Article introduction
In October 2023, the research team of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine published in the journalredox biology（if:11.3997）An article entitled "Cancer-associated fibroblasts impair: the cytotoxic function of NK cells in gastric cancer by inducing ferroptosis via iron regulation" was published.
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Background: background
As the most important immunosuppressive component of the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) inhibit the activity of natural killer cells (NK cells), thereby promoting tumor progression and immune escape. The study found that in human gastric cancer, NK cell levels were inversely proportional to the number of CAFs. Functionally targeting CAFs with a combination of deferoxamine and FSTL1-neutralizing antibodies in a human-derived organoid model significantly alleviates CAF-induced iron phagocytosis in NK cells and enhances NK cell cytotoxicity to GC. This study demonstrates a novel mechanism by which CAF in TME inhibits NK cell activity and provides a potential method to enhance NK cell-mediated anti-GC immune responses.
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Research Methods: methods
It mainly includes the establishment of a co-culture model of gastric cancer spheroids (PDOS) and cancer-associated fibroblasts (CAFs) and natural killer cells (NK cells), the evaluation of the potency of CAR-NK92 cells by flow cytometry and fluorescence microscopy, and the extraction of total RNA using Trizol reagent and quantitative real-time PCR (QRT-PCR) and Western Techniques such as blotting and enzyme-linked immunosorbent assay (ELISA) were used to analyze the expression levels of related proteins and cytokines. In addition, the researchers used ImageJ software to analyze fluorescence microscopy images and statistically analyzed the data using methods such as Student's T-test and Pearson correlation analysis.
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Key results. results
CAF is inversely proportional to NK cell levels in human GC and inhibits NK cell viability and cytotoxicity in vitro
By analyzing the TCGA database and the immunofluorescence staining results of 21 pairs of GC tissue samples, the researchers found that the abundance of CAFS was negatively correlated with the NK cell level. In addition, the researchers also used techniques such as Delifa Eutda cytotoxicity analysis and CCK8 cell viability analysis to demonstrate that CAFS can significantly inhibit the cytotoxicity and viability of NK cells against GC cells.
Figure 1 In human gastric cancer, the level of CAFS is inversely proportional to the level of NK cells and inhibits NK cell viability and cytotoxicity in vitro.
A, Correlation of immune cells in GC tissues with CAFS according to the TCGA database. B-C, representative immunofluorescence staining of -SMA and CD56 in GC tumor tissues and correlation of -SMA and CD56 in 21 GC tumor tissues. Arrows indicate the expression of CD56. D, Determination of the chemotaxis of NFS and CAFS-induced PB-NK cells (labeled with Calcein-AM) by fluorescence intensity. E. Flow cytometry was used to assess cell death of NK92 cells cultured alone (blank) or co-cultured with CAFS. F-G, which was used to detect the proliferation of NK92 PB-NK cells co-cultured with CAFS by CCK8. H-I, the levels of TNF-A and IFN-Y proteins secreted by NK92 PB-NK cells co-cultured with CAFS were determined by enzyme-linked immunosorbent assay. J-M, the effect of CAFS on NK92 and PB-NK cytotoxicity in SNU16 and MKN45 GC cells was determined by the Delfia Eutda cytotoxicity assay. The data represent the results of at least three independent experiments. Data analysis was performed using Student's T-test and Pearson correlation analysis (mean SD; *p < 0.05，**p < 0.01）。
CAFS induces ferroptosis in NK cells by promoting intracellular iron overload
Using techniques such as CCK8 cell viability analysis and fluorescence microscopy, the researchers demonstrated that CAFS can significantly inhibit NK cell viability and cell proliferation, and induce NK cell death through iron overload. In addition, the researchers also used techniques such as quantitative real-time PCR (QRT-PCR) and Western blotting to demonstrate that CAFS can promote iron overload in NK cells by regulating the expression of iron metabolism-related genes and proteins.
Figure 2 CAFS induces ferroptosis by promoting intracellular iron overload in NK.
