex, AJCC stage (4, 3, 2 vs 1), anatomic location (head vs body/tail), pathological features including pathological type and grade (4, 3, 2 vs 1), molecular subtypes, and CD9-based class (“high” vs “low”). Variables with a p-value <0.05 were tested in multivariate analysis. All statistical tests were two-sided at the 5% level of significance. Statistical analysis was done using the survival package (version 3.1-12) in the R software (version 3.5.2; http://www.cran.r-project.org/). We followed the reporting REcommendations for tumor MARKer prognostic studies (REMARK criteria) [33],

Results

Identification of CD9 as a key member of CAFs-derived ANXA6⁺-extracellular vesicles. In a previous study, we demonstrated that ANXA6 dependence for CAFs-derived EVs to improve PC cancer cell aggressiveness was correlated with a modulation of EVs uptake. In order to determine the underlying mechanism of this decreased internalization of ANXA6⁺' EVs in recipient cells, we realized an i-TRAQ quantitative proteomic analysis (data not shown). Using “control” CAFs (CAFs shCtr) and CAFs depleted in ANXA6 (CAFs shANXA6) cultured in physiopathologic conditions as previously reported [22], we extracted EVs from both conditions (data not shown) then determined their specific protein composition. We highlighted 635 common proteins present in EVs produced by control CAFs and shANXA6 CAFs with, among them, 91 proteins exhibiting significant differences (data not shown). While gene ontology (GO) enrichment of the 635 proteins revealed the main biological process (BP) and cellular component (CC) processes involving CAFs-derived EVs (data not shown), a similar analysis restricted to the 91 dysregulated proteins highlighted the ones that are impacted by ANXA6 deletion (data not shown). Interestingly, we figured out that, among the dysregulated biological processes, two of them (GO: 0006928 “Cell Motion” and GO: 0016044 “membrane organization”) were related with the modified biological read-out analyzed in our precedent study [22], We found out that only four proteins were in common between those two clusters (data not shown). We decided to focus our interest on CD9, a tetraspanin membrane protein considered as one of the main exosome surface markers that was recently associated with CAFs-driven cancer cell migration in gastric cancer [34], Interestingly, using “String” (http://versionlO.string-db.org), a protein-protein interaction database, we revealed a molecular link between ANXA6 and CD9 [35], We validated the ANXA6-CD9 interaction in human PC derived CAFs cultured under physiopathologic conditions (data not shown), which suggests CD9 as a member of the ANXA6-TSP1-LRP1 complex previously described and associated with PC improved aggressiveness.

