WO2023082193A1

WO2023082193A1 - Method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using nano-modulator and applications thereof

Info

Publication number: WO2023082193A1
Application number: PCT/CN2021/130411
Authority: WO
Inventors: Dar-Bin Shieh; Yi-Ching Wang; Fu-hsuan SHIH; Chih-Hsiung Hsieh; Hung-Chia HSIEH; Li-xing YANG
Original assignee: Dar-Bin Shieh
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2023-05-19

Abstract

Provided herein is a method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator and applications thereof, which comprises administering an effective dose of the nano-modulator to the subject, and the nano-modulator can be consisted of coated zero-valent iron (ZVI) -containing nanoparticles, for up-regulating or down-regulating expression levels of specific genes in the subject. And provided is a method for treating an angiogenesis-related disease and/or disorder in a subject in need thereof using the nano-modulator. Therefore, the nano-modulator can be applied in the development of anti-cancer precision nanomedicine and immunotherapy.

Description

METHOD FOR MODULATING TUMOR MICROENVIRONMENT TOWARD ANTI-CANCER PHENOTYPE IN A SUBJECT IN NEED THEREOF USING NANO-MODULATOR AND APPLICATIONS THEREOF

BACKGROUND

Field of Invention

The present invention relates to a method for modulating tumor microenvironment. More specifically, the present invention relates to a method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator and applications thereof.

Description of Related Art

Zero-valent-iron (ZVI) has been extensively developed for treatment of groundwater or wastewater contaminated with various organic and inorganic pollutants due to its strong reductive potentials and wide availability. ¹ ZVI can generate massive reactive oxygen species (ROS) through Fenton reaction and other chemical processes. ² In spite of numerous attention on environmental remediation, there is less research on anti-tumor efficacy both in cancer cells per se and in tumor microenvironment immune modulation in both in vitro and in vivo studies. Previously, two types of ZVI nanoparticles (NPs) have been developed by inventors, ^3, 4 including silver coated (ZVI@Ag) and carboxymethylcellulose coated (ZVI@CMC) NPs. ZVI@Ag NPs can be rapidly converted into iron ions by the enhanced lysosomal function of cancer cells. ³ In addition, ZVI@CMC NPs having the biocompatible organic shell, ⁵ not only generate a large amount of ROS, but also induce lipid peroxidation, thus triggering ferroptotic cell death in cancer cells. ⁴ Nevertheless, the underlying molecular mechanism of ZVI-NPs-induced ferroptosis and the effect of ZVI-NPs on tumor-associated immune cells remain elusive.

Ferroptosis is a novel programmed cell death identified in recent years. It is characterized by the intracellular iron accumulation and lipid peroxidation during the cell death process. ⁶ Ferroptotic cells are morphologically characterized by small mitochondria with membrane rupture and vanishing of the crista, ^7, 8 which are obviously different from necrosis, apoptosis, and autophagy. Increasing evidence has shown the great potential of triggering ferroptosis as an effective anti-cancer therapy to eradicate malignancies. ⁹ For example, Sorafenib, an FDA-approved anti-cancer drug, was identified as a ferroptosis inducer by blocking the synthesis of glutathione (GSH) . ¹⁰ However, Sorafenib may cause serious clinical adverse effects, including life-threatening cardiovascular events. ^11, 12 Therefore, developing anti-cancer strategies through inducing ferroptosis with higher efficacy while improving safety is of great importance.

Cancer immunotherapy through enhancing patient's own immune system to eliminate cancer cells has revolutionized the landscape of cancer therapeutics over the past decade. However, substantial benefit of immunotherapy is observed only for a limited fraction of cancer patients. ^13-15 Recently, several NPs have been reported to elicit tumor microenvironment modulation, such as the macrophage reprogramming by manganese dioxide NPs ¹⁶ or by magnetic NPs, ^17, 18 and the promotion of anti-tumor cytotoxic T cells’ function by gold NPs, ¹⁹ as well as the augmented cytotoxic T cells recruitment to tumor site by immune agonist-loaded NPs. ²⁰ Nevertheless, biocompatibility and large-scale production still remained the major challenges in anti-cancer nanomedicine development. Accordingly, ZVI-NPs, efficiently delivering massive iron to cancer cells, might serve as a promising strategy to induce tumor ferroptosis. Besides, how ZVI-NPs modulate immune responses is also worthy of further exploration.

SUMMARY

In an aspect, the invention provides a method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator, which comprises administering an effective dose of the nano-modulator to the subject.

In yet a further aspect, the invention also provides a method for modulating an immune cell behavior in tumor microenvironment in a subject in need thereof using a nano-modulator, which comprises administering an effective dose of the nano-modulator to the subject.

In yet a further aspect, the invention also provides a method for treating an angiogenesis-related disease and/or disorder in a subject in need thereof using a nano-modulator, which comprises administering an effective dose of the nano-modulator to the subject.

According to the aforementioned aspect, the invention provides a method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator. In an embodiment, the method can include the step of administering an effective dose of the nano-modulator to the subject. In the aforementioned embodiment, the nano-modulator can be consisted of coated zero-valent iron (ZVI) -containing nanoparticles.

In the aforementioned embodiment, a metallic coating or an non-metallic coating can be disposed on a surface of the coated ZVI-containing nanoparticles. In some examples, the metallic coating can include silver, gold, iron or copper. In other examples, the non-metallic coating can include carboxymethyl cellulose (CMC) , polystyrene malic acid or mesoporous silica.

In the aforementioned embodiment, the subject can include cancer cells and the tumor microenvironment. In certain examples, the subject can undergo a cancer treatment. The cancer treatment can be for lung cancer. The administration can be performed as a treatment alone or as an adjuvant before, during or after the cancer treatment.

In the aforementioned embodiment, the effective dose of the nano-modulator can be at least 1 μg/mL.

In the aforementioned embodiment, the nano-modulator can up-regulate expression levels of glycogen synthase kinase 3 beta /beta-transducin repeats-containing protein (GSK3β/β-TrCP) and phosphorylation of AMP-activated protein kinase (AMPK) in the subject. In other embodiments, the nano-modulator can down-regulate expression levels of NRF2, mTOR, SLC7A11, GPX4, apoptosis-inducing factor 2 (AIFM2) , AKR1 family genes, PD-L1, cancer stemness genes, and angiogenesis-related genes in the subject. In certain examples, those expression levels can include a transcriptional expression and/or a translational expression.

According to another aspect, the invention provides a method for modulating an immune cell behavior in tumor microenvironment in a subject in need thereof using a nano-modulator. In an embodiment, the method includes the step of administering an effective dose of the nano-modulator to the subject, in which the nano-modulator can be coated ZVI-containing nanoparticles.

In the aforementioned embodiment, the subject is cancer cells and immune cells in the tumor microenvironment. In certain examples, the subject can undergo a cancer treatment. The cancer treatment can be for lung cancer. The administration can be performed as a treatment alone or as an adjuvant before, during or after the treatment.

In the aforementioned embodiment, the immune cell behavior can include promotion of a lymphocyte-mediated cytotoxicity, enhancement of M1-phenotype macrophage population, decrease of M2-macrophage proportion, reduction of a proportion of PD-1 ⁺ cells among CD8 ⁺ lymphocytes, and reduction of a proportion of regulatory T (Treg) cells.

According to yet a further aspect, the invention provides a method for treating an angiogenesis-related disease and/or disorder in a subject in need thereof using a nano-modulator. In an embodiment, the method comprises administering an effective dose of the nano-modulator to the subject. The nano-modulator can have a ZVI core.

In the aforementioned embodiment, the angiogenesis-related disease and/or disorder can be or be caused by tumor, inflammation or inflammatory disease, metabolic disorders, infection, cardiovascular disease, injury, vaccination or age-related degeneration.

With application to the method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator and applications thereof, which comprises administering an effective dose of the nano-modulator of coated ZVI-containing nanoparticles to the subject, for up-regulating or down-regulating expression levels of specific genes in the subject. The nano-modulator can be applied in the development of anti-cancer precision nanomedicine and immunotherapy.

It is to be understood that both the foregoing general description and the following detailed description are by examples, and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure can be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows.

FIG. 1A shows a schematic diagram (left panel) and TEM images (right panel) revealing the round shape morphology and surface coating of both types of NPs according to several embodiments of the invention. (Scale bar: 50 nm. )

FIG. 1B shows line graphs of intracellular concentrations of ZVI@CMC NPs and iron ions determined in A549 (left panel) and MRC-5 (right panel) cells after treatment with ZVI@CMC NPs (10 mg mL ^-1) for 1, 2, 4, 8, or 24 hours according to several embodiments of the invention.

FIG. 1C shows TEM images showing the presence of ZVI@Ag (arrow) and the mitochondrial with damaged cristae 24 hours after NPs (10 μg mL ^-1) treatment (lower panel) compared to the control group (upper panel) according to several embodiments of the invention.

FIGS. 1D and 1E show line graphs of MTT assay showing both ZVI@Ag NPs (FIG. 1D) and ZVI@CMC NPs (FIG. 1E) dose-dependently inhibited cell viability in lung cancer cells H460, A549 and H1299 without affecting normal lung cells MRC-5 and IMR-90 after 48 hours of treatment according to several embodiments of the invention.

FIGS. 1F and 1G show line graphs of both ZVI@Ag NPs or ZVI@CMC NPs without inhibiting cell viability of ex vivo isolated BMDMs (FIG. 1F) and splenic lymphocytes (FIG. 1G) in the test concentrations (5 and 10 μg mL ^-1) in the 72 hours of treatment period according to several embodiments of the invention.

FIGS. 1H and 1I are bar graphs of biodistribution of ZVI@Ag NPs (FIG. 1H) or ZVI@CMC NPs (FIG. 1I) in major organs in nude mice given intravenous injection of a dose (50 mg kg ^-1) according to several embodiments of the invention. The iron concentrations in tissue samples were quantified at indicated time (n=5 per group) . Data were mean ± s.e.m. (n=3 for cell-based assays) . ns: non-significant; *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 2A to 2C show representative images of the size (FIG. 2A) and the number (FIG. 2B) as well as bar graphs of the quantitative analysis of cancer spheres (FIG. 2C) derived from H1299 (left panel) and H460 cells (right panel) treated with ZVI@Ag NPs measured under an inverted light microscope according to several embodiments of the present invention.

FIG. 2D is bar graphs of expression of cancer stemness genes measured by RT-qPCR after ZVI@Ag NPs treatment for 48 hours in H1299 (upper panel) and H460 (lower panel) according to several embodiments of the present invention.

FIGS. 2E and 2F show representative trans-well migration images (FIG. 2E) and its quantitative analysis (FIG. 2F) of HUVEC endothelial cells cultured in CM derived from H460 or H1299 cells treated with or without ZVI@Ag NPs for 8 hours according to several embodiments of the present invention.

FIGS. 2G and 2H show representative tube formation microscopy images (FIG. 2G) and its quantitation (FIG. 2H) of HUVECs treated with ZVI@Ag NPs for 16 hours according to several embodiments of the present invention.

FIG. 2I shows bar graphs of expression of pro-angiogenesis genes measured by RT-qPCR after ZVI@Ag NPs treatment for 48 hours in H1299 (upper panel) and H460 (lower panel) according to several embodiments of the present invention. The expression levels of mRNA are normalized to β-actin. Data were mean ± s.e.m. (n=3) . *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 3A is a bar graph of mitochondrial membrane potential analyzed by flow cytometry after ZVI@Ag NPs treatment according to several embodiments of the present invention.

FIG. 3B shows bar graphs of oxygen consumption rate (OCR) in terms of basal (left panel) or maximum (right panel) respiration examined using seahorse XF24 analyzer after ZVI@Ag NPs (10 μg mL ^-1) treatment for 24 hours according to several embodiments of the present invention.

FIG. 3C is a bar graph of bioluminescence assay showed that total ATP levels dose-dependently suppressed in all three cancer lines by ZVI@Ag NPs treatment for 24 hours according to several embodiments of the present invention.

FIG. 3D is a bar graph of mitochondrial ROS in the three cancer lines analyzed by flow cytometry after ZVI@Ag NPs treatment according to several embodiments of the present invention.

FIG. 3E shows line graphs of intracellular ROS levels measured by flow cytometry after ZVI@Ag NPs (5 μg mL ^-1) treatment with or without Vitamin E (100 μM) according to several embodiments of the present invention.

FIG. 3F is a bar graph of colorimetric analysis performed to detect NADPH levels after ZVI@Ag treatment for 24 hours according to several embodiments of the present invention.

FIG. 3G is a bar graph of flow cytometry analysis of lipid peroxidation for the three cancer cells treated with ZVI@Ag NPs (5 μg mL ^-1) with or without Ferrostatin (10 μM) pre-treatment according to several embodiments of the present invention.