A. The viability of NK92 cells co-cultured with CAFS was detected with CCK8 in the presence of specific inhibitors Z-VAD (CASPASE-3 inhibitor), NEC1 (necrosis inhibitor), or FER 1 (ferrosis, inhibitor). B-C, the effect of CAFS on NK92 and PB-NK cytotoxicity in MKN45 and SNU16 GC cells was examined by Delfia Eutda cytotoxicity assay (FER1 5 M, LiP1 50 nm). D-E, lipid ROS levels in NK92 cells co-cultured with CAFS were detected using the C11 BodyPi 581 591 probe. With 0NK92 cells treated with 5 M RSL3 for 48 h were used as a positive control (scale bar = 20)F, and MDA levels in NK92 cells co-cultured with CAFS were detected using the MDA detection kit. With 0NK92 cells were treated with 5 M RSL3 for 48 h as a positive control. G, Mitochondria of PB-NK cells were analyzed by transmission electron microscopy (scale bar = 1 M 500 nm). H-I, ferrous content in untreated cells by Fe2+ detection probe (scale bar = 20)J in untreated cells or cells co-cultured with CAFS (+CAFS), and ferrous content in NK92 cells in untreated cells by Fe2+ detection probe after co-culture with CAFS in the absence or presence of DFO (10 M, 48 H). K, lipid ROS levels in NK92 cells co-cultured with CAFS or CAFS plus DFO (10 M) alone were detected with the C11 BodyPI 581 591 probe. l, MDA levels in NK92 cells co-cultured with CAFS or CAFS plus DFO (10 M) were detected by MDA assay. m, 4-hne levels in NK92 cells co-cultured with CAFS or CAFS plus DFO (10 M) were detected by ELISA. The data represent the results of at least three independent experiments. Data analysis was performed using the Student's t-test (mean SD; *p < 0.05，**p < 0.01）。
CAFS increases NK cell iron instability levels by exporting iron to TME
Using techniques such as quantitative real-time PCR (QRT-PCR) and Western blotting, the researchers demonstrated that CAFS had higher levels of ferroportin1 and hephaestin and ferritin expression relative to normal fibroblasts (NFS), and higher total iron content was observed in CAFS. In addition, the researchers also used techniques such as Calcein-AM staining and fluorescence microscopy to demonstrate that CAFS can export iron into TME and increase iron instability levels in NK cells by regulating the expression of genes and proteins related to iron metabolism.
Figure 3 CAFS increases iron instability levels in NK cells by exporting iron to TME.
A-B, mRNA levels of - protein (Heph) and ferritin 1 (FPN1) in three pairs of normal fibroblasts (NFS) and CAFS were analyzed by QRT-PCR. C. Protein expression of FTH, FTL, FPN1 and HEPH in NFS and CAFS was analyzed by Western blotting. D-E, iron ions in NFS and CAFS were detected by Calcein-AM staining (scale bar = 100). F-G, after treatment with the indicated dose of DFP (0-100 M, 48 h), iron ions in CAFS were detected by Calcein-AM staining (scale bar = 100) H, and changes in iron ions in CAFS after removal of DFP were detected by Calcein-AM. i. Iron ion levels in CAFS-only or CAFS plus DFP co-cultured with NK92 were detected with Calcein-AM. J, Viability of NK92 cells co-cultured with CAFS or CAFS plus DFP alone is determined by CCK8 assay (FER 1:5 M). K, the ferrous content in NK92 cells co-cultured with CAFS or CAFS plus DFP (100 M) only was detected by Calcein-AM. Determined with a fluorescence microplate reader. L, lipid ROS levels in NK92 cells co-cultured with CAFS or CAFS plus DFP (100 M) alone were detected with the C11 BodyPI 581 591 probe. MDA levels in NK92 cells co-cultured with CAFS or CAFS plus DFP (100 M) alone were detected by MDA assay. n, 4-hne levels in NK92 cells co-cultured with CAFS or CAFS plus DFP (100 M) were detected by ELISA. The data represent the results of at least three independent experiments. Data analysis was performed using the Student's t-test (mean SD; *p < 0.05，**p < 0.01）。
NCOA4-dependent ferrophage mediates CAF-induced intracellular iron overload in NK cells
Using techniques such as Western blotting, the researchers demonstrated that CAFS can significantly increase the expression levels of iron metabolism-related proteins in NK cells, such as ferritin receptor, ferritin 1, heavy and light chains, and promote the degradation of ferritin in NK cells by regulating the expression of NCOa4. In addition, the researchers also used techniques such as fluorescence microscopy to demonstrate that CAFS can induce iron overload in NK cells through NCOA4-mediated ferroptin action.
Figure 4 Ferritin phagocytosis via NCOA4 is involved in CAF-induced intracellular iron overload in NK.
A. Protein expression of TFR, FPN1, FTL, and FTH in PB-NK and NK92 cells cultured with FAS (100 M) was analyzed by Western blotting. Western blot analysis of TFR, FPN1, FTL, and FTH of B-C, PB-NK, and NK92 cells co-cultured with CAFS, CAFS with DFO (10 M), or DFP (100 M) pretreated with CAFS. D, mRNA levels of genes associated with ferritin regulation in NK92 cells co-cultured with CAFS were analyzed by QRT-PCR. E, Western blotting of NCOA4 and FTH in NK92 cells co-cultured with CAFS. F. Western blot analysis of NCOA4 and FTH in NK92SINC or NK92SINCOA4 cells co-cultured with CAFS. G, the viability of NK92SINC or NK92SINCOA4 co-cultured with CAFS was determined by CCK8 assay (FER1 5 M). H, Calcein-Am staining was used to detect ferrous content in NK92SINC or NK92SINCOA4 cultured with or without CAFS. i. Lipid ROS levels were detected with the C11 BodyPi 581 591 probe. j, MDA levels were detected by MDA assay. The data represent the results of at least three independent experiments. Data analysis was performed using the Student's t-test (mean SD; *p < 0.05，**p < 0.01）。
CAF-derived FSTL1 regulates the expression of NCoA4 in NK cells via the DIP2A-P38 pathway
Using techniques such as quantitative real-time PCR (QRT-PCR) and Western blotting, the researchers demonstrated that FSTL1 is highly expressed in CAFS and can significantly increase the expression level of NCoA4 in NK cells. In addition, the researchers also used techniques such as shRNA and rhFSTL1 to demonstrate that FSTL1 can regulate the expression of NCOA4 in NK cells through the DIP2A-P38 pathway. Specifically, the overexpression of FSTL1 can activate the p38 pathway in NK cells, thereby upregulating the expression of NCoA4. Silencing of FSTL1 can inhibit the p38 pathway in NK cells, thereby downregulating the expression of NCOA4.
