CN115372611B

CN115372611B - Application of CD16+ fibroblast in diagnosis, prevention and treatment of monoclonal antibody drug-resistant breast cancer

Info

Publication number: CN115372611B
Application number: CN202210844207.1A
Authority: CN
Inventors: 苏士成; 陆艺文
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2022-07-18
Filing date: 2022-07-18
Publication date: 2023-05-30
Anticipated expiration: 2042-07-18
Also published as: CN115372611A

Abstract

The invention discloses application of CD16+ fibroblasts in diagnosing, preventing and treating monoclonal antibody drug-resistant breast cancer. CD16 based ⁺ Different distribution of fibroblast in monoclonal antibody sensitive breast cancer cells and monoclonal antibody drug resistant breast cancer cells, CD16 ⁺ Fibroblast cells can be used as a marker for predicting, diagnosing and/or pre-treating breast cancer monoclonal antibody resistanceIn the treatment effect of the breast cancer monoclonal antibody, whether the breast cancer of different individuals has potential monoclonal antibody resistance or has monoclonal antibody resistance can be judged in time, so that the appropriate treatment strategy can be adjusted and given in time, and the survival time of a patient can be prolonged. Furthermore, by interfering with the expression of VAV2 in tumor cells, the CD16 can be effectively reversed ⁺ Drug resistance caused by fibroblasts improves the response of breast cancer individuals to monoclonal antibodies, improves the sensitivity of breast cancer to monoclonal antibody treatment, and improves the HER2 targeting therapeutic effect of breast cancer.

Description

Application of CD16+ fibroblast in diagnosis, prevention and treatment of monoclonal antibody drug-resistant breast cancer

Technical Field

The invention relates to the field of tumor molecular biology, in particular to application of CD16+ fibroblasts in diagnosing, preventing and treating monoclonal antibody drug-resistant breast cancer.

Background

Breast cancer has become the most frequently occurring malignancy worldwide. The traditional breast cancer treatment means mainly comprise operation, radiotherapy, chemotherapy and endocrine treatment. But chemotherapy damages normal cells of the body while killing tumors, and has obvious side effects. In recent years, the advent of monoclonal antibody class drugs (mabs) has addressed this challenge. Through the specific combination of antigen-antibody, monoclonal antibody medicine can identify and combine with tumor cell unique marker, so that tumor cell is specifically killed without damaging normal tissue, tumor can be effectively controlled, side effect of treatment is greatly reduced, and the monoclonal antibody medicine is a great breakthrough in the field of tumor treatment. At present, monoclonal antibody drugs have been widely used in various diseases such as tumors, autoimmune diseases, graft versus host reactions, and the like.

Although monoclonal antibodies hold new promise for tumor therapy, some patients cannot benefit from therapy. Trastuzumab is currently the most widely used monoclonal antibody in breast cancer, and targets at epidermal growth factor receptor 2 (human epidermal growth factor receptor, her 2). HER2 positive breast cancer patients account for about 15% -30% of the total number of breast cancers, and HER2 positive breast cancers have the clinical characteristics of strong wettability, rapid progress, high recurrence risk, poor prognosis and the like. Targeted therapy against HER2 molecules can greatly improve survival prognosis for this portion of patients. However, breast cancer is characterized by heterogeneity among patients, and not all HER2 positive patients can benefit from anti-HER 2 targeted therapies. For patients with poor response to HER2 targeting therapy, this treatment not only delays precious treatment time, but also introduces unnecessary adverse effects and treatment costs. It is noted that there are no other molecular markers that can effectively predict the effect of anti-HER 2 targeted therapies, other than the amount of HER2 protein expression and gene amplification. The single judgment means may cause the risks of missed detection and false detection to be increased, which is undoubtedly unfavorable for setting a correct treatment strategy for a breast cancer patient as early as possible so as to prolong the survival time of the patient. In the context of the current diversification of treatment regimens, how to formulate an individualized treatment regimen for a patient is a significant challenge in the field of breast cancer treatment, and therefore, solving the above-mentioned problems is of great clinical significance.

Disclosure of Invention

The present invention is directed to overcoming at least one of the deficiencies of the prior art described above and providing a use of CD16+ fibroblasts for diagnosis, prevention and treatment of monoclonal antibody resistant breast cancer by CD16 ⁺ The fibroblast marker is convenient for predicting and diagnosing whether a patient can benefit from anti-HER 2 targeted therapy, and can promote the anti-HER 2 monoclonal antibody targeted therapy effect by neutralizing or knocking down CD16 antigen and interfering with the expression of breast cancer cell VAV 2.

It is an object of the present invention to provide CD16 ⁺ Use of fibroblasts as a marker for drug resistance in breast cancer monoclonal antibody therapy.

It is a further object of the present invention to provide a drug-resistant marker for breast cancer monoclonal antibody therapy, which is CD16 ⁺ Fibroblasts.

It is a further object of the present invention to provide CD16 ⁺ The application of the fibroblast detection reagent in preparing products for predicting and diagnosing breast cancer monoclonal antibody treatment resistance and/or predicting breast cancer monoclonal antibody treatment effect and/or predicting disease-free survival time of breast cancer patients.

Further, the breast cancer is HER2 positive breast cancer; and/or the monoclonal antibody drug comprises trastuzumab and/or pertuzumab.

Further, CD16 ⁺ Fibroblast detection reagent comprising and CD16 ⁺ Fibroblast cell surfaceAntibodies that react with the antigen-antibody of the surface antigen CD 16. Further, also includes CD16 ⁺ Fibroblast extraction reagent.

It is a further object of the present invention to provide CD16 ⁺ The application of the fibroblast in preparing products for predicting and diagnosing the drug resistance of the breast cancer monoclonal antibody treatment and/or prognosis of the curative effect of the breast cancer monoclonal antibody treatment and/or predicting the disease-free survival period of a breast cancer patient.

Further, the product comprises CD16 ⁺ Fibroblast detection kit.

It is a further object of the present invention to provide a kit for predicting, diagnosing, and/or prognosing the therapeutic efficacy of a breast cancer monoclonal antibody therapy and/or predicting the disease-free survival of a breast cancer patient, comprising a kit for predicting the drug resistance of a breast cancer monoclonal antibody therapy and/or prognosing the disease-free survival of a breast cancer patient ⁺ An antibody which causes an antigen-antibody reaction with the fibroblast surface antigen CD 16.

It is a further object of the present invention to provide the use of a connective tissue formation inhibitor for breast cancer in the manufacture of a product for the prevention and/or treatment of monoclonal antibody resistant breast cancer. Further, inhibitors of connective tissue formation of breast cancer include CAFCD16-SYK-VAV2-RhoA-MLC2-MRTF-A pathway inhibitors; further, the CAFCD16-SYK-VAV2-RhoA-MLC2-MRTF-A pathway inhibitor includes CD16 ⁺ Inhibitors of fibroblast antigen CD16, SYK inhibitors, VAV2 inhibitors, rhoA inhibitors, MLC2 inhibitors and/or MRTF-Sub>A inhibitors; further, CD16 ⁺ Inhibitors of the fibroblast antigen CD16 include and CD16 ⁺ Neutralizing antibodies and/or CD16 binding to fibroblast surface antigen CD16 ⁺ Fibroblast antigen CD16 shRNA; and/or, the SYK inhibitor comprises a SYK shRNA; and/or, the VAV2 inhibitor comprises a VAV2 shRNA; and/or, the RhoA inhibitor comprises a RhoA shRNA; and/or, MLC2 inhibitors include MLC2 shRNA; and/or, the MRTF-A inhibitor includes MRTF-A shRNA.

It is a further object of the present invention to provide a product for the prevention and/or treatment of monoclonal antibody resistant breast cancer comprising a breast cancer connective tissue formation inhibitor.

Further, the monoclonal antibody drug comprises trastuzumab andor pertuzumab; further, the breast cancer is HER2 positive breast cancer. Further, inhibitors of connective tissue formation of breast cancer include CAFCD16-SYK-VAV2-RhoA-MLC2-MRTF-A pathway inhibitors; further, the CAFCD16-SYK-VAV2-RhoA-MLC2-MRTF-A pathway inhibitor includes CD16 ⁺ Inhibitors of fibroblast antigen CD16, SYK inhibitors, VAV2 inhibitors, rhoA inhibitors, MLC2 inhibitors and/or MRTF-Sub>A inhibitors; further, CD16 ⁺ Inhibitors of the fibroblast antigen CD16 include and CD16 ⁺ Neutralizing antibodies and/or CD16 binding to fibroblast surface antigen CD16 ⁺ Fibroblast antigen CD16 shRNA; and/or, the SYK inhibitor comprises a SYK shRNA; and/or, the VAV2 inhibitor comprises a VAV2 shRNA; and/or, the RhoA inhibitor comprises a RhoA shRNA; and/or, MLC2 inhibitors include MLC2 shRNA; and/or, the MRTF-A inhibitor includes MRTF-A shRNA.

It is a further object of the present invention to provide a CD16 ⁺ Use of a neutralizing antibody binding to the fibroblast surface antigen CD16 for the preparation of a product for the prevention and/or treatment of monoclonal antibody resistant breast cancer.

It is a further object of the present invention to provide a CD16 targeting ⁺ Use of shRNA of fibroblast surface antigen CD16 in the preparation of a product for the prevention and/or treatment of monoclonal antibody resistant breast cancer.

It is still another object of the present invention to provide a product for preventing and/or treating monoclonal antibody-resistant breast cancer, comprising a polypeptide which is conjugated with CD16 ⁺ Neutralizing antibodies to fibroblast surface antigen CD16 binding and/or targeting CD16 ⁺ shRNA of fibroblast surface antigen CD 16; further, the monoclonal antibody comprises trastuzumab and/or pertuzumab; further, the breast cancer is HER2 positive breast cancer.