FIG. 3H shows bar graphs of cell viability determined after co-treatment with ZVI@Ag NPs (10 μg mL ^-1) and Vitamin C (100 μM) , Vitamin E (100 μM) , Ferrostatin (10 μM) , or Liproxstatin (10 μM) for 48 hours according to several embodiments of the present invention. Data were mean ± s.e.m. (n=3) . ns: non-significant; *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 4A shows immunoblotting images of NRF2 and GPX4 in four cancer cell lines treated with ZVI@CMC NPs at the indicated doses for 24 hours. GAPDH is used as internal control according to several embodiments of the present invention.

FIG. 4B shows bar graphs of ChIP-qPCR assay performed to measure NRF2 binding ability to the promoter region of SLC7A11, AKR1C1 and AIFM2 in cells treated with ZVI@Ag NPs in H460 (upper panel) and A549 (lower panel) .

FIG. 4C shows a gene heatmap of mRNA expression of NRF2 downstream genes measured by RT-qPCR after ZVI@Ag NPs treatment in H460 and A549 cells according to several embodiments of the present invention. The heat map reflects downregulation of the mRNA levels of these genes compared to the respective untreated controls.

FIG. 4D shows immunoblotting images of p-AMPK, total AMPK, p-mTOR, total mTOR, p-GSK3β and total GSK3β in cells treated with ZVI@Ag NPs (left panel) or ZVI@CMC NPs (right panel) for the indicated time according to several embodiments of the present invention.

FIG. 4E shows immunofluorescence staining images of NRF2, β-TrCP and DAPI in A549 (upper panel) and H460 (lower panel) cells after ZVI@Ag NPs or ZVI@CMC NPs treatment according to several embodiments of the present invention. Data were mean ± s.e.m. (n=3) . *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 5A to 5C show a line graph of the changes in tumor volume over 15 days of observation period (FIG. 5A) , the representative tumor images in the end point (FIG. 5B) , and a bar graph of the end point tumor weight (FIG. 5C) of BALB/c nude mice bearing H460 xenografts treated with 50 mg kg ^-1 ZVI@Ag NPs or PBS by intraperitoneal injection on every other day as indicated by arrows (n=6 for control group, n=7 for ZVI@Ag NPs treated group) according to several embodiments of the present invention.

FIGS. 5D to 5F show a curve graph of the tumor volume (FIG. 5D) , the representative tumor images of the last time point (FIG. 5E) , and a bar graph of the final tumor weight (FIG. 5F) of NOD/SCID mice bearing A549 xenografts treated with ZVI@Ag NPs or PBS by intravenous injection once a week (indicated by arrows) (n=5 for each group) according to several embodiments of the present invention.

FIG. 5G shows Immunohistochemistry images revealed the expression of 4-HNE, NRF2, GPX4 and endothelial cells marker CD31 in tumor tissues from H460 xenografts (left panel) and A549 xenografts (right panel) with or without ZVI@Ag NPs treatment according to several embodiments of the present invention.

FIG. 5H is a bar graph of down regulation of NRF2 targeting genes in H460 xenografts treated with 50 mg kg ^-1 ZVI@Ag NPs determined by RT-qPCR.

FIGS. 5I to 5K show a line graph of the tumor volume (FIG. 5I) , the representative tumor images at the end point of experiment (FIG. 5J) , and a bar graph of the respective tumor weight (FIG. 5K) of NOD/SCID mice bearing H460 xenografts or NRF2 overexpress H460 xenografts treated with ZVI@CMC NPs or PBS by 4 episodes of intravenous injection (indicated by arrows) according to several embodiments of the present invention. Liproxstatin-1 (10 mg kg ^-1) treatment is conducted by daily intraperitoneal injection for 10 days.

FIGS. 5L shows images of lung dissected on day 54 from mouse subcutaneously implanted with A549 cells then subjected to intravenous injection of ZVI@Ag NPs or PBS once a week for four weeks according to several embodiments of the present invention.

FIGS. 5M shows histopathology images of the lung tissues after H&E staining according to several embodiments of the present invention.

FIG. 5N is a bar graph of quantification of the metastatic tumor area according to several embodiments of the present invention. Data were mean ±s.e.m. ns: non-significant; *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 6A is a schematic diagram of dosing regimens of ZVI@CMC in immunocompetent C57BL/6 mice bearing LLC allografts according to several embodiments of the present invention. C57BL/6 mice are treated with intravenous injection of ZVI@CMC (25 mg kg ^-1) or PBS twice a week as indicated by arrows (n= 5 per group) .

FIG. 6B is a line graph of the changes in tumor volume over experimental period according to several embodiments of the present invention.

FIGS. 6C to 6D show the representative images of the dissected tumor (FIG. 6C) and a bar graph of the quantification of tumor weight (FIG. 6D) measured at the endpoint of the experiment according to several embodiments of the present invention.

FIGS. 6E to 6H show immunofluorescent microscopy images of tissue sections stained with antibodies against mouse CD86 (green) and CD206 (red) for observation of tumor-associated macrophages (FIGS. 6E and 6F) and antibodies against mouse CD8 (green) and CD4 (red) to observe infiltrating T cells (FIGS. 6G and 6H) according to several embodiments of the present invention. Scale bar: 100 μm.

FIGS. 6I to 6M show scatter graphs of flow cytometry analysis of the tumor-associated macrophages (FIGS. 6I to 6J) and infiltrating T cells (FIGS. 6K to 6M) in endpoint tumors according to several embodiments of the present invention.

FIG. 6N is a schematic diagram of dosing regimens of ZVI@CMC in xenograft bearing hPBMC reconstituted ASID mice according to several embodiments of the present invention. Mice are treated with ZVI@CMC (25 mg kg ^-1) or PBS by intravenous injection on every other day as indicated by arrows (n=5 per group) .

FIGS. 6O to 6P show the line graphs of the tumor volume (FIG. 6O) and the body weight (FIG. 6P) measured during experiment according to several embodiments of the present invention.

FIG. 6Q is a scatter graph of flow cytometry analysis of Tregs in endpoint tumors according to several embodiments of the present invention. Data are mean ± s.e.m. ns: non-significant; *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 7A to 7B show bar graphs of expression of M1 associated gene (iNOS2) (upper panel, FIG. 7A) and M2 associated gene (Arginase-1) (upper panel, FIG. 7B) measured by RT-qPCR in BMDMs treated with ZVI-NPs while co-cultured with LLC cancer cells (lower panel of FIGS. 7A to 7C) according to several embodiments of the present invention. FIG. 7C is a bar graph of the percentage of CD86 ⁺/CD206 ⁺ (M1/M2) macrophage determined by flow cytometry according to several embodiments of the present invention. The expression levels of mRNA are normalized to β-actin.

FIGS. 7D to 7E show bar graphs of expression of M1 associated gene (TNFα) (upper panel, FIG. 7D) and M2 associated gene (DC-SIGN) (upper panel, FIG. 7E) measured by RT-qPCR in THP-1 macrophages treated with ZVI-NPs while stimulated using IFN-γ plus LPS for M1 polarization and IL-4 for M2 polarization (lower panel of FIGS. 7D to 7E) according to several embodiments of the present invention. The expression levels of mRNA are normalized to β-actin.

FIG. 7F is a bar graph of gene expression of PD-L1 (upper panel) measured in A549 cells that were co-cultured with THP-1 cells (lower panel) according to several embodiments of the present invention.

FIG. 7G shows images of the expression of PD-L1 measured by immunohistochemistry staining in H460 xenografts and LLC allografts according to several embodiments of the present invention.

FIG. 7H is a bar graph of the percentage of Treg cell differentiation (upper panel) of ZVI-NPs-treated splenic T cells with 2 ng mL ^-1 TGF-β stimulation analyzed by flow cytometry (lower panel of FIGS. 7H to 7I) according to several embodiments of the present invention. FIG. 7I is a bar graph of the percentage of PD-1 ⁺ in CD8 ⁺ T cells (upper panel) treated with ZVI-NPs analyzed by flow cytometry (lower panel of FIGS. 7H to 7I) according to several embodiments of the present invention.

FIG. 7J is a bar graph of cancer cell viability (upper panel) measured by luciferase assay, in which Luciferase-LLC cells (LLC-luc) are mixed and cultured with splenic lymphocytes at 1: 10 ratio, and then treated with ZVI-NPs for 24 hours (lower panel) according to several embodiments of the present invention. Data are mean ± s.e.m. (n=3) . ns: non-significant; *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 8 is a schematic diagram of dual synergistic anti-cancer activities and immunomodulation of ZVI-NPs according to several embodiments of the present invention.

DETAILED DESCRIPTION

Reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

As aforementioned, the present invention provides a method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator and applications thereof, which comprises administering an effective dose of the nano-modulator to the subject, for up-regulating or down-regulating expression levels of specific genes in the subject, leading in treatment of an angiogenesis-related disease and/or disorder as well as cancers.

Generally, the nano-modulator recited herein can have a ZVI core or be coated ZVI-containing nanoparticles. A metallic coating or a non-metallic coating can be disposed on a surface of the coated ZVI-containing nanoparticles. In some examples, the metallic coating can include but not be limited to silver, gold, iron or copper. In other examples, the non-metallic coating can include but not be limited to carboxymethyl cellulose (CMC) , polystyrene malic acid or mesoporous silica. In certain examples, the coated ZVI-containing nanoparticles can be selected from the group consisting of silver (Ag) -coated ZVI-containing nanoparticles (or abbreviated as “ZVI@Ag NPs” ) and carboxymethyl cellulose (CMC) -coated ZVI-containing nanoparticles (or abbreviated as “ZVI@CMC NPs” ) . In some embodiments, ZVI@Ag NPs and ZVI@CMC NPs can be synthesized by conventional methods. For examples, ZVI@Ag NPs and ZVI@CMC NPs can be synthesized by chemical reduction method using sodium borohydride (NaBH ₄) as a reducing agent under ambient conditions. In certain examples, the nano-modulator recited herein can refer to the only active ingredient, the primary ingredient or an adjuvant in a composition.

In some embodiments, the nano-modulator can modulate activities of genes in the subject. In these embodiments, the subject can be, for examples, cancer cells and the tumor microenvironment. In some examples, the nano-modulator can up-regulate expression levels of glycogen synthase kinase 3 beta /beta-transducin repeats-containing protein (GSK3β/β-TrCP) and phosphorylation of AMP-activated protein kinase (AMPK) in the subject. In other embodiments, the nano-modulator can down-regulate expression levels of NRF2, mTOR, SLC7A11, GPX4, apoptosis-inducing factor 2 (AIFM2) , AKR1 family genes, PD-L1, cancer stemness genes, and angiogenesis-related genes in the subject. In the aforementioned examples, the cancer stemness genes can include but not be limited to OCT4, Nanog, and SOX2 genes (known as Yamazaki factor) . The angiogenesis-related genes can include but not be limited to Sonic hedgehog, TGF-β and VEGF genes. In certain examples, those expression levels can include a transcriptional expression and/or a translational expression.

Optionally, the subject can undergo a conventional cancer treatment, for example. In these examples, the administration can be performed as a treatment alone or as an adjuvant before, during or after the cancer treatment. The conventional cancer treatment can include but not be limited to chemotherapy, radiation therapy, immunotherapy and targeted therapy. In some examples, the treatment can be for any types of cancer, for examples, for solid tumors, or for lung cancer. In other examples, the administration can be performed before, during or after the treatment.

In some embodiments, the present invention also provides a method for modulating an immune cell behavior in tumor microenvironment in a subject in need thereof using a nano-modulator. In these embodiments, the subject can be, for examples, cancer cells and immune cells in the tumor microenvironment. In certain examples, the subject can undergo a cancer treatment. The cancer treatment can be for lung cancer. The administration can be performed as a treatment alone or as an adjuvant before, during or after the cancer treatment. In certain examples, the immune cell behavior can include but not be limited to promotion of a lymphocyte-mediated cytotoxicity, enhancement of M1-phenotype macrophage population, reduction of a proportion of PD-1 ⁺ cells among CD8 ⁺ lymphocytes, and reduction of a proportion of regulatory T (Treg) cells.

In certain examples, the tumor microenvironment can surround the aforementioned cancer cell (s) and include a precancerous lesion, its apparently normal counterpart or an apparently normal cell (for examples, a lymphocyte) . In some examples, the lymphocyte can be a M1-type macrophage, and an activity of the M1-type macrophage can be promoted after the administration of the nano-modulator.

In other embodiments, the present invention also provides a method for treating an angiogenesis-related disease and/or disorder in a subject in need thereof using a nano-modulator. In an embodiment, the method comprises administering an effective dose of the nano-modulator to the subject. The nano-modulator can have a ZVI core. In this embodiment, the ZVI core can be coated with a metal coating or a non-metallic coating. In some examples, the metallic coating can include but not be limited to silver, gold, iron or copper, and the non-metallic coating can include carboxymethyl cellulose (CMC) , polystyrene malic acid or mesoporous silica.