CD9 expression in human PC tumors correlates with patients’ survival. We assessed the clinical relevance of CD9 expression in human PC by analyzing mRNA expression levels in 951 clinical samples, including 793 PC primary samples and 158 normal pancreatic samples. Briefly, 68% were more than 60 year-olds and 55% were male, 93% of tumors were ductal type, 56% were grade 2, and 81% were classified as AJCC stage II. The most frequent anatomic location was pancreas head (84%). Regarding the molecular classifications, the most frequent Bailey’s subtype was “squamous” (36%), the most frequent Collisson’s subtype was “classical” (49%), and the most frequent Moffitt’s subtypes were “classical” (64%) for the tumor subtype and “normal” (54%) for the stroma subtype. All patients had been treated with primary surgery. CD9 mRNA expression was variable and different between the cancer and normal tissue samples (Figure 1A), with higher expression in PC primary samples (p=2.73E- 19, Student t-test). Such increase in CD9 mRNA expression in PC compared to healthy pancreas was also observed using endogenous mice model of PC (Pdxl-Cre/Kras^G12D/Ink4A^fl/fl mice, Figure 2). We defined two classes of PC samples based upon median expression in tumors (“CD9-high”: N=396; “CD9-low”: N=397), and searched for correlations with clinicopathological and molecular variables (data not shown). No correlation existed with patient’s age and sex, AJCC stage, tumor location, and pathological type and grade. By contrast, correlations (Fisher’s exact test) were found with the molecular subtypes: the “CD9-high” tumors were enriched in squamous then pancreatic progenitor Bailey’s subtypes (p=2.46E-05), in classical then quasi-mesenchymal Collisson’s subtypes (p=9.81E-04), and in classical Moffitt’s tumor subtype (p=1.85E-04). The figures IB, C, D show CD9 expression as continuous value per molecular subtype. Disease-free survival (DFS) data were available for 654 patients. With a median follow-up of 16 months (range, 1 to 156), 465 patients (71%) had a DFS event, the 2-year DFS was 36% (95%CI, 33-41), and the median DFS was 18 months (range, 1 to 156). CD9 expression was associated with DFS with 31% 2-year OS (95%CI, 26- 37) in the “CD9-high” class versus 42% (95%CI, 36-48) in the “CD9-low” class (p=1.35E-02, log-rank test; Figure IE), with hazard ratio (HR) for DFS event equal to 1.26 (95%CI, 1.05- 1.51) in the “CD9-high” class when compared with the “CD9-low” class (p=1.37E-02, Wald test). In univariate analysis (data not shown), the other variables associated with DFS (Wald test) included AJCC stage (p=1.07E-04), and three molecular classifications from Bailey with p=1.48E-07, Collisson with p=8.15E-04, and Moffitt’s tumor type (p=1.54E-04). More importantly, in multivariate analysis (data not shown), CD9 expression remained significant when confronted to AJCC stage (HR=1.37 (95%CI, 1.11-1.69); p=3.68E-03, Wald test) and when confronted to both AJCC stage and the molecular classifications (HR=1.29 (95%CI, 1.04- 1.61); p=1.95E-02, Wald test), suggesting independent prognostic value. All together those data highlight that CD9 expression correlates with the most aggressive PC subtypes from each classification and reveal CD9 as an independent marker of poor prognostic. CD9 is mainly expressed in CAFs within the tumor microenvironment of PC. Regarding our hypothesis that implies CD9 as a member of the ANXA6/LRP1/TSP1 complex involved in CAFs-derived EVs uptake with a consequent improvement of PC cell aggressiveness, we first assessed CD9 expression in human PC samples. Immunohistological analyses revealed a majority of staining in stromal areas in human (data not shown) and mouse (data not shown) PC samples. Interestingly, the increased CD9 expression in pathological tissues seems restricted to tumor samples as not observed in inflammatory tissues such as chronic pancreatitis (Figure 2B). Refining cell type expressing CD9 was done by co-staining of CD9 with aSMA and PDGFR0 markers for CAFs labeling, PKRT (pan-cytokeratin) and CK19 for tumor cells in mouse PC samples. Interestingly, we confirmed a CAFs-restricted CD9 expression similarly to the ANXA6 expression previously reported [22], Indeed, CD9 staining was almost negative in PKRT⁺and CK19⁺ cells while observed in aSMA⁺ CAFs and PDGFR0⁺ CAFs (data not shown). In vitro, CD9 expression was heterogeneous and not correlated with ANXA6 expression in normal human fibroblasts as in primary (pCAFs) and immortalized CAFs (iCAFs) derived from human PC, cultured in classical condition (data not shown). Interestingly, when CAFs were cultured under physiopathologic conditions, CD9 mRNA expression showed a drastic and significant increase of 158 fold (Figure 3). Such increased was confirmed at the protein level (data not shown) and restricted to CAFs, when cultured under physiopathologic culture condition (data not shown). In similar culture conditions, CD9 protein levels were also increased in CAFs-derived EVs (data not shown). Altogether, those data confirm that CD9 is expressed by CAFs in vivo and in vitro, and further reinforced under physiopathologic culture condition, strengthening our hypothesis that suggests CD9 as a pivotal protein involved in ANXA6⁺-EVs mediated CAFs/tumor cells crosstalk.