In one or more embodiments of the invention, a population of CD16 positive non-white blood cells was first found in tumor tissue of a breast cancer patient, and further analysis demonstrated that this population of cells was tumor-associated fibroblasts (Carcinoma-associated fibroblasts, CAFs). The present inventors have found that by analyzing tissue sections of breast cancer patients, compared to treatment-sensitive group patients with trastuzumab,CD16 in tumor specimens of drug-resistant group patients ⁺ The number of CAFs is greater; also, CD16 in tumor tissue ⁺ The number of CAFs is significantly inversely related to the disease-free survival of the patient. In animal models, using a patient-derived xenograft (PDX) model, the inventors found CD16 ⁺ More PDXs of CAFs are less reactive towards trastuzumab. In addition, breast cancer cell lines and CD16 were received ⁺ CAFs co-injected mice were more reactive to trastuzumab than CD16 ^- The CAFs co-injected mice were worse and this phenomenon was also demonstrated in the treatment of pertuzumab. Thus, by detecting breast cancer tissue CD16 ⁺ The cell number of the CAFs can predict and diagnose whether the patient is resistant to monoclonal antibody targeted therapy. Further, the present inventors have found in experiments that the use of neutralizing antibodies to CD16 in tumor-bearing immunodeficient mice can increase the responsiveness of the mice to trastuzumab treatment. In the co-injection model of tumor cells and fibroblasts, mice had increased therapeutic responsiveness to trastuzumab when CD16 expressed by the fibroblasts was selectively knocked out. I.e. by targeting CD16 ⁺ The fibroblast and the downstream passage thereof are beneficial to reversing the drug resistance of the breast cancer to the monoclonal antibody, thereby promoting the treatment of the monoclonal antibody resistant breast cancer.

Compared with the prior art, the invention has the beneficial effects that: CD16 based ⁺ Different distribution of fibroblast in monoclonal antibody sensitive breast cancer cells and monoclonal antibody drug resistant breast cancer cells, CD16 ⁺ The fibroblast can be used as a marker for predicting and diagnosing the drug resistance of the breast cancer monoclonal antibody and/or predicting the curative effect of the breast cancer monoclonal antibody and/or predicting the disease-free survival time of a breast cancer patient, and is helpful for timely judging whether the breast cancer cells of different individuals have the drug resistance of the potential monoclonal antibody or the drug resistance of the existing monoclonal antibody, so that a proper treatment strategy can be timely adjusted and given, invalid or low-effect targeted HER2 treatment can be avoided, and other effective treatment strategies can be timely adopted, so that the survival time of the patient can be prolonged. In one or more embodiments of the invention, the tumor cell V is detected by interventionExpression of AV2 can effectively reverse CD16 ⁺ Drug resistance caused by fibroblasts improves the response of breast cancer individuals to monoclonal antibodies, improves the sensitivity of breast cancer to monoclonal antibody treatment, improves the HER2 targeting treatment effect of breast cancer, and achieves the purpose of prolonging the survival time of patients.

Drawings

Fig. 1 shows: comprehensive profile of fcγr expressing cells in human breast cancer. (A) Flow cytometry analysis of human FcgammaRs (FcgammaRI/CD 64, fcgammaRIIa/CD 32a, fcgammaRIIb/CD 32b, fcgammaRIIC/CD 32c and FcgammaRIII/CD 16) at CD45 ^- Populations and leukocytes (including CD3 ⁺ T cells, CD19 ⁺ B cell, CD56 ⁺ NK cells and CD11b isolated from clinical breast cancer specimens ⁺ Myeloid cells). Quantization of (B) (a) (n=32). Mean ± SEM. (C) Representative images of CD16 and CD45 immunofluorescent staining in human breast cancer tissue (n=26). The asterisks indicate the region of higher left-hand upper corner amplification. Scale bar, 50 μm. Please refer to fig. 9.

Fig. 2 shows: CD16 ⁺ Fibroblast subpopulations and HER2 ⁺ Trastuzumab resistance in breast cancer patients is relevant. (A) Flow cytometry analysis of CD16 expression of CAF in human breast cancer tissue; isolated cells are first gated on Forward (FSC) and Side Scatter (SSC) to exclude debris, then on FVD ^- 、CD45 ^- 、EpCAM ^- 、CD31 ^- Gating was performed to exclude dead cells, leukocytes, tumor cells and endothelial cells, respectively, and CD16 and pdgfrβ expression was further analyzed (n=68). (B) CD16 ⁺ Representative flow cytometry plots of αsma and FAP levels in CAF (n=14). Values represent mean ± SEM. (C) At CD16 ⁺ Representative bright field images and immunofluorescent staining of CD16 and αsma before and after Fluorescence Activated Cell Sorting (FACS) of CAF. Scale bar, 50 μm. (D) Representative immunofluorescence images of CD16 and αsma staining in untreated breast cancer samples. The numerals indicate regions of higher magnification, as shown in the right figure. Scale bar, 50 μm. (E) CD16 of each microscope high power mirror field in breast cancer tissue of patients not receiving neoadjuvant treatment of different molecular subtypes ⁺ CAF number. HR (HR) ⁺ HER2 ^- Hormone receptor positive and HER2 negative, n=488; HER2 ⁺ HER2 positive, n=231; TN, triple negative, n=95; mean.+ -. SEM, # p, by Kruskal-Wallis test<0.05，***p<0.001。(F)HER2 ⁺ Kaplan-Meier curve for disease-free survival of breast cancer patients, comparing CD16 in tumors without neoadjuvant treatment ⁺ CAFs infiltration is low (less than or equal to 9.1 cells/field of view) and high>Patient disease free survival time (n=231) of 9.1 cells/field of view. (G) Trastuzumab pre-adjuvant chemotherapy ^- ) And thereafter (post) ^- ) HER2 of (2) ⁺ CD16 per field in breast cancer samples ⁺ CAF number. Patients with complete remission of pathology (pCR), breast/lymph node only pCR, or Partial Remission (PR) were classified as sensitive (n=262), while patients with Stable Disease (SD) or disease Progression (PD) were classified as resistant (n=165). Mean ± SEM, p of Mann-Whitney U test <0.001. Please refer to fig. 10.

Fig. 3 shows: in a patient-derived xenograft model, CD16 ⁺ Fibroblasts impair the efficacy of trastuzumab. (A) H of human aSMA and CD16 in initial clinical samples and corresponding patient-derived xenografts (PDX) harvested from mice&Representative images of E staining and immunofluorescent staining. The asterisks indicate the region of higher magnification in the lower right corner. Scale bar, 50 μm. (B) Quantification of CD16 in patient primary tumor specimens and corresponding PDXs by immunofluorescent staining (n=16) ⁺ αSMA ⁺ Number of cells; ns, paired Student's t tested that the difference was not significant. (C) Quantitative analysis of CD16 in patient primary tumor specimens and corresponding PDXs by immunofluorescent staining (n=16) ⁺ αSMA ⁺ Number of cells; * P<0.001 was checked by pairing Student's t. (D and E) will have a high or low CD16 ⁺ CAF-infiltrated HER2 ⁺ Primary tumor specimens were implanted into NOD SCID mice; following successful establishment of PDX, mice were treated with trastuzumab for eight weeks. (D) Representative images of tumor growth monitored by positron emission tomography/computed tomography (PET/CT) (left panel) and fold change in tumor size following trastuzumab treatment (right panel); circles represent tumors; each line represents a successfully established PDX. (E) CD16 ⁺ CAFs ^high (n=7) and CD16 ⁺ CAFs ^low (n=18) the response rate of breast cancer sample PDX to trastuzumab; assessing treatment response according to RECIST criteria; * P<0.01, fisher's exact test. (F and G) will have a high CD16 ⁺ CAF-infiltrated HER2 ⁺ Primary tumor specimens were implanted into NOD SCID mice (P-862); after successful establishment of PDX, mice were treated with trastuzumab, anti-human IgG alone, or with CD16 neutralizing antibodies or IgG treatment with trastuzumab along with (trastuzumab plus anti-CD 16 treatment group n=8, other groups n=7). (F) fold change in size of PDXs tumor. (G) response rate of PDX. * Accurate inspection of p by Fisher<0.001. Please refer to fig. 11.

Fig. 4 shows: CD16 ⁺ Fibroblasts impair trastuzumab efficacy in co-injection models. (A) BT-474 breast cancer cells were inoculated alone or co-injected with designated CAF into mammary fat pads of NOD SCID mice; when xenografts are palpable, mice are treated without or with trastuzumab/IgG; tumor growth curves are shown (n=6 per group). (B) CD16 ⁺ CAF transduction was performed without or with shRNA against GFP/CD16 lentivirus; BT-474 tumor cells were inoculated alone or co-injected with designated CAF into mammary fat pads of NOD SCID mice; treatment of mice with trastuzumab when xenografts are accessible; tumor growth curves are shown (n=6 per group). (C) CD16 ^- CAFs express CD 16-FcRgamma (CD 16/gamma) and CD 16-FcRgamma in the absence of empty vector (vec) ^mut (CD16/γ ^mut ) Transduction in the case of lentiviruses of vectors of (1) or in the presence of empty vector (vec) or expression of CD 16-FcRgamma (CD 16/gamma) or CD 16-FcRgamma ^mut (CD16/γ ^mut ) Transduction in the case of lentiviruses of the vector; when xenografts are accessible, BT-474 cells are seeded with designated CAF in NOD SCID mice treated with IgG, trastuzumab, or pertuzumab; tumor size was measured on day 43 (n=6 per group). (D) BT-474 cells and CD16 ⁺ CAF or CD16 ^- CAF co-injected into NOD SCID mice; simultaneously, treating tumor-bearing mice with trastuzumab and/or an anti-CD 16 neutralizing antibody; from day 36, thymidine was administered daily to miceThe analog 5-ethynyl-2' -deoxyuridine (EdU) was continued for 7 days and sacrificed on day 43; representative flow cytometry plots (left) and quantification (right) of the percentage of EdU-labeled specific CAF in tumors of mice treated with IgG or trastuzumab are shown (n=5 per group). Values represent mean ± SD. Mean ± standard deviation; ns, not significant; * P is p<0.05；**p<0.01 and p<0.001 by one-way analysis of variance. Please refer to fig. 12.