In those embodiments, the angiogenesis-related disease and/or disorder can be or be caused by tumor, inflammation or autoimmune diseases, metabolic disorders, infection, cardiovascular disease, injury, vaccination or age-related degeneration.

In the aforementioned embodiments, the terms “cancer” , “cancer cell” , “tumor” and “tumor cell” are used interchangeably herein and refer generally to a group of diseases characterized by uncontrolled, abnormal growth of cells (e.g., a neoplasia) . In some forms of cancer, the cancer cells, which can be drug-sensitive or drug-resistant, can spread locally or through the bloodstream and lymphatic system to other parts of the body ( “metastatic cancer” ) .

In certain examples, the tumor can be selected from the group consisting of lung cancer (squamous) , lung cancer (adenocarcinoma) , small cell lung carcinoma, non-small cell lung carcinoma, skin cancer, head and neck cancer, nasopharyngeal carcinoma, thyroid cancer, breast carcinoma, gastric carcinoma, pancreatic cancer, liver carcinoma, renal cell carcinoma, colorectal carcinoma, urinary bladder carcinoma, cervical carcinoma (squamous) , ovarian carcinoma, prostate cancer, sarcomas, melanoma and hemangioma.

In some examples, the inflammatory or autoimmune diseases can include but not be limited to rheumatoid arthritis, osteoarthritis, diabetic retinopathy, psoriasis, Sjogren's syndrome, acne rosacea, systemic lupus, Wegeners sarcoidosis, polyarteritis, scleroderma, Crohn's disease or Bartonellosis.

In other examples, the metabolic syndrome can include but not be limited to diabetes, high blood pressure (hypertension) and obesity.

In certain examples, the infection can include but not be limited to a bacterial infection, a virus infection, a fungal infection or a protozoan infection.

In some examples, the cardiovascular disease can include but not be limited to atherosclerosis, myocardial angiogenesis, hyperviscosity syndromes, vein occlusion, artery occlusion, carotid obstructive disease or Osler-Weber-Rendu disease.

In other examples, the age-related degeneration can include but not be limited to age-related macular degeneration (AMD) or a chronic wound.

In practice, the nano-modulator the administration can be performed as a treatment alone or as an adjuvant, or optionally in combination with pharmaceutically available excipients and/or carriers, to a subject in need thereof, before, during or after a treatment of the angiogenesis-related disease and/or disorder, depending on the actual requirements.

In some chemotherapy regimens, the nano-modulator can be introduced to the subject via conventional routes, for example, intravenous (i.v. ) , intramuscular (i.m. ) , intraperitoneal (i.p. ) , intrathecal, cutaneous, subcutaneous (s.c. ) , transdermal, sublingual, buccal, rectal, vaginal, ocular, otic, nasal, In some embodiments, there is no limitation on the dose of the nano-modulator administering to the subject in need thereof, for example, such dose without causing unacceptable toxicity, or called as the maximum tolerated dose (MTD) ; however, in some examples, the nano-modulator can be administered in an in vitro effective dose of 0.1 μg/mL to 100 μg/mL, preferably 1 μg/mL to 50 μg/mL, and more preferably 5 μg/mL to 10 μg/mL. In certain examples, an in vivo dose of the nano-modulator can be converted from the in vitro effective dose according to common calculation approaches.

In this embodiment, there is no limitation to the pharmaceutically available excipient and/or carrier, for example, such as water, solution, organic solvent, pharmaceutically available oil or fat or their mixture. In some examples, the pharmaceutically available carrier and/or an excipient can be a saline, sterilized water, a Ringer's solution, a buffered saline, an albumin injection, a dextrose solution, a maltodextrin solution, a glycerol, ethanol, or a mixture of at least one thereof may be used, and conventional additives such as antioxidants, buffers, bacteriostats, etc. may be added when needed.

Typically, the nano-modulator can modulate tumor microenvironment, via increasing a phosphorylation level of AMPK and/or suppressing a phosphorylation level of the mTOR, resulting in modulating Nrf2 activity in a subject in need thereof, which may involve suppression of Nrf2 expression of the subject undergoing a treatment with anti-cancer drug, decrease of the Nrf2-mediated gene activity of the subject after the administration. It should be noted that, ZVI@CMC NPs having biocompatible coating and mass producibility may exert the potential for overcoming drug resistance in cancer and advance in cancer therapy.

Thereinafter, it will be understood that particular configurations, aspects, examples, clauses and embodiments described hereinafter are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Thus, one skilled in the art can easily ascertain the essential characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

EXAMPLE 1

1.1 Preparation of ZVI@Ag and ZVI@CMC NPs

ZVI@Ag and ZVI@CMC NPs were synthesized as described in previous studies ^3, 4. In brief, to synthesize ZVI@Ag, ferrous sulfate (FeSO ₄) and trisodium citrate (Na ₃C ₆H ₅O ₇) dehydrate were mixed within deionized water by magnetic stirring. Next, sodium borohydride (NaBH ₄) was added dropwise into the mixture and stirred at room temperature to form ZVI. Finally, silver nitrate (AgNO ₃) was added with stirring to get ZVI@Ag NPs. To synthesize ZVI@CMC, ferrous sulfate (FeSO ₄) and carboxymethyl cellulose (CMC) were mixed within stirred distilled water followed by adding sodium borohydride (NaBH ₄) to the stirred mixture at room temperature for ZVI@CMC NPs assembly. Ultimately, both NPs solutions were washed with ethanol several times and collected using a magnet platform. Preparation of ZVI@Ag or ZVI@CMC NPs was done in an argon gas environment. The hydrodynamic size distribution of ZVI-NPs measured by dynamic light scattering at room temperature was done using Delsa Nano C Particle analyzer (Beckman Coulter, Brea, CA, USA) .

1.2 Cell lines and culture conditions

Human lung cancer cell lines H1299, H460, A549, mouse Lewis lung carcinoma (LLC) , and normal human lung cell lines MRC-5 and IMR-90 were purchased from American Tissue Culture Company (Rockville, MD, USA) . Luciferase-LLC (LLC-luc) cell line was provided by Dr. Muh-Hwa Yang (Institute of Clinical Medicine, National Yang-Ming University, Taiwan) . These cell lines were maintained in DMEM medium (Gibco, Grand Island, NY, USA) . Human monocytic cell line THP-1 was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) (accession number: BCRC 60430) , or American Type Culture Collection (ATCC, U.S.A. ) (accession number: ATCC TIB-202) , and maintained in RPMI 1640 medium (Gibco) . Both DMEM and RPMI 1640 media were supplemented with 10%Fetal Bovine Serum (FBS; Gibco) and 1%penicillin/streptomycin (Gibco) . Human umbilical vein endothelial cell line (HUVEC) was provided by Dr. Li-Wha Wu (Institute of Molecular Medicine, National Cheng Kung University, Taiwan) . HUVEC cells cultured in dishes, which were coated with 0.1%gelatin for 1 hour, were maintained with endothelial cell growth medium-2 (EBM-2; Lonza, Walkersville, MD, USA) and supplemented with SingleQuots ^TM growth factor kit (Lonza) . All cells were incubated at 37℃ with 5%CO ₂.

1.3 Mouse splenocytes isolation

Following the sacrifice of C57BL/6 mice, spleens were aseptically harvested and washed three times with PBS. To obtain a single cell suspension, the spleens were crushed and passed through a 70-μm nylon cell strainer (FALCON, Corning, NY, USA) , and then red blood cells were lysed and removed. The splenocytes were resuspended thoroughly in RPMI 1640 medium containing 10%FBS and 1%penicillin/streptomycin.

1.4 Regulatory T cells (Tregs) differentiation

Following splenocytes isolation, cells were incubated with RPMI 1640 medium containing 10%FBS, 1%penicillin/streptomycin, plate-bound anti-CD3 and anti-CD28 antibodies (BD Biosciences, San Jose, CA, USA) , recombinant mouse 5 ng ml ^-1 IL-2 and recombinant human TGF-β for Treg differentiation.

1.5 Luciferase cytotoxic T lymphocyte assay

Following splenocytes isolation, 1 × 10 ⁴ lymphocytes were then incubated with 1 × 10 ³ Luciferase-LLC cells (LLC-luc) cells per well (96-well plate) containing 100 μl RPMI 1640 medium supplemented with 10%FBS and 1%penicillin/streptomycin. During the co-cultivation,

lymphocytes were activated by stimulation using plate-bound anti-CD3 and anti-CD28 antibodies, interleukin (IL) -2 (EL-4 culture supernatant) and IL-7 (R&D Systems, Minneapolis, MN, USA) , Insulin-Transferrin-selenium (ITS; Gibco) , and β-mercaptoethanol (SERVA, Heidelberg, Germany) as previously described ⁵⁵. After 24 hours co-cultivation, culture medium was removed, and cell pellets were rinsed twice with PBS. The number of viable LLC-luc cells was determined by luciferase assays using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) as previously described ⁵⁶.

1.6 Isolation of mouse bone-marrow-derived macrophages (BMDMs)

Isolated bones were aseptically harvested from hind legs of C57BL/6 mice, and muscle tissues were removed. Bone marrow was flushed out of the bones using a 25-gauge needle attached to a syringe containing BMDM growth medium, which consists of DMEM, 20%L929 cell-conditioned media to generate M-CSF (macrophage colony-stimulating factor) , 10%FBS, and 1%penicillin/streptomycin. Then, BMDMs were allowed to differentiate for 7 days at 37℃ with 5%CO ₂, and the growth medium was changed every 2 days during ex vivo culture.

1.7 THP-1 macrophage differentiation and polarization

The macrophage-like state was induced by treating THP-1 monocytes for 48 hours with 100 ng ml ^-1 phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in 6 well-plates at a density of 5 × 10 ⁵ cells/well. After washing twice with culture medium, the resting macrophages (M0) were treated with 20 ng ml ^-1 IFNγ plus 1 μg ml ^-1 LPS for 6 hours to differentiate into the M1 phenotype or with 20 ng ml ^-1 IL-4 for 24 hours to the M2 phenotype. Cells were maintained in 5%CO ₂ at 37℃ during differentiation and polarization.

1.8 Co-culture system of macrophages and cancer cells

For LLC/BMDM co-culture system, the lower compartment of a 6-well plate was seeded with BMDMs (1 × 10 ⁶) while the upper compartment with LLC cells (1 × 10 ⁶) . Cells were cultured with BMDM growth medium containing ZVI-NPs or not for 48 hours at 37℃ with 5%CO ₂. BMDMs were collected for further analysis. For A549/THP-1 macrophage co-culture system, the lower compartment of a 6-well plate was seeded with A549 cells (1 × 10 ⁶) while the upper compartment with THP-1 macrophages (M0) (1 × 10 ⁶) . Cells were cultured with RPMI 1640 medium containing 10%FBS and treated with ZVI-NPs or not for 48 hours at 37℃ with 5%CO ₂. A549 cells were collected for further analysis.

1.9 Cell viability assay

Cells viability assay was performed to evaluate the cytotoxicity of ZVI-NPs by using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay or cell counting kit 8 (CCK-8) assay. After ZVI-NPs treatment, cells were replaced with fresh medium containing 1 mg ml ^-1 MTT (Sigma-Aldrich) or CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) and then incubated at 37℃ for 2 hours. For MTT assay, crystals were dissolved in dimethyl sulfoxide, and the optical absorbance at 570 nm was measured. For CCK-8 assay, the optical absorbance at 450 nm was measured.

1.10 Cancer sphere formation assay

H460 and H1299 cells were seeded in ultra-low attachment 6-well plates with DMEM/F12 containing N-2 supplement (Invitrogen, Foster City, CA, USA) , 20 ng ml ^-1 epithelial growth factor (PeproTech Inc., Rocky Hill, NJ, USA) , 20 ng ml ^-1 basic fibroblast growth factor (PeproTech Inc. ) and 1%penicillin/streptomycin at 2 × 10 ⁴ cells per well. Cells were incubated for 7 days and then treated with or without ZVI-NPs for 48 hours. Cancer spheres consisting of 20 or more cells were photographed and counted.

1.11 Endothelial cell transwell migration assay

HUVEC cells (1 × 10 ⁵) were seeded into the upper chambers of transwell (Falcon, Franklin Lakes, NJ, USA) with serum-free DMEM medium. The lower chambers were filled with DMEM medium containing 20%FBS plus ZVI-NPs and then incubated at 37℃ for 24 hours. The cells attached on the reverse side of the membrane were stained with crystal violet and counted under an upright microscope (Nikon E400, Tokyo, Japan) .

1.12 Tube formation assay

Phenol Red-free Matrigel (Corning, New York, NY, USA) was added to 96-well plates and then incubated at 37℃ for 1 hour. HUVEC cells (2 × 10 ⁴ per well) were seeded into 96-well plates with culture medium containing ZVI-NPs or not and then incubated for 8 hours. Tube formation was observed and photographed randomly under microscope (Nikon E400) .