CD9 blockage impairs CAFs-derived ANXA6⁺-EVs uptake by cancer cells and the consequent increased migration. While we associated the stromal expression of CD9 in PC with decreased patients’ survival, its underlying mechanism and biological relevance in PC remain unclear. To assess the direct implication of CD9 in CAFs-derived ANXA6⁺-EVs internalization by pancreatic cancer cells, we incubated EVs preparations with CD9 blocking antibody. To avoid batch or clonal effect, EVs were prepared from either human Normal Fibroblasts (NHF), immortalized human PC-derived CAFs (iCAFs), or primary Human PC- derived CAFs (CAFs), cultured under physiopathologic condition. EVs were extracted and fluorescently labelled (with PKH26) then pancreatic cancer cells were incubated with these PKH26⁺-EVs for 2 hours and PKH26⁺ cells were sorted then quantified (Figure 4A). While variations were observed depending on EVs’ origin, a significant reduction in EVs internalization was systematically revealed following incubation with CD9-blocking antibody. Indeed, NHF-derived EVs revealed a decreased uptake by pancreatic cancer cells of 25% with a maximum of 37.5% (Figure 4B), while for iCAFs-derived EVs a maximum of 56% decrease in EVs uptake was observed (Figure 4C). Interestingly, inhibition of macropinocytosis using EIP A strongly reduced EVs uptake, co-treatment of EIP A and CD9 blocking antibody further reduced EVs uptake confirming that CD9 is involved in a specific EVs internalization process (Figure 4D). As previously reported, CAFs-derived EVs uptake was associated with increased migration abilities in human pancreatic cancer recipient cells [22], We further evaluated the implication of CD9 in such biological outcome and demonstrated that incubation of CAFs derived-EVs with CD9-blocking antibody reduced cancer cell migration by 30% (Figure 4E). Those data confirm that CD9 is involved in CAFs-derived EVs internalization by pancreatic cancer cells and their consequent improvement of migratory abilities. p38MAPK activation is mandatory to improve migratory ability and cellular plasticity of pancreatic cancer cells following CAFs-derived ANXA6⁺CD9⁺-EVs uptake. We next deepened the mechanistic signaling pathway linking CAFs-derived ANXA6⁺-EVs uptake and improved migratory ability through the use of an intracellular signaling array kit and then determined if CD9 was mandatory for the activated pathways. Protein level analyses of human pancreatic cancer recipient cells following uptake of CAFs-derived ANXA6⁺-EVs revealed that p38MAPK was the most activated pathway among the one investigated, while the incubation with shANXA6 CAFs-derived EVs could not lead to p38MAPK activation (Figure 5A). We confirmed this data by comparing five main signaling pathways in human pancreatic cancer cells following incubation with CAFs-derived EVs and observed that phospho- p38MAPK was increased 2.6 fold following EVs uptake compared to 1.3 fold for phospho-Akt, 0.8 fold for phospho-Erk5, 1.6 fold for phospho-Erkl/2 and 0.8 fold for phospho-JNK (data not shown). The use of two different shANXA6 revealed that increased phospho-p38MAPK was not observed following incubation of ANXA6' EVs, with protein levels comparable to recipient cells not incubated with EVs, suggesting the key role of ANXA6 and EVs uptake in p38MAPK activation (data not shown). Interestingly, CD9 blocking antibody induced a similar impairment of p38MAPK activation following ANXA6⁺CD9⁺-CAFs-derived EVs treatment (data not shown), reinforcing our hypothesis that CD9 is mandatory for CAFs- derived EVs uptake and signaling pathway activation in pancreatic cancer recipient cells. In regards to those data, we suspected that p38MAPK activation following uptake of CAFs- derived EVs could lead to enhanced migratory activity in recipient cells. p38MAPK blockage using two chemical inhibitors (SB203580 and SB202190) and their mock control (SB202474) in pancreatic cancer cells following CAFs-derived EVs uptake (Figure 5B) impaired the consequent increase in migratory ability (Figure 5C). We confirmed such data using a p38MAPK siRNA in recipient pancreatic cancer cells (Figure 5D) as ANXA6⁺CD9⁺-CAFs- derived EVs could no longer improve pancreatic cancer cell migration when cancer cells were transfected with p38MAPK siRNA prior to EVs treatments (Figure 5E). Supporting the enhanced migratory ability of tumor cells following CAFs-derived EVs uptake, we observed that Epithelial-to-Mesenchymal (EMT) markers such as SNAIL, TWIST and N-CADHERIN were increased in pancreatic cancer cells following CAFs-derived EVs uptake, with a concomitant reduction of the epithelial marker E-CADHERIN (data not shown). The use of shANXA6 CAFs-derived EVs could no longer drive such changes on epithelial and EMT markers in pancreatic cancer cells (data not shown), in accordance with the absence of improved migratory abilities [22], Interestingly, the increased CD9 mRNA level during PC development in endogenous mouse model (Figure 2A) was concomitant to an increased mRNA level of EMT markers (Figure 6). These data suggest the mandatory activation of p38MAPK pathway by CAFs-derived ANXA6⁺CD9⁺-EVs to improve pancreatic cancer cell migration and cellular plasticity.