Fig. 5 shows: CD16 ⁺ CAF is closely related to connective tissue hyperplasia and trastuzumab resistance in patients. (a and B) analyzed HER2 sensitive (n=21) or resistant (n=13) to trastuzumab neoadjuvant therapy ⁺ Tumor samples after breast cancer patient treatment. (A) Representative images of sirius red staining in tumor sections (left) and quantification (right). Scale bar, 50 μm. Mean ± SEM; * P<0.001,Student's t. (B) Representative images (left) and quantification (right) of tumor stroma hardness distribution measured by Atomic Force Microscopy (AFM); scale bar, 5 μm; mean ± SEM; * P<0.001,Student's t. (C and D) HER2 was collected with trastuzumab neoadjuvant therapy ⁺ Paired samples before and after breast cancer patient treatment (n=34); CD16 ⁺ The abundance of CAF and the hardness of the matrix are respectively quantified by immunofluorescence and AFM; showing CD16 in the post-treatment (C) and pre-treatment (D) samples ⁺ Correlation between CAF abundance and elastic modulus (r, pearson correlation coefficient). (E and F) collection of HER2 receiving trastuzumab neoadjuvant therapy ⁺ Paired paraffin-embedded tumor sections before and after breast cancer patient treatment (n=5 patients). (E) Representative immunofluorescence images of CD16, α -SMA and CK staining in paired pre-and post-treatment samples. The numerals indicate enlarged areas in the illustrations. Scale bar, 50 μm. (F) Calculating the distance between the tumor cells and the nearest specific CAF in three randomly selected areas of each patient; a distribution curve (upper) and a scatter plot (lower) showing the distance from tumor cells to a specific CAF; each dot represents one tumor cell (average n=98 tumor cells per image); mean ± SEM; * P <0.001, one-way analysis of variance.

Fig. 6 shows: CD16 ⁺ Fiber formingCells reduce vascular perfusion and intratumoral drug delivery by enhancing connective tissue formation. (A-E) BT-474 cells and CD16 ^- Or CD16 ⁺ CAF was injected into mammary fat pads of NOD SCID mice, which were treated with trastuzumab or IgG when xenografts were palpable; after 6 weeks, tumors were harvested (n=6 per group). (A) Micro sirius red staining and immunohistochemical staining representative images of collagen I, collagen III and hyaluronic acid in tumor sections; scale bar, 50 μm. (B) Representative images of tumor stroma stiffness (hardness) distribution measured by AFM; scale bar, 5 μm. (C) Mice were injected with lectin-FITC and anti-CD 31-PE via the tail vein prior to sacrifice; shows CD31 in tumors ⁺ Representative immunofluorescence images of lectin perfusion in blood vessels; scale bar, 50 μm. (D) Mice were injected with Hoechst and anti-CD 31-PE via tail vein prior to sacrifice; representative immunofluorescence images showing Hoechst leakage in tumor vessels; scale bar, 100 μm. (E) Mice were injected with fluorescence-labeled trastuzumab and anti-CD 31-FITC prior to sacrifice; displaying a 3D visual representative image of trastuzumab drug distribution in a tumor; scale bar, 50 μm. (F-I) Co-injection of EO771 cells expressing human ErbB2 and mouse fibroblasts that force FcgammaRIII into immunocompetent pWAP-human ErbB2 (ErbB 2) ⁺ ) In the fat pad of mice, tumors were reached when treated with 4D5 or IgG. (F) tumor growth curve (n=6 per group). (G) representative images of micro sirius red staining in tumors; scale bar, 50 μm. Quantification of (H) (G) (n=6 per group). (I) Tumor invasive CD49b ⁺ NK cells and CD8 ⁺ Quantification of T cells (n=6 per group); mean ± standard deviation; ns, not significant; * P is p<0.05,***p<0.001 by one-way analysis of variance. See fig. 13 and 14.

Fig. 7 shows: CD16 ⁺ Fibroblast-induced connective tissue proliferation is triggered by the SYK-VAV2-RhoA-ROCK-MLC2-MRTF-A pathway. (A) Treatment of CD16 with HER 2-tagged magnetic beads in the presence of IgG or trastuzumab ^- Or CD16 ⁺ CAF. Phosphorylation of SYK, VAV2 and MLC2, GTP-bound form of RhoA, αsma and levels of collagen I (n=3) were determined by western blot. (B) Marking phalloidinCD16 ^- Or CD16 ⁺ CAF was embedded in FITC conjugated fibrous collagen matrix and treated with HER2 labeled magnetic beads in the presence of IgG or trastuzumab. Representative immunofluorescence images (left) and quantification (right) of collagen compression levels are shown (n=6). Scale bar, 50 μm. (C) Representative immunofluorescence images of F-actin and p-MLC2 staining in CAF (left) (A) and quantification (right). Scale bar, 50 μm (n=6). (D) Treatment of CD16 with HER 2-labeled magnetic beads and trastuzumab ^- CAFs, and treatment of CD16 with HER 2-labeled magnetic beads and trastuzumab ⁺ CAFs (transduced without or with lentiviral shRNA against SYK, VAV2, RHOA or MLC 2); specific proteins were determined by western blot (n=3). (E) CD16 ^- /CD16 ⁺ CAF was treated with HER 2-labeled magnetic beads in the presence of IgG/trastuzumab; representative immunofluorescence images of F-actin and MRTF-A; scale bar, 50 μm. Quantization of (F) (E) (n=6). (G) CD16 without shRNA/shGFP/MRTF-A shRNA transduction with HER 2-labeled magnetic beads in the presence/absence of trastuzumab ⁺ CAF processing. Representative immunoblots corresponding to MRTF-A, alphSub>A SMA and collagen I in nuclear (Nuc) or cytoplasmic (Cyto) fractions. Histone H1 and GAPDH were used as controls for nuclear and cytoplasmic proteins, respectively (n=3). Mean ± standard deviation; * P<0.001, one-way analysis of variance. Please refer to fig. 15.

Fig. 8 shows: targeting VAV2 to inhibit CD16 ⁺ Fibroblast-induced connective tissue proliferation. (A and B) CD16 transduced with GFPshRNA or VAV2 shRNA ⁺ CAF, treated with HER2 labeled magnetic beads and trastuzumab. (A) Representative immunofluorescence images of F-actin and p-MLC2 staining. Scale bar, 50 μm. (B) CAF was embedded in FITC conjugated fibrous collagen matrix and treated with HER2 labeled magnetic beads and trastuzumab. Representative images of extracellular collagen matrix. Scale bar, 50 μm. (C) BT-474 cells were labeled with CellTrackerTM CTDR and co-cultured with NK cells transduced with VAV2 shRNA in the presence of trastuzumab. Representative flow cytometry plots of PI and CTDR are shown (n=4). Numerical representation CTDR ⁺ PI ⁺ Cell in Total CTDR ⁺ Percentage in cells (mean ± SD). Ns, through a single sheetAnalysis of variance of factors, no significant difference compared to the third group. (D) Macrophages were transduced with VAV2 shRNA, labeled with CMFDA, and co-cultured with BT-474 cells stained with CTDR in the presence of trastuzumab. Representative flow cytometry plots of CTDR and CMFDA are shown (n=4). Numerical representation CTDR ⁺ CMFDA ⁺ Cell in Total CMFDA ⁺ Percentage in cells (mean ± SD). Ns, by one-way analysis of variance, did not differ significantly from the third group. (E) ^- K) Inoculating BT-474 cells into fat pad of NOD SCID mouse, wherein CD16 ⁺ CAF transduces VAV2 shRNA. Mice were treated with trastuzumab when xenografts were accessible. After 6 weeks, tumors were harvested (n=6 per group). (E) tumor growth curve. (F) representative images of IVIS Lumina imaging. (G) quantification of micro-sirius red staining in tumors. (H) quantifying the Young's modulus of elasticity of the tumor stroma. (I) Hoechst and CD31-PE were injected into mice via the tail vein prior to sacrifice. CD31 ⁺ Hoechst leakage representative image of blood vessels. Scale bar, 100 μm. Quantification of (J) (I). (K) CD31 in tumor ⁺ Quantification of lectin perfusion in blood vessels. Mean ± standard deviation; * P is p <0.05；***p<0.001, one-way analysis of variance. Please refer to fig. 16.

Fig. 9 shows: a comprehensive overview of Fc gamma R expressing cells in human breast cancer is shown in FIG. 1. (A) CD45 in breast cancer tissue of patient ⁺ Quantification of cell percentages (n=32). Mean ± SEM. (B) CD16 in breast cancer tissue of patient ⁺ CD45 ^- Quantification of cell percentages (n=32). Mean ± SEM. (C) Representative immunofluorescence images of CK and CD32b/c staining in human breast cancer tissue. The asterisks indicate the region of higher upper right corner magnification (n=26). Scale bar, 50 μm. (D) Representative immunofluorescence images of CD16, CD45 and CK staining in human breast cancer tissue. The asterisks indicate the region of higher upper right corner magnification (n=26). Scale bar, 50 μm.