1.13 Intracellular ROS and lipid peroxidation measurement

To measure intracellular ROS level, cells (8 × 10 ⁴ per well) were seeded in 12-well plates. After treatment with ZVI-NPs, cells were detached with trypsin, washed twice with PBS, and then incubated with 10 μM H ₂DCFDA (Sigma-Aldrich) at 37℃ for 30 min in the dark. After washing with PBS, the intracellular ROS levels were analyzed by flow cytometry (CytoFLEX ^TM, Beckman coulter, Brea, CA, USA) . To detect lipid peroxides level, cells were incubated with 5 μM Liperfluo (Dojindo) at 37℃ for 30 min in the dark. Sample fluorescence was measured by flow cytometry (CytoFLEX ^TM) .

1.14 Mitochondrial ROS and mitochondrial membrane potential assay

To measure mitochondrial ROS, cells were incubated with 5 μM MitoSOX (Invitrogen) at 37℃ for 30 min in the dark. Sample fluorescence was measured by flow cytometry (CytoFLEX ^TM) . To detect mitochondrial membrane potential, cells were incubated with 20 nM 3,3-Dihexyloxacarbocyanine iodide (DiOC6) (Enzo, New York, NY, USA) at 37℃ for 15 min in the dark and then analyzed by flow cytometry (CytoFLEX ^TM) .

1.15 Mitochondrial respiration function measurement

Cell monolayers were cultured in XF Cell Culture Microplates (Seahorse Bioscience, North Billerica, MA, USA) at a density of 2.5 × 10 ⁴ cells per well. The sensor cartridge (Seahorse Bioscience) was polarized overnight and calibrated. After ZVI-NPs treatment, the medium was replaced with appropriate assay medium without sodium bicarbonate and serum, and cells were then incubated for 30 min at 37℃ without CO ₂. The compounds were injected sequentially: 1 μM oligomycin; 2 μM FCCP; 2 μM Rotenone (all from Sigma-Aldrich) . The basal OCR and OCR responses toward compounds injection were performed in a Seahorse XF24 analyzer (Seahorse Bioscience) according to the manufacturer’s instructions.

1.16 Total ATP and NADPH determination assay

Total ATP level was measured by ATP determination kit (Invitrogen) . Cells (8 × 10 ⁴ per well) were seeded in 12-well plates. After ZVI-NPs treatment, cells were lysed and mixed with 1X reaction solution, and then incubated for 5 to 15 min. Then, the sample analysis was performed according to the manufacturer's instructions.

To detect NADPH level, NADPH determination assay kit (Biovision, San Francisco, CA, USA) was employed. Cells (8 × 10 ⁵) were seeded in 10 cm dish. After ZVI-NPs treatment, cells were lysed by extraction buffer and then heated 60℃ for 30 min. The extracted samples were then applied to each well of a 96-well plate and mixed with NADPH developer at room temperature for 1 to 4 hours incubation. To determine the intracellular NADPH level, the optical absorbance at 450 nm was measured once an hour according to the manufacturer's instructions.

1.17 Transfection of plasmids

NRF2 plasmid (pCMV3-C-OFP/NFE2L2) was purchased from Sino Biological Inc. (Beijing, China) , and transfection was conducted using Turbofect reagent (Invitrogen) according to the manufacture’s protocol. After 24 hours transfection, cells were harvested for animal experiments.

1.18 RNA extraction and RT-qPCR assays

After ZVI-NPs treatment, total RNA was extracted using Trizol reagent (Invitrogen) . Purified RNA was converted into cDNA by reverse transcription. RT-qPCR was performed with SYBR Green Master Mix (Invitrogen) using the StepOnePlus ^TM Real-Time PCR system (Life Technologies, Carlsbad, CA, USA) . Expression levels were normalized with internal control β-actin. The primer sets were listed in SEQ ID NOs: 1-58, as disclosed in Theranostics 11 (14) : 7072-7091 (2021) and its supplementary information, which were incorporated entirely herein by reference.

1.19 Protein extraction and Western blotting

Cells were lysed in 1x RIPA buffer (0.05 M Tris-HCl, 0.15 M sodium chloride, 0.25%deoxycholic acid, 1%Nonidet P-40, 1 mM EDTA, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 5 μg ml ^-1 leupeptin, and 10 μg ml ^-1 aprotinin) containing protease inhibitors cocktail (Sigma-Aldrich) . Lysates were centrifuged at 13,200 r.p.m. for 15 min. Protein extracts were solubilized in loading buffer (60 mM Tris-base, 2%SDS, 10%glycerol, and 5%β-mercaptoethanol) . Equal amounts of lysate were separated on 8%SDS-PAGE and transferred onto a polyvinyl difluoride (PVDF) membrane. The protein was identified by incubating the membrane with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The antibodies conditions were described in Table 1.

Table 1

^a ChIP: chromatin immunoprecipitation

^b Molecular weight is not applicable to immunohistochemistry, immunofluorescence, or flow cytometry analysis of this antibody.

1.20 Chromatin immunoprecipitation assay (ChIP assay)

Cells (5 × 10 ⁶) were cross-linked followed by preparation of nuclear lysates using Magna ChIP ^TM protein G Kit (Millipore, Burlington, MA, USA) . Nuclear lysates were sonicated to shear DNA to around 500 bp followed by immunoprecipitation for 16 hours at 4℃ using IgG or anti-NRF2 antibody (Genetex, San Antonio, TX, USA) . The levels of targeted genes in ChIP products were determined by RT-qPCR. Primers used were listed in SEQ ID NOs: 1-58, as disclosed in Theranostics 11 (14) : 7072-7091 (2021) and its supplementary information, which were incorporated entirely herein by reference.

1.21 Immunofluorescence (IF) and immunochemistry (IHC) assay

For immunofluorescence (IF) staining, Opal stain kit (PerkinElmer, Waltham, MA, USA) was employed. After cell fixation, antigen retrieval was performed with citrate buffer (pH 6.0) in a microwave oven. The slides were then washed, blocked, and incubated with β-TrCP primary antibody at 4℃ overnight followed by incubation with secondary antibody polymer HRP for 10 min and subsequently with Opal fluorophore for 10 min at room temperature. To further stain with anti-NRF2 antibody, the slides were again placed in citrate buffer (pH 6.0) and heated in a microwave oven. After Opal staining process, DAPI was applied for nuclei staining.

For immunochemistry (IHC) staining, Novolink Max Polymer kit (Leica Biosystems, Wetzlar, Germany) was employed. All slides were dewaxed with xylene/ethanol, and then antigen retrieval was performed with TRS buffer (pH 6.0) in a microwave oven. After blocking, the slides were reacted with primary antibodies. The peroxidase activity was visualized with diaminobenzidine tetrahydroxychloride (DAB) solution. The sections were counter stained with hematoxylin. Dark brown staining was considered positive. The antibodies conditions are described in Table 1.

1.22 Transmission electron microscopy (TEM) imaging

The ZVI-NPs were characterized by TEM as previously described. ^3, 4 For intracellular structure observation, cells were collected and incubated overnight with fix solution (2.5%glutaraldehyde, 3 mM CaCl ₂, and 0.1 M cacodylate) . Each sample was diluted with absolute alcohol and then applied onto copper grids followed by vacuum drying. The digital images were acquired using a JEOL JEM1400 TEM (JEOL, Tokyo, Japan) .

1.23 Animal experiment –immunodeficient and immunocompetent models

All animal experiments were performed in compliance with institutional guidelines for use and care of animals. For H460 xenograft model of immunodeficient mouse, 5-6-week-old BALB/c nude mice (ZVI@Ag treatment) or NOD/SCID mice (ZVI@CMC treatment) were subcutaneously implanted with 1 × 10 ⁶ H460 cells. For A549 xenograft model of immunodeficient mouse and spontaneous lung metastasis model, 5-6-week-old NOD/SCID mice were subcutaneously implanted with 5 × 10 ⁶ A549 cells. For subcutaneous model of immunocompetent mouse, LLC cells (5 × 10 ⁵) were injected into both flank of 6-week-old C57BL/6 mice.

When tumor volume reached 40-50 mm ³, the mice were injected with PBS or ZVI-NPs at the doses and times indicated. Mice were weighed, and the volumes of the xenografts or allografts were measured and quantified during experiment. The tumor volume was calculated as (length × width square) /2 in mm ³. For the tumor metastasis assay, lung was excised and fixed with 4%formaldehyde (Sigma-Aldrich) at the end of experiment. Area of metastatic lung tumor nodules was analyzed by image J.

1.24 Animal experiment –humanized mice animal model in vivo

To generate humanized mice, human peripheral blood mononuclear cells (hPBMCs) were intravenously injected into 6-8-week-old advanced severe immunodeficiency (ASID) mice (NOD. Cg-Prkdc ^scidIl2rg ^tm1Wjl/YckNarl; National Laboratory Animal Center, Taipei, Taiwan) . Eight days after hPBMC engraftment, H460 cells (5 × 10 ⁶) were subcutaneously injected into the right flank of hPBMC-ASID mice. When tumor xenograft volume reached 50-200 mm ³, the mice were treated intravenously with 25 mg kg ^-1 ZVI@CMC four times every other day. Mice were weighed, and the xenografts were measured and quantified as described above.

1.25 Single cell suspension of tissue and flow cytometry analysis

Tumor tissues were digested with 0.1 mg ml ^-1 collagenase (Sigma-Aldrich) and 1 mg ml ^-1 dispase II (Sigma-Aldrich) in serum-free DMEM for 30 min at 37℃, and then crushed through mesh for single cell suspension. To determine macrophage polarization, cells from BMDMs, tumor tissues or peripheral blood mononuclear cells were stained with anti-mouse CD11b, CD86, and CD206. To measure the proportion of Tregs, cells from splenocytes, tumor tissues or peripheral blood mononuclear cells were stained with anti-mouse CD25, Foxp3, and CD4. To determine the presentation of PD-1 and CTLA4 on CD8 ⁺ lymphocytes, cells from splenocytes, tumor tissues or peripheral blood mononuclear cells were stained with anti-mouse PD-1, CTLA4, and CD8. For humanized hPBMC-ASID mice, cells from tumor tissues or peripheral blood mononuclear cells were stained with anti-human CD45, CD3, CD4, CD8, and CD25. The antibodies conditions are described in Table 1. After staining process by following the manufacturer's instructions, samples were analyzed by flow cytometry (CytoFLEX ^TM) .

1.26 Organ distribution of iron

BALB/c nude mice (5-6-week-old) were intravenously injected with 25 mg kg ^-1 ZVI-NPs or PBS. After treatment, major organs (heart, lungs, spleen, liver, and kidney) , tumors and blood were collected at the indicated time. Each organ was homogenized and dissolved in nitrohydrochloric acid. The sample solutions were continuously shaken for 2 days to ensure iron dissociation. All samples were filtered and analyzed using an inductively coupled plasma mass spectrometry (Agilent technology, Santa Clara, CA, USA) provided by Chia Nan University of Pharmacy and Science, Taiwan.

1.27 Statistical analysis

Three independent experiments for cell studies and six mice per group for animal studies were analyzed unless indicated otherwise. Two-tailed Student’s t-test was used in cell and animal studies. Data represent mean ±s.e.m. The levels of statistical significance were expressed as p-values, *, p<0.05; **, p<0.01; ***, p<0.001; ns: non-significant.

Example 2: ZVI-NPs exhibit cancer-specific cytotoxicity

ZVI-NPs including silver coated (ZVI@Ag) and carboxymethylcellulose coated (ZVI@CMC) NPs were developed. ZVI@Ag NPs were prepared and synthesized as described in previous studies, ^3, 21 ZVI@CMC NPs were produced by reducing ferrous ions under anaerobic environment followed by polymer coating with 0.2%carboxymethyl cellulose. ⁴ Reference was made to FIGS. 1A to 1I, which were morphological and biological characterization of ZVI-NPs in cancer-specific cytotoxicity according to several embodiments of the present invention. As shown in the TEM images of FIG. 1A, both types of NPs exhibited core/shell structure and the mean physical diameters of ZVI@Ag NPs and ZVI@CMC NPs were 84.9 ± 17.4 nm and 60.6 ± 5.3 nm, respectively. On the other hand, the dynamic light scattering analysis revealed that both the freshly prepared ZVI@Ag and ZVI@CMC NPs were monodispersed and the mean hydrodynamic sizes were 157.6 ± 14.7 nm and 119.5 ± 8.6 nm, respectively.