Discussion

We previously demonstrated that, under hostile environment, CAFs within intra- tumoral microenvironment of PC release specific ANXA6⁺-EVs that improve cancer cell aggressiveness [22], While this study shed some light on the biological relevance of such cellular crosstalk, the mechanism involved in the modulation of EVs’ uptake as well as the molecular changes induced in tumor cells following EVs signals remained unknown. In the present study, we purified EVs from Cancer-Associated Fibroblasts cultured in physiopathologic conditions (mimicking PC hostile environment) to investigate their internalization process and deepen their molecular impact on pancreatic cancer recipient cells. We determined the tetraspanin CD9 as a new cargo of CAFs-derived ANXA6⁺-EVs, member of the previously described ANXA6/LRP1/TSP1 complex and involved in their internalization. We further highlighted that, following uptake of CAFs-derived ANXA6⁺/CD9⁺-EVs, pancreatic cancer recipient cells activate p38MAPK pathway as a mandatory process to improve their cellular plasticity and aggressive phenotype highlighted by an enhanced migratory ability. REFERENCES:

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13. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell. 2012;151 : 1542-56. 14. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014;74:2913-21.

15. Neesse A, Bauer CA, Ohlund D, Lauth M, Buchholz M, Michl P, et al. Stromal biology and therapy in pancreatic cancer: ready for clinical translation? Gut. 2019;68: 159-71.

16. Nicolle R, Blum Y, Marisa L, Loncle C, Gayet O, Moutardier V, et al. Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived Xenografts. Cell Rep. 2017;21 :2458-70.

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Claims

- 49 - WO 2023/089159 PCT/EP2022/082555 CLAIMS:

1. A method for predicting the survival time of a patient suffering from a cancer with preponderant stroma, comprising i) determining the expression level of CD9 in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

2. The method for predicting the survival time of a patient suffering from a cancer with preponderant stroma according to claim 1, comprising i) isolating extracellular vesicles from a sample obtained from the patient ii) determining the expression level of CD9 in extracellular vesicles from the sample iii) providing a good prognosis when the expression level determined at step ii) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step ii) is higher than its predetermined reference value.

3. A method for predicting the aggressiveness of a cancer with preponderant stroma, comprising i) determining the expression level of CD9 in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

4. The method for predicting the aggressiveness of a cancer with preponderant stroma according to claim 3, comprising i) isolating extracellular vesicles from a sample obtained from the patient ii) determining the expression level of CD9 in extracellular vesicles from the sample iii) providing a good prognosis when the expression level determined at step ii) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step ii) is higher than its predetermined reference value. - 50 -

WO 2023/089159 PCT/EP2022/082555

5. The method according to claim 2 or 4, wherein the extracellular vesicles are Cancer- Associated Fibroblast (CAF)-derived extracellular vesicles.

6. The method according to claim 2 or 4, wherein the extracellular vesicles are Cancer- Associated Fibroblast (CAF)-derived ANXA6⁺ extracellular vesicles.

7. The method according to any of claims 1 to 6, wherein the cancer with preponderant stroma is a pancreatic cancer, a cholangiocarcinoma, a squirrhus of breast, Hodgkin Lymphoma, colorectal cancer or prostate cancer.

8. A CD9 inhibitor for use in the treatment of a cancer with preponderant stroma in a subject in need thereof.

9. The CD9 inhibitor according to claim 8, wherein the CD9 inhibitor is an antibody.

10. The CD9 inhibitor according to claim 8 or 9, and at least one anti-cancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a cancer with preponderant stroma.

11. The CD9 inhibitor according to claim 10, wherein the at least one anti-cancer agent is gemcitabine and/or FOLFIRINOX and/or a p38MAPK inhibitor.

12. The CD9 inhibitor for use according to claim 8 to 11, wherein the cancer with preponderant stroma is a pancreatic cancer, a cholangiocarcinoma, a squirrhus of breast, Hodgkin Lymphoma, colorectal cancer or prostate cancer.

13. The CD9 inhibitor for use according to claim 8 to 12, wherein the cancer with preponderant stroma is a metastatic cancer.

14. A method to perform the differential diagnostic between pancreatic cancer and pancreatitis, comprising a step of determining the expression level of CD9 in a sample obtained from the patient. - 51 -

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15. A method for treating a pancreatic cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CD9 inhibitor.