Fig. 10 shows: CD16 ⁺ Fibroblast subpopulations and HER2 ⁺ Trastuzumab resistance in breast cancer patients is related to figure 2. (A and B) detection of FVD in tumor samples of breast cancer patients by flow cytometry ^- CD45 ^- EpCAM ^- CD31 ^- PDGFRβ ⁺ CD16 ⁺ Gated CD16 ⁺ CAF, as shown in fig. 2A (n=68). (A) Tumor specimens with/without CD16 ⁺ Percentage of breast cancer patients with CAF. (B) CD16 ⁺ Proportion of CAF in total CAF subpopulations. Mean ± SEM. (C) CD16 ⁺ Flow cytometry analysis of CD10 and GPR77 expression in CAF (n=14). Values represent mean ± SEM. (D) CD16 ⁺ Flow cytometry analysis of CD16a and CD16b expression in CAF (n=14). Values represent mean ± SEM. (E) For CD16 isolated from breast cancer patient tumor samples by FACS (n=6) ⁺ PDGFRβ ⁺ Cells were analyzed for αsma and CD45 expression by flow cytometry. Values represent mean ± SEM. (F) Structured Illumination Microscope (SIM) image display: CD16 isolated from breast cancer patient (n=3) tumor samples ⁺ CD16 and N-cadherin staining in CAF. Scale bar, 2 μm. (G) Isolation of CD16 from primary breast cancer tissue by FACS ⁺ CAF and maintained in culture for up to 10 passages. Shown is CD16 per passage as determined by flow cytometry ⁺ Percentage of population (n=15). Mean ± standard deviation; ns, one-way analysis of variance shows no significant difference. (H and I) T cells, B cells, NK cells, bone marrow cells, endothelial cells, tumor cells, CD16 isolated from clinical breast cancer samples ⁺ CAF and CD16 ^- CAF. (H) The relative expression level of CD16 in specific cells was detected by RT-qPCR (n=3). (I) The expression level of CD16 in the indicated cells was determined by western blotting (n=3). (J) HR without adjuvant treatment ⁺ HER2 ^- Non-disease survival Kaplan-Meier curves for (hormone receptor positive and HER2 negative, n=488) and TN (triple negative, n=95) breast cancer patients, CD16 in tumor samples ⁺ The infiltration of CAF is divided into low (. Ltoreq.9.1 cells/field of vision) and high (. About.9 cells/field of vision)>9.1 cells/field of view). (K) Collection of HER2 ⁺ Paired tumor samples before and after breast cancer patients receive trastuzumab neoadjuvant chemotherapy; h in tumor sections from treatment-sensitive or drug-resistant patients&Representative images of E staining and αsma, CD16 immunofluorescence staining. The asterisks indicate the region of higher right-upper corner amplification. Scale bar, 50 mum. (L) collecting HER2 ⁺ Paired tumor samples before and after the breast cancer patient does not use trastuzumab neoadjuvant chemotherapy; immunofluorescent staining of paired tumor samples from treatment-sensitive (n=55) or drug-resistant (n=36) patients, CD16 for each field of view is shown ⁺ CAF number. Mean ± SEM; ns, mann-Whitney U test is not significant.

Fig. 11 shows: in a patient-derived xenograft model, CD16 ⁺ The efficacy of trastuzumab was compromised by fibroblasts, as related to figure 3. (A) Flow cytometry analysis of spontaneous mammary tumors of MMTV-PyMT mice and isogenic 4T1/EO771 grafts of BALB/C or C57BL/6J mice to obtain CD45 ⁺ Population and alpha SMA ⁺ Fcγrii/iii and fcγrvi expression in the population (n=6 per group). Values represent mean ± SD. (B) Will have a high/low CD16 ⁺ CAF-infiltrated HER2 ⁺ The primary tumor specimens were implanted into NOD SCID mice. After successful establishment of PDX, mice were treated with docetaxel. Fold change in tumor volume (left) and response rate to PDX (right) are shown. CD16 ^high Group n=7, cd16 ^low Group n=18. (C) Will have a high/low CD16 ⁺ CAF-infiltrated HER2 ⁺ The primary tumor specimens were implanted into NOD SCID mice. After successful establishment of PDX, mice were treated with IgG. Assessment of CD16 ^high (n＝7)、CD16 ^low Tumor volume fold change (left) and PDX response rate (right) for (n=18) group. (D) Will have a high CD16 ⁺ CAF-infiltrated HER2 ⁺ Primary tumor specimens were implanted into NOD SCID mice (P-540). After successful establishment of PDX, mice were treated with trastuzumab or anti-human IgG alone, and with CD16 neutralizing antibodies or IgG simultaneously with trastuzumab, n=8 per group. Fold change in tumor volume (left) and response rate (right) were evaluated. (E) Will have a low CD16 ⁺ CAF-infiltrated HER2 ⁺ Primary tumor specimens were implanted into NOD SCID mice (P-029). After successful establishment of PDX, mice were treated with trastuzumab or anti-human IgG alone, and with CD16 neutralizing antibodies or IgG simultaneously with trastuzumab, n=8 per group. Fold change in tumor volume (left) and response rate (right) were evaluated. (F) Will have a high CD16 ⁺ CAF-infiltrated HER2 ⁺ Primary tumor specimens were implanted into NOD SCID mice (P-133). After successful establishment of PDX, mice were treated with either no or docetaxel alone, and with CD16 neutralizing antibodies or IgG at the same time as docetaxel, with n=8 per group. Fold change in tumor volume (left) and response rate (right) were evaluated. Ns, insignificant and p<0.001, by Fisher exact test.

Fig. 12 shows: CD16 ⁺ Fibroblasts impair trastuzumab efficacy in co-injection models, as related to fig. 4. (A) BT-474 breast cancer cells were inoculated alone or co-injected with designated CAF at a ratio of 1:3 into mammary fat pads of NOD SCID mice. When the in situ xenograft is accessible, mice are treated with PBS or docetaxel. Tumor growth curves are shown (n=6 per group). (B) CD16 ⁺ shRNA in CAF silences CD 16. Efficiency was assessed by western blot (n=3). (C) SK-BR-3 breast cancer cells were vaccinated alone or co-injected with designated CAF at a ratio of 1:3 into mammary fat pads of NOD SCID mice. Mice were treated with trastuzumab or IgG when in situ xenografts were accessible. Tumor growth curves are shown (n=6 per group). (D) CD16 ⁺ CAF was transduced without/with shRNA against GFP/CD16 lentivirus. SK-BR-3 tumor cells were inoculated alone or co-injected with designated CAF at a ratio of 1:3 into mammary fat pads of NOD SCID mice. Mice were treated with trastuzumab when in situ xenografts were accessible. Tumor growth curves are shown (n=6 per group). (E) CD16 ^- CAFs express CD 16-FcRgamma and CD 16-FcRgamma without empty vector ^mut Is transduced in the presence of lentiviruses of vectors of (1) or in the presence of empty vector (vec) or in the expression of CD 16-FcRgamma (CD 16/gamma) or CD 16-FcRgamma ^mut (CD16/γ ^mut ) Transduction in the case of lentiviruses of the vector of (2). Expression of CD16 in designated cells was determined by western blot (n=3). (F) Post-treatment CD16 with IgG/trastuzumab in the presence of HER2 beads using CCK-8 assay (n=3) evaluation ⁺ CAF and CD16 ^- Proliferation of CAF. Mean ± standard deviation; ns, not significant, # p<0.05 and p<0.001 by one-way analysis of variance.

Fig. 13 shows: CD16 ⁺ Fibroblasts were associated with fig. 6 by enhancing connective tissue formation to reduce vascular perfusion and intratumoral drug delivery. (a) quantification of fig. 6A (n=6 per group). (B) Quantification of young's modulus of elasticity of tumor stroma in fig. 6B (n=6 per group). (C) BT-474 tumor cells and CD16 ^- /CD16 ⁺ CAF was co-injected into mammary fat pads of NOD SCID mice at a ratio of 1:3. When the in situ xenograft was accessible, mice were treated with PBS/docetaxel. After 6 weeks, mice were sacrificed and tumors were harvested (n=6 per group). Quantification of young's modulus of elasticity of tumor stroma in the indicated group is shown. (D) Tumor CD31 in FIG. 6C ⁺ Quantification of lectin perfusion in blood vessels (n=6 per group). (E) Quantification of tumor vascular Hoechst leakage in fig. 6D (n=6 per group). (F and G) BT-474 tumor cells and CD16 ^- /CD16 ⁺ CAF was co-injected into mammary fat pads of NOD SCID mice at a ratio of 1:3. When the in situ xenograft is accessible, the mice are treated without/with docetaxel. After 6 weeks, mice were sacrificed and tumors were harvested (n=6 per group). (F) lectin-FITC and anti-CD 31-PE were injected into mice via tail vein prior to sacrifice. Shows the tumor and CD31 ⁺ Vascular co-stained lectin pixel percentages (n=6 per group). (G) Hoechst and PE-anti-CD31 were injected into mice via the tail vein prior to their sacrifice. Quantification of CD31 in tumors ⁺ Hoechst leakage of blood vessels (n=6 per group). (H) quantification of fig. 6E (n=6 per group). (I and J) BT-474 tumor cells and CD16 ^- /CD16 ⁺ CAF was co-injected into mammary fat pads of NOD SCID mice at a ratio of 1:3. Mice were treated with docetaxel (doc) and IgG/trastuzumab when tumors were accessible. (I) Mice were injected with rhodamine-B-conjugated docetaxel (purchased from Qi Yue Biology, cat#Q-0031418) and anti-CD 31-FITC prior to sacrifice. A 3D visual representative image of rhodamine-B-conjugated docetaxel drug distribution in the tumor is displayed. Scale bar, 50 μm. Quantification of (J) (I) (n=6 per group). Mean ± standard deviation; ns, insignificant and p<0.001 (by one-way analysis of variance).

Fig. 14 shows: fcγriii expressing fibroblasts inhibit infiltration of cytotoxic effector cells,in connection with fig. 6. (A) Mouse fibroblasts were transduced with lentiviruses without vector, with empty vector or fcyriii expressing vector. Efficiency was assessed by western blot (n=3). (B) EO771 cells were transduced without vector and with lentiviruses carrying empty vector or vector expressing human ErbB 2. Efficiency was assessed by western blot (n=3). (C) Co-injection of EO771 cells expressing human ErbB2 and mouse fibroblasts forced to express FcgammaRIII into immunocompetent pWAP-human ErbB2 (ErbB 2) ⁺ ) In the fat pad of the mice. When the tumor is accessible, the mice are treated with 4D5 or IgG. Representative immunofluorescence images of CD49b and CD8 staining in tumor sections are shown. Asterisks indicate the enlarged area in the inset.