The anti-tumor efficacy of ZVI-NPs had been further examined in lung cancer models and their influence on tumor microenvironment. Firstly, the uptake of ZVI@CMC in human lung cancer cells (A549) versus normal human lung cells (MRC-5) were evaluated by measuring the intracellular iron ion levels compared to that of the untreated control group (FIG. 1B) . Although the concentrations of ZVI@CMC NPs were detected in both lung cancer and normal lung cells at the early time points (1 hour and 2 hours) (left panel, FIG. 1B) , the intracellular iron ions increased rapidly in A549 cells, but not in MRC-5 cells (right panel, FIG. 1B) . Similar results were observed for ZVI@Ag NPs where the conversion of ZVI to intracellular iron ions has been reported to be processed rapidly via acidification of endosome-lysosome system in oral squamous cell carcinoma lines than in normal cells, ³ suggesting a cancer cell specific enhanced conversion of these ZVI-NPs.

The ultrastructure of ZVI-NPs inside cancer cells was next examined. The TEM images illustrated the presence of ZVI@Ag NPs in lysosomes (arrow, FIG. 1C) and the release of iron particles into cytoplasm in NPs-treated A549 cancer cells. Notably, these cells exhibited ruptured mitochondrial outer membrane and reduced mitochondrial crista with normal nuclear size and lack of chromatin condensation (lower panel, FIG. 1C) . These organelle alterations resembled the morphological characteristics of the novel iron-dependent programmed cell death, namely ferroptosis, as described previously. ^6, 7, 22

To evaluate the cytotoxic effects induced by ZVI@Ag or ZVI@CMC NPs, human non-small cell lung cancer cell lines (H460, A549, and H1299) , murine Lewis lung carcinoma cell line (LLC) , and normal human lung cell lines (MRC-5 and IMR-90) were treated with various doses of ZVI@Ag or ZVI@CMC NPs for 48 hours and then subjected to MTT cell viability assay (FIGS. 1D to 1E) . Both ZVI@Ag and ZVI@CMC NPs treatments significantly inhibited the viability of lung cancer cells without showing apparent cytotoxicity toward normal lung cell lines. Given the observation of cancer-specific cytotoxicity, the viability of additional normal cells including ex vivo isolated bone-marrow-derived macrophages (BMDMs) and splenic lymphocytes from C57BL/6 mice were further examined after ex vivo treatment of ZVI@Ag or ZVI@CMC NPs (FIGS. 1F to 1G) . Neither treatment of the ZVI-NPs affected the viability of ex vivo cultured BMDMs or lymphocytes, supporting that ZVI-NPs exerted cancer-specific cytotoxicity.

Certain amount of iron is essential for organismal functions. However, excessive amounts of iron are associated with ROS generation and cytotoxicity responses. ^23, 24 To dissect the biodistribution of iron from the NPs in vivo, the concentrations of iron in organs of nude mice bearing subcutaneous A549 xenografts treated with a single intravenous (i.v. ) dose of ZVI-NPs (25 mg kg ^-1) were measured (FIGS. 1H to 1I) . The results indicated that both ZVI@Ag and ZVI@CMC NPs retained in the tumors for 120 hours after single dose of i.v. injection. By contrast, previous studies showed much higher concentration of iron accumulated in the liver, spleen, kidneys, and other tissues after intravenous administration of iron oxide superparamagnetic NPs. ^25-27 Since concentrated accumulation of NPs focused on targeting cancer cells is of great importance, further augmentation of ZVI-NPs to accumulate inside tumor tissues could be engineered by ameliorating the physical, chemical properties and assembly of active targeting moieties on the particles. Collectively, these results revealed that ZVI-NPs treatment exerted cancer-specific cytotoxicity in lung cancer models in vitro and ZVI-NPs were preferentially retained in the tumor lesions in vivo.

EXAMPLE 3. ZVI-NPs suppress cancer stemness and angiogenesis

In addition to the observed cancer-specific cytotoxicity induced by ZVI-NPs, their inhibitory effects on cancer stemness and angiogenesis in H460 and H1299 cells were further evaluated. Reference was made to FIGS. 2A to 2I, which were ZVI-NPs showing anti-cancer stemness and anti-angiogenesis effects in vitro according to several embodiments of the present invention. As shown in FIG. 2A, both ZVI@Ag NPs and ZVI@CMC NPs treatments decreased the size of tumor sphere as compared to the control. The number of spheres was significantly reduced by ZVI@Ag NPs treatment (FIGS. 2B to 2C) . Importantly, RT-qPCR analysis showed that ZVI@Ag NPs treatment significantly decreased the expression levels of cancer stemness genes, including OCT4, Nanog, and SOX2 (FIG. 2D) . Together, these findings showed that ZVI-NPs treatment exerted anti-cancer stemness effects on lung cancer cells.

Further, it was found that conditioned medium (CM) derived from ZVI@Ag NPs-treated cancer cells for 8 hours significantly decreased the migration ability of human umbilical vein endothelial cells (HUVECs) (FIG. 2E to 2F) and the number of tube formation (FIG. 2G to 2H) . In addition, the expressions of pro-angiogenesis genes such as Sonic hedgehog, TGF-β and VEGF were downregulated after ZVI@Ag NPs treatment (FIG. 2I) . Collectively, these results indicated that ZVI-NPs treatment inhibited both cancer stemness and angiogenesis in vitro, which may contribute to their integrated anti-cancer efficacy.

EXAMPLE 4. ZVI-NPs cause lipid peroxidation and ferroptosis in cancer

Given the observation of morphological evidence of ferroptosis shown in FIG. 1C, how the function of mitochondria affected by ZVI-NPs treatment was further determined. Reference was made to FIGS. 3A to 3H, which were ZVI-NPs causing mitochondria dysfunction, oxidative stress, and lipid peroxidation in vitro according to several embodiments of the present invention. As shown in FIG. 3A, ZVI@Ag NPs treatment significantly decreased the fluorescence intensity of DiOC6, indicating the loss of mitochondrial membrane potential in treated cancer cells H1299, H460 and A549. Subsequently, seahorse assay and ATP production analysis demonstrated reduced oxygen consumption rate (OCR) (FIG. 3B) and ATP level after ZVI@Ag NPs treatment (FIG. 3C) . Furthermore, the MitoSOX fluorescence intensity was significantly augmented in ZVI@Ag NPs-treated cells, indicating the accumulation of mitochondrial reactive oxygen species (mtROS) and increased oxidative stress in mitochondria (FIG. 3D) . Together, these findings confirmed that ZVI@Ag NPs could induce mitochondria dysfunction and overproduction of mtROS in lung cancer cells.

Since damaged mitochondria could release high levels of ROS into the cytoplasm, and intracellular ZVI ions may also generate ROS through the Fenton reaction, the intracellular ROS level in cancer cells after treatment was next determined. As shown in FIG. 3E, ZVI@Ag NPs treatment drastically increased the intensity of DCF fluorescence, an index of intracellular ROS. Similar result was found after treatment with ZVI@CMC NPs (data not shown) . In addition, intracellular NADPH levels, indicating the ROS detoxification power, had also declined in cancer cells after ZVI@Ag treatment (FIG. 3F) , supporting the notion that ZVI-NPs treatment diminished antioxidant defense systems and induced intracellular ROS and mtROS levels to augment oxidative stress.

To confirm whether iron overload and oxidative stress triggered peroxidation of membrane lipid, flow cytometry analysis of lipid peroxidation status was conducted (FIG. 3G) . The level of lipid peroxidation was significantly increased after ZVI-NPs treatment, and this increment was inhibited by the addition of canonical ferroptosis inhibitor, Ferrostatin-1, in all lung cancer cells examined. Together, these findings demonstrated that ZVI-NPs treatment caused excessive production of ROS and led to ferroptotic lipid peroxidation in cancer cells.

To further verify whether ZVI-NPs treatment-induced cancer-specific cell death was attributed to high levels of ROS and ferroptotic lipid peroxidation, ZVI-NPs-treated cancer cells were incubated with antioxidant vitamins (vitamin C or vitamin E) , ferroptosis inhibitor (Ferrostatin-1) or lipid peroxidation inhibitor (Liproxstatin-1) and then subjected to lipid peroxidation or cell viability assay. Notably, ZVI-NPs-induced intracellular ROS level was significantly suppressed by the addition of antioxidant vitamin E (FIG. 3E) . Moreover, ZVI-NPs-induced cell death was attenuated by vitamin C, vitamin E, Ferrostatin-1 or Liproxstatin-1 treatments (FIG. 3H) , indicating that the cancer-specific cytotoxicity induced by ZVI-NPs depends on excessive oxidative stress and could be mainly attributed to ferroptosis. Collectively, a ferroptotic cancer cell death model was identified by which ZVI-NPs induced excessive ROS and resulted in peroxidation of membrane lipid, thereby causing ferroptosis of lung cancer cells.

EXAMPLE 5. ZVI-NPs suppress NRF2-mediated cytoprotection program

the molecular mechanism underlying the anti-cancer efficacy through ferroptosis induction by ZVI-NPs treatment was further dissected. NRF2, an essential transcription factor, plays an important role in the maintenance of the cellular redox status via regulating detoxification, antioxidant, and NADPH regeneration enzymes. ²⁸ Reference was made to FIGS. 4A to 4E, which were ZVI-NPs inhibiting NRF2-regulated antioxidant activity via enhancement of GSK3β/β-TrCP degradation pathway according to several embodiments of the present invention. Interestingly, protein expression levels of NRF2 and glutathione peroxidase 4 (GPX4) , a major scavenger of phospholipid peroxides, were both reduced in lung cancer cells after ZVI-NPs treatment (FIG. 4A) . Furthermore, the binding activities of NRF2 to the targeting promoter region of SLC7A11, AKR1C1 and apoptosis-inducing factor 2 (AIFM2) genes were significantly attenuated upon ZVI@Ag treatment in H460 and A549 cells as suggested by chromatin immunoprecipitation (ChIP) -qPCR assay (FIG. 4B) . It is worth to mention that AIFM2 has recently been identified as a potent ferroptosis-resistance factor. ^29, 30 These results provide first ChIP binding evidence that NRF2 bound at transcriptional target gene AIFM2 and ZVI-NPs treatment reduced the NRF2-mediated expression of AIFM2.

Furthermore, the expression levels of NRF2 targeting antioxidant gene SLC7A11 and ROS detoxification genes AKR1B1, AKR1C1, AKR1C2 and AKR1C3 were decreased after ZVI-NPs treatment (FIG. 4C) . In addition, ZVI@Ag NPs attenuated the expression of genes coding for NADPH-production enzymes such as IDH1, ME1 and 6PGD and NADPH-dependent enzymes NDUFAF4 and AIFM2 (FIG. 4C) , consistent with the aforementioned observation of reduced intracellular NADPH levels (FIG. 4F) . Collectively, these findings revealed a disruption of NRF2-dependent transcription program by which ZVI-NPs decreased NRF2 protein level and suppressed its transcription activity. Subsequently, the expression of NRF2-regulated ROS detoxification genes was decreased, leading to accumulation of intracellular ROS and peroxidation of membrane lipid.

EXAMPLE 6. ZVI-NPs enhance GSK3β/β-TrCP-dependent degradation of NRF2

NRF2 protein level is regulated through degradation pathways. The major pathway is localized in the cytoplasm and governed by KEAP1 E3 ubiquitin ligase. ³¹ The second pathway is in the nucleus and is regulated by GSK3β/β-TrCP phosphorylation-dependent ubiquitination system. ³² The KEAP1-dependent degradation of NRF2 is deficient in A549 and H460 cells. ^33, 34 Thus, GSK3β/β-TrCP-dependent degradation system may be probably activated by ZVI-NPs treatment. As shown in FIG. 4D, ZVI-NPs treatment induced GSK3β phosphorylation on Tyr216, which is positively correlated with GSK3β activity. Further, immunofluorescence staining illustrated that β-TrCP translocated into the nucleus along with reduced NRF2 protein expression after ZVI-NPs treatment (FIG. 4E) . These findings indicated that both ZVI@Ag and ZVI@CMC NPs could enhance NRF2 degradation through the GSK3β/β-TrCP pathway.

To further dissect the upstream signaling that triggered phosphorylation and activation of GSK3β, the change in phosphorylation level of AKT, the major kinase that regulates phosphorylation and inactivation of GSK3β were examined. ³⁵ However, the level of AKT phosphorylation was not reduced by ZVI-NPs treatment (data not shown) . In addition to AKT, mammalian target of rapamycin (mTOR) , a central regulator of cell growth, has been reported to be involved in metabolic reprogramming through suppression of GSK3-mediated substrate phosphorylation. ³⁶ Interestingly, the immunoblotting results showed that ZVI-NPs treatment decreased mTOR phosphorylation on Ser2448 (FIG. 4D) , indicating an antagonistic relationship between mTOR and GSK3β in ZVI-NPs-treated A549 and H460 cells. This finding is quite important, as several studies have reported that mTOR negatively regulates the GSK3β-dependent pathways. ^37-39 Conversely, the phosphorylation level of AMP-activated protein kinase (AMPK) , a pivotal cellular energy sensor that negatively regulates the mTOR pathway, was increased after ZVI-NPs treatment (FIG. 4D) . Recently, phosphorylated AMPK was observed to be involved in the initiation of lysosome-dependent ferroptosis while phosphorylated mTOR was suppressed. ⁴⁰ Interestingly, it has been demonstrated that iron overload promotes AMPK phosphorylation. ⁴¹ Here, the first evidence showed that AMPK presents a mechanistic link between ZVI-NPs-induced iron overload and NRF2 degradation. Taken together, those results elucidated a novel phosphorylation-dependent NRF2 protein degradation mechanism that ZVI-NPs disrupt AMPK/mTOR pathway to activate p-GSK3/β-TrCP and in turn degrade NRF2, leading to cell death under oxidative stress and subsequent lipid peroxidation.