Fig. 15 shows: CD16 ⁺ Fibroblast-induced connective tissue proliferation was triggered by the SYK-VAV2-RhoA-ROCK-MLC2 pathway, as shown in FIG. 7. (A) Treatment of CD16 with HER 2-tagged magnetic beads in the presence of IgG or trastuzumab ^- Or CD16 ⁺ CAF. Hyaluronic Acid (HA) levels in the designated CAF supernatants were measured by ELISA (n=5). (B) quantification of collagen alignment level in fig. 7B (n=6). (C) p-MLC2 staining in CAF shown in fig. 7C (n=6) was quantified. (D and E) use of CellTrackerTM CTDR and CMFDA labeled BT-474 cells and CD16, respectively ⁺ CAF, and BT-474 tumor cells with or without CD16 without antibody, igG or trastuzumab ⁺ CAF co-cultivation. (D) Cytotoxicity of BT-474 cells was assessed by flow cytometry (n=4). Numerical representation CTDR ⁺ PI ⁺ Cell in Total CTDR ⁺ Percentage in cells (mean ± SD). Ns, compared to the third group, single-factor analysis of variance, the difference was not significant. (E) Phagocytosis of BT-474 cells was assessed by flow cytometry (n=4). Numerical representation CTDR ⁺ CMFDA ⁺ Cell in Total CMFDA ⁺ Percentage in cells (mean ± SD). Ns, one-way analysis of variance, the difference was not significant compared to untreated groups. (F) Expression profiles of VAV1, VAV2 and VAV3 in different cell types from human protein profile datasets. (G) Determination of human Peripheral Blood Mononuclear Cells (PBMC) and CD16 by Western blotting ⁺ CAFIn VAV1, VAV2 and VAV3 (n=3). (H) Treatment of CD16 without lentiviral shRNA transduction with HER 2-tagged magnetic beads in the presence of trastuzumab ^- CAF, and treatment of CD16 transduced with lentiviral shRNA without lentiviral shRNA transduction or with corresponding SYK, VAV2, RHOA or MLC2 ⁺ CAF. Hyaluronic acid levels produced by the specified CAF were measured by ELISA (n=5). With CD16 without transduction ⁺ CAF compared with p<0.01 and p<0.001. (I) MRTF-A at CD16 ⁺ CAF is silenced by shRNA. Efficiency was assessed by western blot (n=3). (J) Relative expression levels of αsma and collagen I were quantified in CAF shown in fig. 7G (n=3). (K) Three HER 2's from receiving trastuzumab neoadjuvant chemotherapy ⁺ Isolation of CD16 in tumor samples from breast cancer patients ⁺ And CD16 ^- CAF. Phosphorylation of VAV2 and MLC2 in the corresponding cells was determined by western blotting (n=3) and activation of RhoA. (L) on-line RNA-seq data from breast cancer cohorts receiving trastuzumab neoadjuvant therapy was analyzed. A Gene Set Enrichment Analysis (GSEA) analysis showed that the genes associated with the production of collagen-containing extracellular matrix were significantly enriched in the residual disease patient samples compared to the fully remitted patients. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are shown. Mean ± standard deviation; ns, not significant, p by one-way analysis of variance <0.01 and p<0.001。

Fig. 16 shows: targeting VAV2 inhibition by CD16 ⁺ Fibroblast-induced connective tissue proliferation, as related to figure 8. (a) quantification of p-MLC2 levels in fig. 8A (n=6). (B) Quantification of F-actin levels in fig. 8A (n=6). (C) Quantification of collagen compression (left) and alignment (right) levels in fig. 8B (n=6). (D) NK cells were transduced without/with lentiviral shRNA against GFP/VAV 2. Efficiency was assessed by western blot (n=3). (E) Macrophages were transduced without/with lentiviral shRNA against GFP or VAV 2. Efficiency was assessed by western blot (n=3). (F) schematic representation of the main findings of this study. Mean ± standard deviation; * P<0.01 and p<0.001 by one-way analysis of variance.

Fig. 17 shows: HER2 ⁺ Patient survival without diseaseCox regression analysis, associated with FIG. 2

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The invention will now be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention. The test specimens and test procedures used in the following examples include those (if the specific conditions of the experiment are not specified in the examples, generally according to conventional conditions or according to the recommended conditions of the reagent company; the reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified).

Description of the samples: the breast cancer patient samples and data of the embodiment of the invention are derived from the university of Zhongshan Sun Yixian commemorative hospital, and wherein for HER2 positive patients, the new adjuvant chemotherapy regimen is as follows: adriamycin 60mg/m every 21 days ² And cyclophosphamide 600mg/m ² For a total of 4 cycles, followed by weekly administration of paclitaxel (80 mg/m ² ) Treatment was 3 months. For patients receiving trastuzumab, the patient is concurrently receiving trastuzumab therapy at an initial dose of 8mg/kg (at the beginning of paclitaxel therapy) followed by a 6mg/kg dose every three weeks for one year. The treatment effect was evaluated according to the criteria of RECIST (solid tumor response evaluation criteria). Complete remission of pathology (pCR) is defined as the absence of any residual invasive carcinoma or Ductal Carcinoma In Situ (DCIS) in any resected breast or lymph node tissue detected under a microscope after neoadjuvant treatment; breast/lymph node only pCR means pCR is limited to only the breast or armpit; partial Remission (PR) means a decrease in tumor size of greater than or equal to 30%; disease Progression (PD) refers to tumor enlargement of > 20%; disease Stabilization (SD) refers to tumor size progression<20% increase to<30% reduced range. patients with pCR, breast/lymph node only pCR or PR were classified as sensitive, while patients with SD or PD were classified as resistant to neoadjuvant therapy. All samples were from patient informed consent, all The relevant procedures were all approved by the national institutes of review and ethics committee, university of Zhongshan Sun Yixian commemorative hospital. In this example, CD16 ⁺ And CD16 ^- CAF can be sorted by flow cytometry (BD Influx) by flow cytometry sorting method (FACS) (Su et al 2018 a).

Example 1

1. Research profile of fcγr expression in human breast cancer

Although it is well recognized that fcγr mediated responses play a significant role in mAb therapy (mAb therapy), there is no current discussion of which cell populations play a major role. And for the research of specific mechanism, the method is beneficial to optimizing the related cancer treatment strategy. For this reason, the present embodiment makes a specific study.

In this example, a single cell suspension of a clinical breast cancer specimen isolated from a living cell was examined by flow cytometry analysis (n=32 patients), and expression of human fcγr therein was examined. As a result, it was found that CD64 (FcgammaRI) and CD32a (FcgammaRIIa) were mainly expressed on myeloid cells, while CD32B/c (FcgammaRIIb/IIc) was mainly expressed on myeloid cells and B cells in tumors. In addition, CD16 (fcγrii) is expressed on tumor-infiltrating NK cells and myeloid cells (fig. 1a,1b and 9A). Notably, a portion of CD32b/c was observed ⁺ Cells and CD16 ⁺ The cells were CD45 ^- I.e. it appears negative for CD45 (fig. 1a,1B and 9B). Immunofluorescent staining showed that in human breast cancer tissue, some Cytokeratins (CK) with nuclear atypical properties ⁺ Cells were positive for CD32b/C (FIG. 9C). These data indicate CD32b/c ⁺ CD45 ^- The cells were tumor cells, consistent with the disclosure of the prior studies.

According to FIG. 9D, CD45 ^- CD16 ⁺ The cells are expressed as CK ^- (FIG. 9D).

Based on the elongated morphology, the inventors speculated that CD16 ⁺ CD45 ^- The cells may be CAFs (fig. 1C).

Overall, the data of this example demonstrate that in human breast cancer, fcγrs can be expressed in some tumor cell subsets and in stromal cells in addition to leukocytes.

2. CD16 ⁺ Fibroblast subpopulations and HER2 ⁺ Trastuzumab drug resistance correlation in breast cancer patients

To assess whether human CAFs express CD16, this example isolated primary CAF from tumors of 68 breast cancer patients and performed flow cytometry. Flow cytometry results showed that in 45.6% (31/68) of cancer specimens, expression of CD16 was observed in fibroblast subpopulations positive for the fibroblast surface marker pdgfrβ and negative for the pan lymphocyte marker CD45, negative for the endothelial marker CD31 and negative for the epithelial marker EpCAM (fig. 2A and 10A). CD16 ⁺ Fibroblasts account for 20.13±3.38% (mean±sem) of the total CAF population (fig. 10B). Several sub-populations of fibroblasts in human tumors have been reported in the prior art. To study these CD16 ⁺ Whether fibroblasts belong to any of the prior art subsets of fibroblast subpopulations was determined, the inventors examined CD16 ⁺ Expression of α SMA, FAP, CD10 and GPR77 in fibroblasts (from n=14 patients). Over 90% of CD16 was observed ⁺ Fibroblasts exhibit alpha SMA ⁺ FAP ^- CD10 ^- GPR77 ^- Phenotypes (as shown in figures 2B and 10C).

Because CD16 is divided into two subtypes, CD16a and CD16 b. To further evaluate the subtype of CD16 on CAF, this example was analyzed using corresponding specific antibodies. Flow cytometry analysis showed that PDGFR beta ⁺ The CD16 and CD16a stained areas in the cell population overlap almost completely; in contrast, few signals were detected with the CD16 b-specific antibody (fig. 10D).

Next, this example evaluates whether expression of CD16 on CAF is sustainable in vitro. Consistent with flow cytometry analysis, immunostaining results showed: partial alpha SMA ⁺ Cells expressed CD16 at high levels (fig. 2C). And CD16 isolated by Fluorescence Activated Cell Sorting (FACS) ⁺ αSMA ⁺ The cells appear bipolar or multipolar, have an elongated shape, with a typical activated fibroblast morphology (fig. 2C). CD16 pair by flow cytometry ⁺ PDGFRβ ⁺ Cells were phenotypedThe results show that over 95% of the sorted cells are αsma ⁺ CD45 ^- (FIG. 10E). And the Structural Illumination Microscope (SIM) image further confirmed the presence of CD16 signal on plasma membrane (fig. 10F).

Under conventional fibroblast culture conditions (10% FBS-DMEM), CD16 was found ⁺ The percentage of CAFs in the overall CAFs remained unchanged for at least 10 passages (fig. 10G).

To determine CD16 in breast cancer ⁺ Relative levels of CD16 on CAFs and other cells, T cells, B cells, NK cells, myeloid cells, endothelial cells, tumor cells, CD16 isolated from clinical breast cancer specimens by RT-qPCR and western blotting ⁺ CAFs and CD16 ^- CAFs were tested for the corresponding CD16 levels (fig. 10H and 10I). The results showed that endothelial cells, tumor cells and CD16 ^- In CAFs, CD16 is not detected; CD16 ⁺ The CD16 level of fibroblasts is lower than NK cells and myeloid cells, but much higher than T cells and B cells.

To study this CD16 ⁺ Clinical significance of CAF subpopulations this example performed double immunofluorescent staining for CD16 and αsma in untreated breast cancer samples (n=814). Although CD16 was observed in all subtypes of breast cancer ⁺ Presence of CAF, but HER2 ⁺ CD16 in subtype and triple negative subtype breast cancers ⁺ The number of CAF is significantly higher than HR (hormone receptor) ⁺ HER2 ^- CD16 in subtype breast cancer ⁺ CAF number (fig. 2D and 2E)). In HER2 ⁺ And triple negative breast cancer subtypes, high CD16 ⁺ CAF invasive tumor (CD 16) ⁺ CAFhigh).

Importantly, CD16 ⁺ Number of CAF and HER2 ⁺ Long-term prognosis dysplasia of subtype is significantly correlated (fig. 2F), but in triple negative or HR ⁺ HER2 ^- The subtypes are uncorrelated (fig. 10J).

Given the important role CD16 plays in antibody-mediated immune responses, several risk factors such as intratumoral immune infiltration were analyzed for their impact on patient survival using the Cox regression model. Multivariate analysis showed CD16 ⁺ CAF abundance is HER2 ⁺ Independent prognostic factors for breast cancer (fig. 17). To study CD16 ⁺ Whether CAFs is associated with HER2 ⁺ The response to breast cancer treatment is relevant, HER2 for the present example, receiving neoadjuvant therapy ⁺ The pre-treatment biopsy sample and the post-treatment excision sample of the breast cancer patient are evaluated to obtain CD16 ⁺ Infiltration of CAFs.

We observed that CD16 in pre-treatment biopsy samples and post-treatment excision samples for patients with resistance to trastuzumab neoadjuvant therapy compared to patients with response to trastuzumab neoadjuvant therapy (i.e., patients susceptible to trastuzumab neoadjuvant therapy) ⁺ CAF infiltration was significantly higher than for responders (figures 2G and 10K). Whereas in the absence of trastuzumab, HER2 is resistant and sensitive to neoadjuvant chemotherapy ⁺ CD16 in biopsy samples or post-treatment excision samples between breast cancer patients ⁺ There was no significant difference in CAF number (fig. 10L).

In general, this example identifies CD16 ⁺ αSMA ⁺ PDGFRβ ⁺ FAP ^- CD10 ^- GPR77 ^- Fibroblast, a subset of fibroblasts, and which is resistant to trastuzumab, HER2 ⁺ The long-term survival rate of breast cancer patients is closely related.

3. CD16 ⁺ Therapeutic effects of trastuzumab on fibroblast damage in vivo

In view of CD16 ⁺ CAF and HER2 ⁺ The present example discusses whether CD16 in fibroblasts induces trastuzumab resistance in vivo in breast cancer patients with poor response to trastuzumab. However, only a few FcgammaR were detected in spontaneous breast tumors of MMTV-PyMT mice or in isogenic 4T1/EO771 grafts of BALB/C/C57BL/6J mice ⁺ CAF (fig. 11A). Human and mouse CAF are shown to be different in fcγr expression profile.

On this basis, further studies were performed. This example in situ implantation of fresh primary tumor samples (excised in situ from breast cancer patients) into immunocompromised NOD SCID mice in the removal of mammary fat pads and in patient-derived xenografts (PDX) up to 100-200mm ³ After that, the processing unit is configured to,trastuzumab is intravenously injected weekly at clinically relevant exposure levels. The inventors found that it is derived from CD 16-rich ⁺ In PDX of the primary tumor of fibroblasts, most of the fibroblasts remained CD16 positive (fig. 3A and 3B), consistent with the previous experiment of serial passage after ex vivo (fig. 10G). In contrast, PDX rarely retains cd16+ a SMA-cells in the primary tumor (fig. 3A and 3C), consistent with previous reports that human leukocytes were lost after PDX implantation. More importantly, has a high CD16 ⁺ The response rate of CAF-infiltrated PDX to trastuzumab is significantly lower than with low CD16 ⁺ CAF infiltrated PDX (fig. 3D and 3E). In contrast, no significant difference in tumor growth was found when mice were treated with either chemotherapy alone (fig. 11B) or control IgG (fig. 11C).

Whereas human CD16 is expressed predominantly in fibroblasts of PDX (fig. 3A), this example uses specific neutralizing antibodies (available from bioleged, including Cat # 302001) against human CD16 along with trastuzumab therapy. The results show that blocking of CD16 significantly enhanced trastuzumab enrichment with CD16 ⁺ Therapeutic effects in PDX of CAF (FIGS. 3F, 3G and 11D), but in CD16 ⁺ There was no significant enhancement in PDX with little CAF (fig. 11E). Furthermore, in the case of chemotherapy alone and with CD16 ⁺ There was no synergy of CD16 neutralizing antibodies in CAF PDX (fig. 11F).

To further evaluate CD16 ⁺ Effect of fibroblasts on in vivo trastuzumab response, isolation of human CD16 from clinical samples of breast cancer by FACS ⁺ PDGFRβ ⁺ CD45 ^- CD31 ^- EpCAM ^- Fibroblasts and CD16 ^- PDGFRβ ⁺ CD45 ^- CD31 ^- EpCAM ^- Fibroblasts and contacting these cells with HER2 ⁺ Breast cancer BT-474 cells were co-injected into NOD SCID mice. Co-injection of BT-474 cells with CD16 in mice treated with trastuzumab ⁺ CAF (rather than autologous CD 16) ^- CAF), the tumor showed continued growth (fig. 4A). In contrast, in tumor-bearing mice treated with control IgG (fig. 4A) or chemotherapy alone (fig. 12A), no injection of CD16 was observed on BT-474 xenograft size ⁺ Fibroblasts or CD16 ^- Any significant differences between the fibroblast groups, indicating CD16 ⁺ The differential effect brought by fibroblasts is trastuzumab-specific.

Furthermore, when CD16 was silenced by shRNA lentiviral vectors prior to coinjection ⁺ CD16 in fibroblasts, then CD16 can be eliminated ⁺ Fibroblast-induced trastuzumab resistance (fig. 4B and 12B). Furthermore, in using different HER2 ⁺ Similar results were observed when the above experiment was repeated with human breast cancer cell line SK-BR-3 (FIGS. 12C and 12D).

Overall, these data indicate that CD16 signaling in fibroblasts mediates resistance to trastuzumab in vivo.

The lentiviral vectors used for gene silencing in this example included pGLV2-U6-Puro; lentiviral shrnSub>A targeting CD16, SYK, VAV2, RHOA, MLC2, MRTF-Sub>A and GFP were obtained from genepharmSub>A Inc (Shanghai, chinSub>A), and specifically shrnSub>A corresponds to: CD16: shRNA1:5' -GTTTACTTCCTCCTGTCTAGT ^-3’ ；shRNA2：5’-GAGACTGGAAGGACCATAAAT ^-3’ 。SYK：shRNA1：5’-CCTTAGCATGTGACTCCTGAA ^-3’ ；shRNA2：5’-GCCCACAACTTGTCACCCAAA ^-3’ 。VAV2：shRNA1：5’-CAATAAGCATCAAGTTCAATG ^-3’ ；shRNA2：5’-CCATGCAGAGGGTGCTCAAAT ^-3’ 。RHOA：shRNA1：5’-GAAAGCAGGTAGAGTTGGCTT ^-3’ ；shRNA2：5’-GTACATGGAGTGTTCAGCAAA ^-3’ 。MLC2：shRNA1：5’-GGCTGATTACGTTCGGGAAAT ^-3’ ；shRNA2：5’-GGAGGAGGTTGACCAGATGTT ^-3’ 。MRTF-A：shRNA1：5’-GACTATCTCAAACGGAAGATT ^-3’ ；shRNA2：5’-GCTCAAGTACCACCAGTACAT ^-3’ 。GFP：shRNA1：5’-TAGCGACTAAACACATCAA ^-3’ 。)

To investigate whether CD16 on CAF reduces trastuzumab efficacy by antibody isolation, CD16 was transduced with human CD 16-fcrgamma or CD 16-fcrgamma with mutant fcrgamma ^- CAF for correlation studies. Wherein, for CD 16-FcRgamma with mutant FcRgamma, its immune receptor tyrosine based activation motif (ITAM) cannot mediate downstream with SYKA signal (fig. 12E). Recording CD 16-FcRgamma with mutant FcRgamma as CD 16-FcRgamma ^mut 。

BT-474 cells are combined with CD 16-FcRgamma or CD 16-FcRgamma ^mut Transduced by an overexpression vector or otherwise without CD 16-FcRgamma, CD 16-FcRgamma ^mut Overexpression vector transduced CD16 ^- CAF was co-injected into the fat pad of NOD SCID mice. After tumor palpable, mice were treated with IgG, trastuzumab, or pertuzumab (another anti-HER 2 antibody).

As a result, trastuzumab was found to be effective against CD16 which forced expression of CD 16-FcRgamma ^- The CAF co-injected tumors were significantly attenuated. However, when injected CD16 ^- CD 16-FcRgamma for CAF ^mut This effect (reduced trastuzumab efficacy) was eliminated during transduction (fig. 4C). And similar results were also demonstrated in the pertuzumab assay, validated in the pertuzumab setting (fig. 4C).

These data indicate that the reduced efficacy of anti-HER 2 treatment is due to intracellular signaling of CD16 in fibroblasts, rather than antibody isolation.