Example 7. ZVI-NPs inhibit NRF2 activity and lung metastases in vivo

To determine whether ZVI-NPs treatment could induce ferroptosis and suppress NRF2-mediated transcriptional regulation of antioxidant functions in vivo, subcutaneous xenograft animal models were established. Reference was made to FIGS. 5A to 5N, which were ZVI-NPs inhibiting NRF2-regulated antioxidant transcription program in vivo and suppressing lung metastases according to several embodiments of the present invention. As shown in FIGS. 5A to 5C, tumor volume, tumor image and tumor weight of H460 xenografts were significantly reduced after intraperitoneal (i.p. ) injection of ZVI@Ag NPs as compared to the PBS control. Similar results were obtained in A549 xenograft model treated through i.v. injection of ZVI@Ag NPs (FIGS. 5D to 5F) . Body weight, blood biochemistry analysis and H&E staining of tissue sections from major organs showed no apparent pathological effects of ZVI@Ag NPs treatment via either i.p. or i.v. route (data not shown) .

The IHC staining of xenograft tumor tissues showed that 4-HNE, a biomarker of lipid peroxidation, was dramatically increased after ZVI-NPs treatment (FIG. 5G) . Conversely, protein levels of NRF2 and GPX4 of xenograft tumor tissues were significantly reduced by ZVI-NPs exposure (FIG. 5G) . Interestingly, CD31 staining in ZVI@Ag NPs-treated group showed reduced endothelial cell infiltration (FIG. 5G) , consistent with the foregoing observation of reduced in vitro migration and angiogenesis of HUVECs affected by ZVI-NPs (FIG. 2E to 2I) . Furthermore, mRNA expression levels of NRF2 target genes including SCL7A11, GPX4, SLC40A1 and AKR1 family genes were downregulated in ZVI@Ag NPs-treated xenografts as compared to the control group (FIG. 5H) . Similar results were observed in ZVI@CMC NPs-treated xenografts model (data not shown) . These findings together indicated that ZVI-NPs treatment effectively reduced tumor growth and suppressed cytoprotective NRF2-regulated transcriptional regulatory functions in vivo.

To verify whether ZVI-NPs treatment-induced anti-tumor effects were attributed to downregulation of the NRF2 pathway and ferroptotic lipid peroxidation in vivo, overexpression of NRF2 or Liproxstatin-1 treatment were conducted in H460 subcutaneous xenograft model treated with or without ZVI@CMC NPs. As shown in FIGS. 5I to 5K, tumor volume, tumor size and tumor weight were significantly reduced after ZVI@CMC NPs treatment as compared to PBS control. Importantly, the addition of Liproxstatin-1 or overexpression of NRF2 significantly attenuated the anti-tumor growth effects of ZVI@CMC NPs, confirming that NRF2 degradation involved in ZVI@CMC NPs-induced ferroptotic cell death signaling in vivo.

Spontaneous lung metastasis model was further employed to investigate the anti-metastasis effect of ZVI-NPs in vivo. Large tumor nodules were observed in lung of the control mice (FIGS. 5L to 5N) . In contrast, ZVI@Ag NPs treatment reduced the metastatic tumor nodules, suggesting that ZVI@Ag NPs could suppress metastatic tumors in the lung. These in vivo results indicated that ZVI-NPs could downregulate the NRF2 pathway and induce ferroptosis in cancer cells, while effectively suppressing both tumor growth and distant metastasis without apparent adverse effects in vivo. Those findings highlight the role of NRF2 in regulating ZVI-NPs-induced ferroptosis. In fact, transient NRF2 activation can protect cell from external stress; however, persistent NRF2 activation in cancer cells (known as NRF2 addiction) confers therapeutic resistance and aggressive tumorigenicity. ^42-44 Hence, ZVI-NPs could be a promising anti-cancer strategy for NRF2-addicted cancers.

Example 8. ZVI-NPs modulate immune cell profile in mouse model in vivo

The observations of cancer-specific cytotoxicity while sparing the normal lung cells, BMDMs and lymphocytes upon ZVI-NPs treatment (FIGS. 1D to 1G) encouraged the hypothesis that ZVI-NPs may modulate immunity in vivo in addition to their endogenous anti-tumor efficacy. Reference was made to FIGS. 6A to 6Q, which were ZVI-NPs treatments inhibiting tumor growth and modulating immune cell profile in vivo according to several embodiments of the present invention. Therefore, a syngeneic mouse model was established by subcutaneous injection of LLC cells into immunocompetent C57BL/6 mice and the tumor growth and immune cell profile were observed with or without i.v. injection of ZVI@CMC (FIG. 6A) . As shown in FIGS. 6B to 6D, tumor growth measured by tumor volume, tumor image and tumor weight was significantly reduced after ZVI@CMC NPs treatment as compared to the PBS control. Additionally, body weight, blood biochemistry analysis and H&E stained tissue sections of major organs showed no obvious difference between the control and ZVI@CMC NPs-treated groups (data not shown) , indicating that ZVI@CMC inhibited tumor growth in immunocompetent mice without apparent adverse effects.

Recently, targeting iron homeostasis in immune cells has received substantial interest as it involves anti-cancer immunity. M2 macrophages contain lower intracellular iron and promote tumor growth, while M1 macrophages are “iron-retaining” with proinflammatory activity to limit tumor progression and even to kill tumor cells. ^45, 46 Iron metabolism also plays an important role in T cell activation and proliferation, and T cell activation can be boosted by iron–dextran NPs. ⁴⁷ Therefore, the endpoint LLC allografts from the mice were collected to analyze tumor-infiltrating macrophages and T cells by immunofluorescence microscopy (FIGS. 6E to 6H) and flow cytometry analysis (FIGS. 6I to 6M) . The immunofluorescence images showed that ZVI@CMC treatment increased the infiltration of anti-tumor M1 macrophages (CD86 ⁺) and cytotoxic T cells (CD8 ⁺) in the center of tumor lesions (region 2, FIGS. 6F and 6H) as compared to the control group which showed predominantly peri-tumor localization of M1 macrophages and CD8+ T cells (region 2, FIGS. 6E and 6G) . In addition, flow cytometry analysis demonstrated that ZVI@CMC treatment decreased the proportion of M2-like macrophages (FIG. 6I) but increased that of M1-like macrophages (FIG. 6J) among tumor associated macrophages. Also, among tumor-infiltrating CD8 ⁺ T cells, the proportion of PD-1 ⁺ cells and that of CTLA4 ⁺ cells were decreased by ZVI@CMC treatment (FIGS. 6K and 6L) . Concomitantly, the proportion of tumor-infiltrating regulatory T cells (Tregs, CD25 ⁺ FoxP3 ⁺) , a subset of CD4 ⁺ T cells that have pro-tumor influences, was reduced after ZVI@CMC treatment (FIG. 6M) . Moreover, similar results were observed in the circulating blood of the treated mice (data not shown) , indicating that ZVI@CMC-induced anti-tumor immunity was systemic.

To further investigate how ZVI@CMC modulated the human immune system, the advanced severe immunodeficiency (ASID) mice were implanted with human peripheral blood mononuclear cells (hPBMCs) . After hPBMC engraftment, hPBMC mice bearing subcutaneous H460 tumor xenografts were treated with i.v. injection of ZVI@CMC (25 mg kg ^-1) (FIG. 6N) . As shown in FIG. 6O, tumor volume was significantly reduced after ZVI@CMC treatment as compared to the PBS control. Body weight, H&E stained tissue sections of major organs, and blood biochemistry analysis of the mice showed no obvious difference between PBS control and ZVI@CMC NPs treatment groups (FIG. 6P) . Notably, flow cytometry analysis showed that ZVI@CMC treatment decreased the proportion of Tregs among tumor-infiltrating T cells (FIG. 6Q) . Collectively, these results suggested that ZVI-NPs treatment could modulate human immunity and provide anti-tumor efficacy in vivo.

Example 9. ZVI-NPs stimulate macrophage and lymphocyte immunity ex vivo

Reference was made to FIGS. 7A to 7J, which were ZVI-NPs modulating immune cell profile in vitro and ex vivo according to several embodiments of the present invention. To verify whether cancer cell-induced polarization of macrophages can be reprogrammed by ZVI-NPs, ex vivo isolated BMDMs co-cultured with LLC cells were treated with ZVI-NPs (FIGS. 7A and 7B) . The RT-qPCR results demonstrated that both ZVI@Ag and ZVI@CMC NPs were able to enhance the expression of M1 marker iNOS but reduced the level of M2 marker Arginase-1 (Arg1) in BMDMs under the cancer cell co-culture condition. Consistently, flow cytometry analysis indicated that the ratio of M1-like/M2-like macrophages among the co-cultured BMDMs increased after ZVI-NPs treatment (FIG. 7C) . To further determine the effects of ZVI-NPs treatment on macrophage polarization, THP-1 macrophages were treated with ZVI-NPs while stimulated with IFN-γ plus LPS for M1 polarization and IL-4 for M2 polarization (FIGS. 7D and 7E) . RT-qPCR results revealed that ZVI-NPs treatments promoted the M1 polarization induction derived overexpression of TNF-α, while attenuated the expression of the M2 polarization gene DC-SIGN.

Further, how the PD-L1 expression in A549 cancer cells co-cultured with THP-1 macrophages affected by ZVI-NPs treatment were examined (FIG. 7F) . RT-qPCR results showed that the level of PD-L1 in A549 cells was significantly increased after co-cultured with THP-1 macrophages, and the PD-L1 overexpression could be attenuated by ZVI-NPs treatment. Importantly, the IHC staining of allograft and xenograft tumor tissues showed that PD-L1 expression was dramatically downregulated in ZVI-NPs-treated groups (FIG. 7G) . Together, these findings suggested that ZVI-NPs treatment improved anti-cancer immunoresponses by modulating macrophage polarization toward M1 phenotype and inhibiting the expression of PD-L1 on cancer cells.

Next, the T cell signaling and expression of inhibitory immune checkpoint protein PD-1 in splenic lymphocytes were investigated after ZVI-NPs treatment. Firstly, TGF-β stimulation was used to induce Treg differentiation in ex vivo isolated splenic lymphocytes (FIG. 7H) . Flow cytometry analysis showed that ZVI@CMC NPs treatment decreased the proportion of Tregs (FIG. 7H) . Notably, the results of flow cytometry analysis demonstrated that both ZVI@Ag and ZVI@CMC NPs reduced the proportion of PD-1 ⁺ cells among CD8 ⁺ lymphocytes (FIG. 7I) . Furthermore, luciferase-expressing LLC cell line was used for measuring lymphocyte-mediated cytotoxicity against cancer cells (FIG. 7J) . The luminescence intensity was lower in lane 2 as compared to lane 1, indicating that the population of viable LLC cells was reduced after co-culture of LLC cells with splenic lymphocytes. In particular, the lymphocyte-mediated cytotoxicity was further promoted by both ZVI@Ag NPs and ZVI@CMC NPs (

lanes

4, 6, 8, 10, FIG. 7J) . Collectively, these results showed that iron regulation plays a significant role in anti-cancer responses of both macrophages and T cells.

Notably, cystine/glutamate antiporter xCT (encoded by SLC7A11) , mediating intracellular redox balance and preventing ferroptosis, is dispensable for T cell proliferation and antitumor immunity in vivo. ⁴⁸ This suggests that ZVI-NPs-induced downregulation of SLC7A11 may have an impact on tumor growth but not on T cells. Indeed, ZVI-NPs did not adversely affect the proliferation of lymphocytes and BMDMs in ex vivo culture system. These results revealed that ZVI-NPs treatment decreased the proportion of Treg cells and enhanced the cytolytic activity of CD8 ⁺ lymphocytes. Interestingly, immunotherapy and radiotherapy have recently been demonstrated to activate CD8 ⁺ T cells and to modulate tumor ferroptosis through IFNγ pathway. ^49, 50 The synergistic effect of ZVI-NPs and other cancer therapeutics such as radiotherapy or immunotherapies may worth further investigation.