To detect CD16 in vivo ⁺ Proliferation of CAF, thymidine analog 5-ethynyl-2' -deoxyuridine (EdU) was administered daily to tumor-bearing mice 7 days prior to tissue harvesting. Flow cytometry analysis showed that IgG treated EdU-labeled CD16 in tumors ⁺ Percentage of CAFs with IgG or trastuzumab for treatment of EdU-labeled CD16 in tumors ^- The percentage of CAFs was comparable (fig. 4D); however, CD16 of EdU was retained after trastuzumab treatment ⁺ The proportion of CAF increases significantly and this trend can be reversed again by blocking CD16 (fig. 4D). Likewise, the cell count kit-8 (CCK-8) assay showed that: trastuzumab and HER2 microbeads significantly increased CD16 ⁺ Proliferation of CAF without increasing CD16 ^- Proliferation of CAF (fig. 12F).

These findings indicate that trastuzumab triggers CD16 by CD16 ⁺ Proliferation of CAF.

4. CD16 ⁺ Fibroblast to reduce vascular perfusion and intratumoral drug delivery by enhancing connective tissue production

Fiber formationThe vitamin cells play an important role in coordinating tumor connective tissue formation. Thus, the present inventors have discovered HER2 that is sensitive or resistant to trastuzumab neoadjuvant chemotherapy ⁺ Breast cancer patients were subjected to relevant evaluations, in particular, the extracellular matrix (ECM) production and hardness of tumors. Sirius red staining showed a significant increase in collagen levels in the drug-resistant patient samples compared to the single drug-sensitive patients (fig. 5A). Samples from drug resistant patients were found to have significantly harder matrices by calculating young's modulus of elasticity of fresh tumor samples with Atomic Force Microscopy (AFM) (fig. 5B). Importantly, trastuzumab post-treatment HER2 ⁺ The extent of connective tissue-promoting proliferative response in tumor samples and CD16 therein ⁺ CAF abundance is closely related (fig. 5C). In contrast, in the initial tumor sample, the level of desmoplasia response was comparable to CD16 ⁺ There was no obvious correlation between CAFs abundance (fig. 5D).

Furthermore, HER2 was obtained via trastuzumab neoadjuvant chemotherapy ⁺ Breast cancer patient samples, including corresponding pre-treatment biopsies and post-treatment resected samples, were measured for CD16 ⁺ CAF and tumor cell proximity. As a result, it was found that CD16 was in close contact with tumor cells in the post-treatment sample ⁺ The amount of CAF is significantly higher than CD16 ^- Amount of CAF. In addition, CD16 after trastuzumab treatment ⁺ The distance between CAF and tumor cells was significantly reduced (fig. 5E and 5F).

These data indicate that, following trastuzumab treatment, connective tissue proliferation promoting matrix increases with CD16 ⁺ CAFs related, CD16 ⁺ CAFs are located near tumor cells.

To study CD16 ⁺ The potential mechanism by which fibroblasts induced trastuzumab resistance, collagen deposition in xenografts was assessed using a variety of methods, including sirius red staining and immunostaining of human collagen i, collagen iii and hyaluronic acid. The data of this example demonstrate co-injection of CD16 in trastuzumab-treated mice ^- Co-injection of CD16 compared to fibroblasts ⁺ Connective tissue production was significantly increased in the fibroblast tumors but not in the IgG control treated miceThis is the case (fig. 6A and 13A).

Furthermore, we found that CD16 after trastuzumab was used ⁺ Is not CD16 ^- Is shown) significantly increased young's modulus of elasticity of the xenografts (fig. 6B and 13B).

To evaluate CD16 ⁺ Whether fibroblasts also induced connective tissue proliferation upon chemotherapy alone, the inventors treated tumor-bearing mice weekly with docetaxel. The results showed that no post-chemotherapy CD16 was observed compared to trastuzumab group ⁺ Or CD16 ^- There was a significant difference in matrix stiffness between fibroblast xenografts (fig. 13C).

Taken together, our data indicate that CD16 ⁺ Fibroblasts can enhance tumor connective tissue formation (i.e., promote tumor connective tissue proliferation) in the presence of trastuzumab.

Connective tissue hyperplasia has been reported to cause vascular compression, resulting in inhibition of drug delivery. To assess vascular compression, tumor-bearing mice were injected with lectin-FITC and Hoechst, and vascular perfusion and permeability were analyzed, respectively. After trastuzumab treatment, and CD 16-containing ^- Fibroblast-containing tumor compared with CD16 ⁺ In the tumors of fibroblasts, both lectin perfusion (FIGS. 6C and 13D) and Hoechst permeability (FIGS. 6D and 13E) were significantly reduced. In contrast, co-injection of CD16 following chemotherapy alone ⁺ Or CD16 ^- Vascular perfusion and permeability levels in xenografts were comparable between fibroblasts mice (fig. 13F and 13G).

To determine if vascular collapse would impair trastuzumab delivery, we examined trastuzumab (fluorescently labeled) tumor uptake and observed CD16 ⁺ Co-injection of fibroblasts significantly reduced tumor uptake of trastuzumab (FIGS. 6E and 13H).

To explore the administration of chemotherapeutic agents in combination with trastuzumab, tumor-bearing mice were administered docetaxel and trastuzumab simultaneously. With co-injection of CD16 ^- In comparison to those of CAF, in comparison to CD16 ⁺ In CAF co-injected tumors, trastuzumab was concomitantly presentTumor uptake of rhodamine-B-conjugated docetaxel was significantly reduced at the time of anti-treatment (fig. 13I and 13J).

Taken together, these results demonstrate that CD16 during trastuzumab-based therapy ⁺ Is not CD16 ^- Is a fibroblast) that induces matrix connective tissue formation and reduces drug delivery.

To evaluate CD16 ⁺ The function of fibroblasts in an immunocompetent model was performed using pWAP-human ErbB2 (ErbB 2 ⁺ ) Mice were subjected to experiments, which are a mouse model of huErbB2 knock-in C57BL/6J, with the previously described human HER2 immune tolerance. In view of the absence of fcγr in mammary tumors of immunocompetent mouse models ⁺ CAF (fig. 11A), the inventors isolated mouse fibroblasts from normal breast tissue and transduced them with mouse fcyriii homologous to human CD16 (fig. 14A).

Then, EO771 cells expressing human ErbB2 and fibroblasts expressing FcgammaRIII were co-injected into ErbB2 ⁺ In the fat pad of the mice (fig. 14B). Mice were treated with 4D5 when the graft was palpable, 4D5 being the parent drug for the mouse anti-human HER2 antibody and trastuzumab. Consistent with the findings of NOD SCID mouse experiments, the efficacy of 4D5 was significantly impaired in mice vaccinated with fcyriii expressing fibroblasts (fig. 6F). At the same time, collagen deposition levels increased significantly (fig. 6G and 6H). In addition, tumor infiltrating NK cells and CD8 ⁺ The number of T cells was significantly reduced (fig. 6I and 14C).

These data indicate that for fcyriii expressing fibroblasts, connective tissue proliferation induced by them inhibits infiltration of cytotoxic effector cells in immunocompetent mice.

5. CD16 ⁺ Fibroblast-induced connective tissue proliferation is triggered by SYK-VAV2-RhoA-ROCK-MLC2-MRTF-A pathway

Animal experimental data confirm that CD16 is incubated with HER 2-labeled magnetic beads ⁺ Fibroblasts had significantly higher levels of αsma and collagen type i (fig. 7A), and secreted more hyaluronic acid after addition of trastuzumab (15A). Next, we have been prepared by embedding fibroblasts Collagen gel remodeling assays were performed in FITC conjugated fibrous collagen matrix. And contains CD16 ^- Fibroblasts or cells containing CD16 treated with IgG ⁺ Random oriented fibril distribution in samples of fibroblasts is different for embedded CD16 ⁺ The matrix of fibroblasts and trastuzumab showed a distinct ordered arrangement of collagen fibrils, and in the presence of HER 2-labeled magnetic beads, these collagen fibrils had a higher density around the cell viability pole, indicating ECM remodeling (fig. 7B and 15B).

In addition, in CD16 treated with HER 2-labeled magnetic beads and trastuzumab ⁺ In fibroblasts, there was a stronger actin bundle (fig. 7C). These data indicate CD16 ⁺ Fibroblasts increase connective tissue formation by producing ECM components and driving contractility.

Notably, ADCC and ADCP have been considered to be the major fcγr-mediated influencing functions in tumors. To study CD16 ⁺ Whether or not the fibroblast performs ADCC and ADCP, BT-474 cells are combined with CD16 in the presence of trastuzumab ⁺ Fibroblast co-culture. Flow cytometry analysis showed that trastuzumab post-treatment CD16 ⁺ Neither the tumoricidal nor phagocytic activity of fibroblasts was enhanced (fig. 15D and 15E).

Fcγr is reported to be involved in stimulating spleen tyrosine kinase (SYK) activation, followed by phosphorylation of VAV guanine nucleotide exchange factor (VAV). The VAV proteins have 3 family members, including VAV1, VAV2 and VAV3. On-line cytogram databases (HUMAN protein profile databases) showed that fibroblasts highly expressed VAV2 instead of VAV1 or VAV3 (fig. 15F).

Consistently, our data shows CD16 ⁺ Fibroblasts expressed considerable levels of VAV2 instead of the other two VAV members (15G). We observed that CD16 after HER 2-labeled magnetic beads and trastuzumab treatment ⁺ Phosphorylation of both SYK and VAV2 was significantly increased in fibroblasts (fig. 7A).

VAV2 induces guanine nucleotide exchange on RhoA (Crespo et al, 1997; van Aelst and D' Souza-Schorey, 1997). RhoA can activate myosin light chain 2 #MLC 2) (Fukata et al, 2001; vicenter-Manzanares et al 2009), which is important for actin polymerization and ECM production in fibroblasts (Chen et al, 2008; totsukawa et al 2009), 2000). We observed that CD16 treated with trastuzumab ⁺ In CAF, levels of RhoA and phosphorylated MLC2 in GTP-bound form were significantly increased (fig. 7A, 7C and 15C). More importantly, silencing SYK, VAV2, rhoA and MLC2 reduced activation of their downstream mediators and the levels of αsma, type i collagen and hyaluronic acid, respectively (fig. 7D and 15H).