In summary, those embodiments identify a dual mechanism of anti-cancer activities of ZVI-NPs that spares non-malignant cells. The first mechanism involves enhanced GSK3β/β-TrCP-dependent degradation of NRF2 through activation of the AMPK/mTOR signaling pathway, and thereby triggering ferroptosis selectively in lung cancer cells. The second mechanism is through activating anti-tumor immune responses. It involves both modulation of macrophage polarization toward anti-tumor M1 phenotype and boosting the cytolytic activity of CD8 ⁺ lymphocytes as well as decreasing the proportion of Treg cells. In addition, the cancer-specific cytotoxicity and in vivo anti-tumor effects highlight the promising potential of ZVI-NPs for anti-cancer treatment. Through understanding of the molecular mechanism, NRF2 or associated proteins may serve as biomarkers for lung cancer or NRF2-addicted cancer patients that may benefit from ZVI-NPs treatment. These results provide an insight into development of novel anti-cancer precision nanomedicine that synergistically targets both cancer cells and tumor microenvironment.

In the aforementioned embodiments, the potential roles of newly developed ZVI-NPs in tumor microenvironment were evaluated. The dual synergistic anti-cancer activities of ZVI-NPs were discovered through inducing cancer cell ferroptotic death and modulating cancer microenvironment favorable to antitumor immune responses (FIG. 8) . As shown in FIG. 8, ZVI-NPs triggered ferroptosis selectively in cancer cells by suppressing NRF2-mediated cytoprotection program, which was attributed to the ZVI-NPs-induced disruption of AMPK/mTOR signaling and activation of GSK3β/β-TrCP-dependent degradation system. Of note, ZVI-NPs treatment reprogrammed the polarization of tumor-associated macrophages toward anti-tumor M1 phenotype and increased cytotoxic function of CD8 ⁺ T cells as well as reduced regulatory T cell (Treg) proportion to augment anti-tumor immunity in ex vivo and in vivo models. In addition, ZVI-NPs treatment could downregulate PD-L1 expression on cancer cells, inhibit both cancer stemness genes (including OCT4, Nanog, and SOX2) and angiogenesis genes (such as Sonic hedgehog, TGF-β and VEGF) , as shown in FIG. 8. Integrated with dual targeting to both cancer cells and tumor microenvironment, ZVI nanotherapeutics have profoundly opened up the potential for new advanced cancer therapy with reduced side effects and augmented efficacy.

In summary, specific coated ZVI-containing nanoparticles, specific sequences of primers, specific antibodies, specific patient groups, specific analysis models or specific evaluating methods are exemplified for clarifying the method for modulating AMPK activity, mTOR activity and PD-L1 expression in a subject. However, as is understood by a person skilled in the art, other coated ZVI-containing nanoparticles, other sequences of primers, other antibodies, other patient groups, other analysis models or other evaluating methods can be also adopted in the method for modulating AMPK activity, mTOR activity and PD-L1 expression in a subject without departing the spirit and scope of the present invention rather than being limited as aforementioned. For example, the coated ZVI-containing nanoparticles can be combined with other known pharmaceutically available excipients and/or carriers, or the coated ZVI-containing nanoparticles can be modified without altering its characteristic, thereby beneficially modulating Nrf2 activity in cancer cells or tumor microenvironments.

According to the embodiments of the present invention, the method for modulating AMPK activity, mTOR activity and PD-L1 expression in a subject, which comprises administering an effective dose of a nano-modulator of coated ZVI-containing nanoparticles to the subject, thereby increasing a phosphorylation level of the AMPK, suppressing a phosphorylation level of the mTOR, downregulating an expression level of PD-L1, suppressing cancer stemness gene expression, modulating immune cell behavior, modulating angiogenesis-related genes, and treating an angiogenesis-related disease and/or disorder. The nano-modulator can be applied in the development of anti-cancer precision nanomedicine and immunotherapy.

Although the present invention has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained herein.

References:

1. Zou, Y.; Wang, X.; Khan, A.; Wang, P.; Liu, Y.; Alsaedi, A.; Hayat, T.; Wang, X., Environmental remediation and application of nanoscale zero-valent iron and its composites for the removal of heavy metal ions: a review. Environ. Sci. Technol. 2016, 50 (14) , 7290-304.

2. Nam, S.; Tratnyek, P.G., Reduction of azo dyes with zero-valent iron. Water Res. 2000, 34 (6) , 1837-1845.

3. Yang, L.X.; Wu, Y.N.; Wang, P.W.; Huang, K.J.; Su, W.C.; Shieh, D.B., Silver-coated zero-valent iron nanoparticles enhance cancer therapy in mice through lysosome-dependent dual programed cell death pathways: triggering simultaneous apoptosis and autophagy only in cancerous cells. J. Mater. Chem. B 2020, 8 (18) , 4122-4131.

4. Huang, K.J.; Wei, Y.H.; Chiu, Y.C.; Wu, S.R.; Shieh, D.B., Assessment of zero-valent iron-based nanotherapeutics for ferroptosis induction and resensitization strategy in cancer cells. Biomater. Sci. 2019, 7 (4) , 1311-1322.

5. Miyamoto, T.; Takahashi, S.; Ito, H.; Inagaki, H.; Noishiki, Y., Tissue biocompatibility of cellulose and its derivatives. J. Biomed. Mater. Res. 1989, 23 (1) , 125-33.

6. Dixon, S.J.; Lemberg, K.M.; Lamprecht, M.R.; Skouta, R.; Zaitsev, E.M.; Gleason, C.E.; Patel, D.N.; Bauer, A.J.; Cantley, A.M.; Yang, W.S.; Morrison, B., 3rd; Stockwell, B.R., Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012, 149 (5) , 1060-72.

7. Xie, Y.; Hou, W.; Song, X.; Yu, Y.; Huang, J.; Sun, X.; Kang, R.; Tang, D., Ferroptosis: process and function. Cell Death Differ. 2016, 23 (3) , 369-79.

8. Tang, D.; Kang, R.; Berghe, T.V.; Vandenabeele, P.; Kroemer, G., The molecular machinery of regulated cell death. Cell Res. 2019, 29 (5) , 347-364.

9. Dixon, S.J.; Stockwell, B.R., The hallmarks of ferroptosis. Annu. Rev. Cancer Biol. 2019, 3 (1) , 35-54.

10. Dixon, S.J.; Patel, D.N.; Welsch, M.; Skouta, R.; Lee, E.D.; Hayano, M.; Thomas, A.G.; Gleason, C.E.; Tatonetti, N.P.; Slusher, B.S.; Stockwell, B.R., Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife 2014, 3, e02523.

11. Brose, M.S.; Frenette, C.T.; Keefe, S.M.; Stein, S.M., Management of sorafenib-related adverse events: a clinician's perspective. Semin. Oncol. 2014, 41 Suppl 2, S1-S16.

12. Kudo, M.; Finn, R.S.; Qin, S.; Han, K.H.; Ikeda, K.; Piscaglia, F.; Baron, A.; Park, J.W.; Han, G.; Jassem, J.; Blanc, J.F.; Vogel, A.; Komov, D.; Evans, T.R. J.; Lopez, C.; Dutcus, C.; Guo, M.; Saito, K.; Kraljevic, S.; Tamai, T.; Ren, M.; Cheng, A.L., Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet 2018, 391 (10126) , 1163-1173.

13. Topalian, S.L.; Hodi, F.S.; Brahmer, J.R.; Gettinger, S.N.; Smith, D.C.; McDermott, D.F.; Powderly, J.D.; Carvajal, R.D.; Sosman, J.A.; Atkins, M.B.; Leming, P.D.; Spigel, D.R.; Antonia, S.J.; Horn, L.; Drake, C.G.; Pardoll, D.M.; Chen, L.; Sharfman, W.H.; Anders, R.A.; Taube, J.M.; McMiller, T.L.; Xu, H.; Korman, A.J.; Jure-Kunkel, M.; Agrawal, S.; McDonald, D.; Kollia, G.D.; Gupta, A.; Wigginton, J.M.; Sznol, M., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 2012, 366 (26) , 2443-54.

14. Ribas, A.; Wolchok, J.D., Cancer immunotherapy using checkpoint blockade. Science 2018, 359 (6382) , 1350-1355.

15. Topalian, S.L.; Drake, C.G.; Pardoll, D.M., Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 2015, 27 (4) , 450-61.

16. Song, M.; Liu, T.; Shi, C.; Zhang, X.; Chen, X., Bioconjugated manganese dioxide nanoparticles enhance chemotherapy response by priming tumor-associated macrophages toward M1-like phenotype and attenuating tumor hypoxia. ACS Nano 2016, 10 (1) , 633-647.

17. Wu, P.C.; Hsiao, H.T.; Lin, Y.C.; Shieh, D.B.; Liu, Y.C., The analgesia efficiency of ultrasmall magnetic iron oxide nanoparticles in mice chronic inflammatory pain model. Nanomedicine 2017, 13 (6) , 1975-1981.

18. Gu, Z.; Liu, T.; Tang, J.; Yang, Y.; Song, H.; Tuong, Z.K.; Fu, J.; Yu, C., Mechanism of iron oxide-induced macrophage activation: the impact of composition and the underlying signaling pathway. J. Am. Chem. Soc. 2019, 141 (15) , 6122-6126.

19. Zhou, Q.; Zhang, Y.; Du, J.; Li, Y.; Zhou, Y.; Fu, Q.; Zhang, J.; Wang, X.; Zhan, L., Different-Sized Gold Nanoparticle Activator/Antigen Increases Dendritic Cells Accumulation in Liver-Draining Lymph Nodes and CD8+ T Cell Responses. ACS Nano 2016, 10 (2) , 2678-92.

20. Kim, S.Y.; Kim, S.; Kim, J.E.; Lee, S.N.; Shin, I.W.; Shin, H.S.; Jin, S.M.; Noh, Y.W.; Kang, Y.J.; Kim, Y.S.; Kang, T.H.; Park, Y.M.; Lim, Y.T., Lyophilizable and Multifaceted Toll-like Receptor 7/8 Agonist-Loaded Nanoemulsion for the Reprogramming of Tumor Microenvironments and Enhanced Cancer Immunotherapy. ACS Nano 2019, 13 (11) , 12671-12686.

21. Carroll, K.J.; Hudgins, D.M.; Spurgeon, S.; Kemner, K.M.; Mishra, B.; Boyanov, M.I.; Brown, L.W.; Taheri, M.L.; Carpenter, E.E., One-pot aqueous synthesis of Fe and Ag core/shell nanoparticles. Chem. Mater. 2010, 22 (23) , 6291-6296.

22. Yu, H.; Guo, P.; Xie, X.; Wang, Y.; Chen, G., Ferroptosis, a new form of cell death, and its relationships with tumourous diseases. J. Cell. Mol. Med. 2017, 21 (4) , 648-657.

23. Dixon, S.J.; Stockwell, B.R., The role of iron and reactive oxygen species in cell death. Nat. Chem. Biol. 2014, 10 (1) , 9-17.

24. Okada, S., Iron-induced tissue damage and cancer: the role of reactive oxygen species-free radicals. Pathol. Int. 1996, 46 (5) , 311-32.

25. Pham, B.T.T.; Colvin, E.K.; Pham, N.T.H.; Kim, B.J.; Fuller, E.S.; Moon, E.A.; Barbey, R.; Yuen, S.; Rickman, B.H.; Bryce, N.S.; Bickley, S.; Tanudji, M.; Jones, S.K.; Howell, V.M.; Hawkett, B.S., Biodistribution and clearance of stable superparamagnetic maghemite iron oxide nanoparticles in mice following intraperitoneal administration. Int. J. Mol. Sci. 2018, 19 (1) , 205.

26. Hu, Y.; Wang, R.; Li, J.; Ding, L.; Wang, X.; Shi, X.; Shen, M., Facile synthesis of lactobionic acid-targeted iron oxide nanoparticles with ultrahigh relaxivity for targeted MR imaging of an orthotopic model of human hepatocellular carcinoma. Particle &Particle Systems Characterization 2017, 34 (1) , 1600113.

27. Gobbo, O.L.; Wetterling, F.; Vaes, P.; Teughels, S.; Markos, F.; Edge, D.; Shortt, C.M.; Crosbie-Staunton, K.; Radomski, M.W.; Volkov, Y.; Prina-Mello, A., Biodistribution and pharmacokinetic studies of SPION using particle electron paramagnetic resonance, MRI and ICP-MS. Nanomedicine (Lond) 2015, 10 (11) , 1751-60.

28. Hayes, J.D.; Dinkova-Kostova, A.T., The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem. Sci 2014, 39 (4) , 199-218.

29. Bersuker, K.; Hendricks, J.M.; Li, Z.; Magtanong, L.; Ford, B.; Tang, P.H.; Roberts, M.A.; Tong, B.; Maimone, T.J.; Zoncu, R.; Bassik, M.C.; Nomura, D.K.; Dixon, S.J.; Olzmann, J.A., The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 2019, 575 (7784) , 688-692.

30. Doll, S.; Freitas, F.P.; Shah, R.; Aldrovandi, M.; da Silva, M.C.; Ingold, I.; Goya Grocin, A.; Xavier da Silva, T.N.; Panzilius, E.; Scheel, C.H.; Mourao, A.; Buday, K.; Sato, M.; Wanninger, J.; Vignane, T.; Mohana, V.; Rehberg, M.; Flatley, A.; Schepers, A.; Kurz, A.; White, D.; Sauer, M.; Sattler, M.; Tate, E.W.; Schmitz, W.; Schulze, A.; O'Donnell, V.; Proneth, B.; Popowicz, G.M.; Pratt, D.A.; Angeli, J.P.F.; Conrad, M., FSP1 is a glutathione-independent ferroptosis suppressor. Nature 2019, 575 (7784) , 693-698.

31. Itoh, K.; Wakabayashi, N.; Katoh, Y.; Ishii, T.; Igarashi, K.; Engel, J.D.; Yamamoto, M., Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 1999, 13 (1) , 76-86.

32. Rada, P.; Rojo, A.I.; Chowdhry, S.; McMahon, M.; Hayes, J.D.; Cuadrado, A., SCF/ {beta} -TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner. Mol. Cell. Biol. 2011, 31 (6) , 1121-33.

33. Singh, A.; Misra, V.; Thimmulappa, R.K.; Lee, H.; Ames, S.; Hoque, M.O.; Herman, J.G.; Baylin, S.B.; Sidransky, D.; Gabrielson, E.; Brock, M.V.; Biswal, S., Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med. 2006, 3 (10) , e420.

34. Taguchi, K.; Shimada, M.; Fujii, S.; Sumi, D.; Pan, X.; Yamano, S.; Nishiyama, T.; Hiratsuka, A.; Yamamoto, M.; Cho, A.K.; Froines, J.R.; Kumagai, Y., Redox cycling of 9, 10-phenanthraquinone to cause oxidative stress is terminated through its monoglucuronide conjugation in human pulmonary epithelial A549 cells. Free Radic. Biol. Med. 2008, 44 (8) , 1645-55.

35. Cross, D.A.; Alessi, D.R.; Cohen, P.; Andjelkovich, M.; Hemmings, B.A., Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995, 378 (6559) , 785-9.

36. He, L.; Gomes, A.P.; Wang, X.; Yoon, S.O.; Lee, G.; Nagiec, M.J.; Cho, S.; Chavez, A.; Islam, T.; Yu, Y.; Asara, J.M.; Kim, B.Y.; Blenis, J., mTORC1 promotes metabolic reprogramming by the suppression of GSK3-dependent Foxk1 phosphorylation. Mol. Cell 2018, 70 (5) , 949-960 e4.

37. Zhang, S.; Qian, G.; Zhang, Q.Q.; Yao, Y.; Wang, D.; Chen, Z.G.; Wang, L.J.; Chen, M.; Sun, S.Y., mTORC2 suppresses GSK3-dependent Snail degradation to positively regulate cancer cell invasion and metastasis. Cancer Res. 2019, 79 (14) , 3725-3736.

38. Koo, J.; Yue, P.; Gal, A.A.; Khuri, F.R.; Sun, S.Y., Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth. Cancer Res. 2014, 74 (9) , 2555-68.

39. Zhang, H.H.; Lipovsky, A.I.; Dibble, C.C.; Sahin, M.; Manning, B.D., S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt. Mol. Cell 2006, 24 (2) , 185-97.

40. Han, D.; Jiang, L.; Gu, X.; Huang, S.; Pang, J.; Wu, Y.; Yin, J.; Wang, J., SIRT3 deficiency is resistant to autophagy-dependent ferroptosis by inhibiting the AMPK/mTOR pathway and promoting GPX4 levels. J. Cell. Physiol. 2020, 235 (11) , 8839-8851.

41. Zheng, Q.; Zhao, Y.; Guo, J.; Zhao, S.; Fei, C.; Xiao, C.; Wu, D.; Wu, L.; Li, X.; Chang, C., Iron overload promotes mitochondrial fragmentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway. Cell Death Dis. 2018, 9 (5) , 515.

42. Kitamura, H.; Motohashi, H., NRF2 addiction in cancer cells. Cancer Sci. 2018, 109 (4) , 900-911.

43. Hammad, A.; Namani, A.; Elshaer, M.; Wang, X.J.; Tang, X., "NRF2 addiction" in lung cancer cells and its impact on cancer therapy. Cancer Lett. 2019, 467, 40-49.

44. Takahashi, N.; Cho, P.; Selfors, L.M.; Kuiken, H.J.; Kaul, R.; Fujiwara, T.; Harris, I.S.; Zhang, T.; Gygi, S.P.; Brugge, J.S., 3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis. Mol. Cell 2020.

45. Recalcati, S.; Locati, M.; Marini, A.; Santambrogio, P.; Zaninotto, F.; De Pizzol, M.; Zammataro, L.; Girelli, D.; Cairo, G., Differential regulation of iron homeostasis during human macrophage polarized activation. Eur. J. Immunol. 2010, 40 (3) , 824-35.

46. Costa da Silva, M.; Breckwoldt, M.O.; Vinchi, F.; Correia, M.P.; Stojanovic, A.; Thielmann, C.M.; Meister, M.; Muley, T.; Warth, A.; Platten, M.; Hentze, M.W.; Cerwenka, A.; Muckenthaler, M.U., Iron induces anti-tumor activity in tumor-associated macrophages. Front. Immunol. 2017, 8, 1479.

47. Perica, K.; Tu, A.; Richter, A.; Bieler, J.G.; Edidin, M.; Schneck, J.P., Magnetic field-induced T cell receptor clustering by nanoparticles enhances T cell activation and stimulates antitumor activity. ACS Nano 2014, 8 (3) , 2252-60.

48. Arensman, M.D.; Yang, X.S.; Leahy, D.M.; Toral-Barza, L.; Mileski, M.; Rosfjord, E.C.; Wang, F.; Deng, S.; Myers, J.S.; Abraham, R.T.; Eng, C.H., Cystine-glutamate antiporter xCT deficiency suppresses tumor growth while preserving antitumor immunity. Proc. Natl. Acad. Sci. U.S. A. 2019, 116 (19) , 9533-9542.

49. Lang, X.; Green, M.D.; Wang, W.; Yu, J.; Choi, J.E.; Jiang, L.; Liao, P.; Zhou, J.; Zhang, Q.; Dow, A.; Saripalli, A.L.; Kryczek, I.; Wei, S.; Szeliga, W.; Vatan, L.; Stone, E.M.; Georgiou, G.; Cieslik, M.; Wahl, D.R.; Morgan, M.A.; Chinnaiyan, A.M.; Lawrence, T.S.; Zou, W., Radiotherapy and immunotherapy promote tumoral lipid oxidation and ferroptosis via synergistic repression of SLC7A11. Cancer Discov. 2019, 9 (12) , 1673-1685.

50. Wang, W.; Green, M.; Choi, J.E.; Gijon, M.; Kennedy, P.D.; Johnson, J.K.; Liao, P.; Lang, X.; Kryczek, I.; Sell, A.; Xia, H.; Zhou, J.; Li, G.; Li, J.; Li, W.; Wei, S.; Vatan, L.; Zhang, H.; Szeliga, W.; Gu, W.; Liu, R.; Lawrence, T.S.; Lamb, C.; Tanno, Y.; Cieslik, M.; Stone, E.; Georgiou, G.; Chan, T.A.; Chinnaiyan, A.; Zou, W., CD8 (+) T cells regulate tumour ferroptosis during cancer immunotherapy. Nature 2019, 569 (7755) , 270-274.

Claims

A method for modulating tumor microenvironment toward anti-cancer phenotype in a subject in need thereof using a nano-modulator, comprising administering an effective dose of the nano-modulator to the subject, wherein the nano-modulator is consisted of coated zero-valent iron (ZVI) -containing nanoparticles.
The method of claim 1, wherein a metallic coating or a non-metallic coating is disposed on a surface of the coated ZVI-containing nanoparticles.
The method of claim 2, wherein the metallic coating includes silver, gold, iron or copper.
The method of claim 2, wherein the non-metallic coating includes carboxymethyl cellulose (CMC) or mesoporous silica.
The method of claim 1, wherein the subject comprises cancer cells and the tumor microenvironment.
The method of claim 5, wherein the subject undergoes a cancer treatment.
The method of claim 6, wherein the cancer treatment is for solid tumors.
The method of claim 6, wherein the administration is performed as a treatment alone or as an adjuvant before, during or after the cancer treatment.
The method of claim 1, wherein the effective dose of the nano-modulator is at least 1 μg/mL.
The method of claim 5, wherein the nano-modulator up-regulates expression levels of glycogen synthase kinase 3 beta /beta-transducin repeats-containing protein (GSK3β/β-TrCP) and phosphorylation of AMP-activated protein kinase (AMPK) in the subject.
The method of claim 10, wherein the expression levels include a transcriptional expression and/or a translational expression.
The method of claim 5, wherein the nano-modulator down-regulates expression levels of NRF2, mTOR, SLC7A11, GPX4, apoptosis-inducing factor 2 (AIFM2) , AKR1 family genes, PD-L1, cancer stemness genes, and angiogenesis-related genes in the subject.
The method of claim 12, wherein the cancer stemness genes include OCT4, Nanog and SOX2 genes.
The method of claim 12, wherein the angiogenesis-related genes include Sonic hedgehog, TGF-β and VEGF genes.
The method of claim 12, wherein the expression levels include a transcriptional expression and/or a translational expression.
A method for modulating an immune cell behavior in tumor microenvironment in a subject in need thereof using a nano-modulator, comprising administering an effective dose of the nano-modulator to the subject, wherein the nano-modulator is consisted of coated ZVI-containing nanoparticles.
The method of claim 16, wherein a metallic coating or a non-metallic coating is disposed on a surface of the coated ZVI-containing nanoparticles.
The method of claim 16, wherein the subject includes cancer cells and immune cells in the tumor microenvironment.
The method of claim 16, wherein the subject undergoes a cancer treatment.
The method of claim 19, wherein the cancer treatment is for lung cancer.
The method of claim 19, wherein the administration is performed as a treatment alone or as an adjuvant before, during or after the cancer treatment.
The method of claim 16, wherein the effective dose of the nano-modulator is at least 1 μg/mL.
The method of claim 16, wherein the immune cell behavior includes promotion of a lymphocyte-mediated cytotoxicity, enhancement of M1-phenotype macrophage population, decrease of M2-macrophage proportion, reduction of a proportion of PD-1 ⁺ cells among CD8 ⁺ lymphocytes, and reduction of a proportion of regulatory T (Treg) cells.
A method for treating an angiogenesis-related disease and/or disorder in a subject in need thereof using a nano-modulator, comprising administering an effective dose to the subject, wherein the nano-modulator has a ZVI core.
The method of claim 24, wherein the angiogenesis-related disease and/or disorder is or is caused by tumor, inflammation or autoimmune diseases, metabolic disorders, infection, cardiovascular disease, injury, vaccination or age-related degeneration.
The method of claim 25, wherein the tumor is selected from the group consisting of lung cancer (squamous) , lung cancer (adenocarcinoma) , small cell lung carcinoma, non-small cell lung carcinoma, skin cancer, head and neck cancer, nasopharyngeal carcinoma, thyroid cancer, breast carcinoma, gastric carcinoma, pancreatic cancer, liver carcinoma, renal cell carcinoma, colorectal carcinoma, urinary bladder carcinoma, cervical carcinoma (squamous) , ovarian carcinoma, prostate cancer, sarcomas, melanoma and hemangioma.
The method of claim 25, wherein the inflammatory or autoimmune diseases include rheumatoid arthritis, osteoarthritis, diabetic retinopathy, psoriasis, Sjogren's syndrome, acne rosacea, systemic lupus, Wegeners sarcoidosis, polyarteritis, scleroderma, Crohn's disease or Bartonellosis.
The method of claim 25, wherein the metabolic syndrome includes diabetes, high blood pressure (hypertension) and obesity.
The method of claim 25, wherein the infection includes a bacterial infection, a virus infection, a fungal infection or a protozoan infection.
The method of claim 25, wherein the cardiovascular disease includes atherosclerosis, myocardial angiogenesis, hyperviscosity syndromes, vein occlusion, artery occlusion, carotid obstructive disease or Osler-Weber-Rendu disease.
The method of claim 25, wherein the age-related degeneration includes age-related macular degeneration (AMD) or a chronic wound.
The method of claim 24, wherein the administration is performed as a treatment alone or as an adjuvant before, during or after a treatment of the angiogenesis-related disease and/or disorder.
The method of claim 24, wherein the ZVI core is coated with a metal coating or a non-metallic coating.
The method of claim 33, wherein the metallic coating includes silver, gold, iron or copper.
The method of claim 33, wherein the non-metallic coating includes carboxymethyl cellulose (CMC) , polystyrene malic acid or mesoporous silica.
The method of claim 24, wherein the effective dose of the nano-modulator is at least 1 μg/mL.