The cardiomyocyte-associated transcription factor-A (MRTF-A) mediates the expression of fibrotic genes (Luchsinger et al, 2011; small,2012; small et al, 2010). MLC2 phosphorylation induces actin polymerization, which recruits G-actin to F-actin stress fibers, releasing MRTF-A from G-actin to the nucleus (Fan et al, 2007; yokotSub>A et al, 2017).

While immunofluorescent staining of this example showed CD16 treated with HER2 magnetic beads and trastuzumab ⁺ In fibroblasts, MLC2 phosphorylation and nuclear translocation of MRTF-A were increased (FIGS. 7C, 7E, 7F and 15C). Silencing MRTF-A eliminates CD16 induced by trastuzumab ⁺ Upregulation of fibrotic genes in fibroblasts (FIGS. 7G,15I and 15J).

To verify the intracellular signaling pathway of CD16 in clinical samples, HER2 was neoadjuvant chemotherapy from trastuzumab ⁺ CD16 separation from breast cancer patient tumor sample ^- CAF and CD16 ⁺ CAF performs correlation detection. Western blot showed that, in CD16 ⁺ Levels of p-VAV2, rhoA-GTP and p-MLC2 in CAFs were significantly higher than CD16 ^- Levels in CAFs (15K). In addition, this example also analyzed online RNA-seq data (data from GEO database) containing trastuzumab neoadjuvant therapy breast cancer cohorts; analysis of the Gene Set Enrichment (GSEA) showed significant enrichment of residual disease patient samples with genes involved in the production of collagen-containing extracellular matrix compared to fully-relieved patients (False Discovery Rat) <0.25 (fig. 15L).

Taken together, these data indicate that trastuzumab activates CD16 ⁺ Fibroblast cellsCD16-SYK-VAV2-RhoA-MLC2-MRTF-A pathway mediating connective tissue proliferation.

6. Targeting VAV2 to inhibit CD16 ⁺ Fibroblast-induced connective tissue proliferation

VAV2 has been shown to be unnecessary for ADCC of NK cells or ADCP of macrophages. In view of the CD16 ⁺ VAV2 in fibroblasts is shown to be high and not VAV1 or VAV3 is shown to be high, the inventors propose: whether the different demands of immune cells and fibroblasts on VAV family members can be exploited to enhance the therapeutic efficacy of trastuzumab.

For this purpose, the inventors have made CD16 ⁺ VAV2 silencing in fibroblasts was observed and trastuzumab-induced MLC2 phosphorylation (fig. 8A and 16A), actin polymerization (fig. 8A and 16B), and collagen gel contraction (fig. 8B and 16C) were significantly inhibited. In contrast, knockdown VAV2 in NK cells and macrophages had no significant effect on trastuzumab-induced ADCC (fig. 8C and 16D) or ADCP (fig. 8D and 16E), respectively.

Overall, these data indicate that VAV2 is essential for CD16 function in fibroblasts, but not immune cells.

To explore the therapeutic potential of VAV2 in vivo, we have BT-474 cells and CD16 with VAV2 knockdown ⁺ Fibroblasts were co-injected into mammary fat pads of mice receiving trastuzumab treatment.

CD16 ⁺ Co-injection of fibroblasts severely limited the responsiveness of BT-474 tumors to trastuzumab (FIGS. 8E and 8F). At the same time, accommodate CD16 ⁺ The fibroblast xenografts exhibited significantly increased tissue fibrosis and matrix stiffness, and reduced vascular permeability and perfusion (fig. 8G-8K). But more importantly, CD16 ⁺ VAV2 silencing in fibroblasts restored trastuzumab sensitivity to corresponding tumor tissues and significantly reversed tumor connective tissue formation (fig. 8E-8K). Taken together, these data indicate that targeting VAV2 is a potential therapeutic modality that increases trastuzumab efficacy.

Generally, currently, more than 20 mabs have been approved for use in cancer indications. Successful mAbThe key to treatment is the ability to accurately determine which patient populations can obtain the greatest benefit from a certain treatment setting. To date, the prior art reports that fcγr is expressed predominantly on leukocytes in TME. In the present invention, however, the inventors have found that human breast cancer often contains a portion of CD16 ⁺ CAF. HER2 in treatment with trastuzumab ⁺ In breast cancer patients, this CAF subpopulation is associated with poor therapeutic response and long-term prognosis. Given the importance of mabs in a variety of diseases, the identification of this fcγr expressing fibroblast subset represents an important prognostic biomarker and therapeutic target.

In the present invention, the pro-neoplastic effect of CAF fcγr signals is unexpected. It is well known that the signal participation mechanism after ligand binding is very different between different fcγr expressing effector cells, including ADCC, ADCP, antigen presentation, and adaptive immune response modulation. However, these previously established fcγr mediated functions are always anti-tumor. For example, a trisome consisting of two HER 2-specific variables and one anti-CD 16 Fab fragment triggers direct lysis of HER2 by NK cells and γδ T cells expressing CD16 after trastuzumab therapy ⁺ Tumor cells. Whereas the data of the present invention show that binding of mAb to CD16 promotes fibroblast production and ECM contraction; the research proposed by the invention expands the current understanding of pleiotropic fcγr reactions.

Mechanistically, the inventors found that after trastuzumab stimulation, the CD16-SYK-VAV2-RhoA-MLC2-MRTF-A signaling cascade was activated in CAF. VAV2 has been reported to have guanosine nucleotide exchange factor activity for RhoA, but not for the other two members of the Rho family, rac1 and Cdc 42. RhoA may activate MLC2 via Rho-associated coiled-coil containing kinase (ROCK). MLC2 activation promotes the interaction of myosin with actin filaments (which is critical for the contractile properties caused by cytoskeletal rearrangement). MRTF-A is involved in the expression of various fibrosis genes, is sequestered in the cytosol by binding G-actin, and is released during cytoskeletal reorganization as Sub>A result of the recruitment of G-actin to F-actin stress fibers. The present data shows that trastuzumab-CD 16 is involved in initiating the SYK-VAV2-RhoA-MLC2 pathway. MLC2 phosphorylation induces cytoskeletal remodeling and enhances contractility of fibroblasts. Furthermore, actin stress fiber formation leads to nuclear shuttling of MRTF-A, which promotes the production of ECM components. Thus, the present invention finds a previously unrecognized mechanism of how fcγr signals in fibroblasts regulate matrix stiffness, bringing these signal events into the context of fibroblasts.

More notably, in one embodiment of the invention, inhibition of VAV2 was shown to eliminate fcγr signals in fibroblasts without significant impact on ADCC of NK cells or ADCP of macrophages. SYK-related receptors activate many signaling pathways through VAV proteins, which consist of 3 family members; among them, VAV1 expression is mainly restricted to leukocytes and plays a key role in fcγr mediated immune responses; in contrast, VAV2 and VAV3 expressions are more widely distributed. Our data shows CD16 ⁺ Fibroblasts express high levels of VAV2, but do not express VAV1 or VAV3. More importantly, we demonstrate that VAV2 in fibroblasts is essential for ECM production and contraction induced by trastuzumab-CD 16 signal in vitro and in vivo. Furthermore, the previous data and experiments of the present invention also show that the lack of VAV2 alone does not significantly affect ADCC of NK cells or ADCP of macrophages. Overall, our studies indicate that selective targeting of VAV2 can eliminate the deleterious effects of fcγr and maintain its therapeutic efficacy during mAb treatment.

Specifically, the invention further constructs a complex animal model to determine fcγr function in fibroblasts and demonstrates that inhibition of fibroblast CD16 enhances the efficacy of trastuzumab in PDX and CAF tumor cell coinjection xenograft models.

Drug delivery and permeation are critical to the efficacy of anticancer drugs. Drug delivery to tumors encounters various disorders such as tortuous vasculature, increased interstitial fluid pressure, and dense ECM. Most tumor masses are composed of ECM, which determines the physicochemical properties of tumor cells. Collagen fibers in ECM are a major obstacle to molecular diffusion, especially for larger particles. In addition, ECMs exhibit fast update characteristicsThis provides the possibility to modify connective tissue formation by blocking ECM component synthesis. In the present invention, a portion of CD16 was found ⁺ Fibroblasts induce trastuzumab resistance by producing ECM components and for this process can be effectively alleviated by targeting VAV 2. Thus, the present invention also provides a new strategy to increase penetration of anti-tumor drugs by modifying ECM.

Overall, this study showed that fcγr on a portion of CAF promotes mAb resistance by inducing connective tissue formation (fig. 16F). Moreover, the results of the present study reveal a previously unrecognized role for fcγr in tumor ECM remodeling and its underlying mechanism. Clinically, VAV2 in CAF can be identified as a potential therapeutic target to improve the therapeutic effect of mabs.

It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and are not intended to limit the present invention to the specific embodiments thereof. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1.CD16 ⁺ Application of fibroblast detection reagent in preparing products for predicting and diagnosing breast cancer monoclonal antibody treatment resistance and/or prognosis breast cancer monoclonal antibody treatment effect and/or predicting disease-free survival time of breast cancer patients; the breast cancer is HER2 positive breast cancer; the monoclonal antibodies include trastuzumab.

2. The use according to claim 1, wherein CD16 ⁺ Fibroblast detection reagent comprising and CD16 ⁺ An antibody which causes an antigen-antibody reaction with the fibroblast surface antigen CD 16.

3.CD16 ⁺ The application of the fibroblast in preparing a product for predicting and diagnosing the drug resistance of the breast cancer monoclonal antibody treatment and/or prognosis of the curative effect of the breast cancer monoclonal antibody treatment and/or predicting the disease-free survival period of a breast cancer patient; the milkAdenocarcinomas are HER2 positive breast cancers; the monoclonal antibodies include trastuzumab.

4. The use according to claim 3, wherein the product comprises CD16 ⁺ Fibroblast detection kit.