Figure 5 CAF-derived FSTL1 upregulates the expression of NCOa4 in NK cells via the DIP2A-P38 pathway.
A. Correlation of NCOA4 and FSTL1 expression in GC in the TCGA database. B. Analysis of FSTL1 content in NK cells (PB-NK, NK92), GC cell lines (MKN-45, N87, SNU5, SNU16, and AGS) and paired GC patient NF and CAF cell supernatants by ELISA. c. mRNA levels of NCOA4 in NK92 cells treated with RhFSTL1 (20 ng ML) for 48 h by QRT-PCR. D. Western blotting analysis of rhfstl1 (20 ng ml; 48 H) protein expression of NCOA4 and FTH in NK92 treated cells. With or without RhFSTL1 (20 ng ML; 48 h) in treated NK92 cells. F. Protein expression of NCOA4 and FTH in NK92 cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC for 48 h by Western blotting. G, Calcein-Am staining was used to detect the ferrous content in NK92 cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC for 48 h. H, Lipid ROS levels in NK92 cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC were detected using the C11 BodyPI 581 591 probe. i. MDA levels in NK92 cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC were detected by MDA assay. J, Enzyme-linked immunosorbent assay (ELISA) was used to detect 4-hne levels in NK92 cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC. K. Western blotting analysis by rhFSTL1 (20 ng ml; 48 h) protein expression of NCOa4 in NK92SIDIP2A or control cells. l, protein expression of p-p38 and p38 in NK92 cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC by western blotting. m, analyzed by western blotting after rhfstl1 (20 ng ml; 48 H) protein expression of p-p38 and p38 in nk92siDIP2A or control cells. n, protein expression of p-p38, p38, and ncoa4 in nk92 cells treated with rhfstl1 (20 ng ml) or p38 inhibitor (p38-mapk-in-1 20 m) was analyzed by western blotting. o. Protein expression of DIP2A, P-P38, P38, NCOa4, and FTH in PB-NK cells co-cultured with CAFS-SHFSTL1 or CAFS-SHNC by Western blotting. The data represent the results of at least three independent experiments. Data analysis was performed using Student's T-test and Pearson correlation analysis (mean SD; *p < 0.05，**p < 0.01）。
In addition, the combination of DFO and FSTL1-neutralizing antibodies attenuates CAF-induced ferroptosis in NK cells and enhances the cytotoxicity of CAR-NK to GC.
Figure 6 The combination of DFO and FSTL1-neutralizing antibody attenuates CAF-induced ferroptosis in NK cells and enhances the cytotoxicity of NK cells to GC.
A, Ferrous content in NK92 cells co-cultured with or without FSTL1-neutralizing antibody (FNab, 1 g mL) and or DFO (10 M) was detected by Calcein-Am staining. B, Lipid ROS levels with or without FNAB (1 g mL) and DFO (10 M) in NK92 cells co-cultured with CAFS were detected with the C11 BodyPi 581 591 probe. C, MDA levels of NK92 cells co-cultured with CAFS with or without FSTL1-neutralizing antibody (FNAB, 1 g mL) and DFO (10 M) were detected by MDA assay. D, 4-Hne content of NK92 cells co-cultured with or without FSTL1-neutralizing antibody (FNAB, 1 g mL) and DFO (10 M) was measured by ELISA. E, Flow cytometry analysis of cell death in NK92 cells co-cultured with CAFS with or without FNAB (1 g mL) and DFO (10 M). F-G, the cytotoxicity of NK92 cells and PB-NK on MKN45 and SNU16 GC cells after co-culture with or without CFab (1 g mL) and DFO (10 M) was detected with the Delfia Eutda cytotoxicity assay. H, Protein expression of MSLN in GC organoids (PDO1T and PDO3T) and GC cells (MKN45 and SNU16) was analyzed by Western blotting. i, Flow cytometry was performed to analyze cell death in co-cultures of PDO1T with NK92 or CAR-NK cells (PDO1T: NK92 or CAR-NK = 1:5). J-K, cell death co-cultured with PDO1T and CAFS in the presence of DFO FNab and/or CAR-NK cells (PDO1T: CAFS: NK = 1:2:5, DFO: 10 M, FNab: 1 G ML) was analyzed by flow cytometry. L-M was used to analyze cell death co-cultured with PDO3T co-cultured with CAFS in the presence of DFO1T: CAFS: NK = 1:2:5, DFO: 10 M, FNaB: 1 G ML (scale bar = 20) in the presence of DFO1T: CAFS: NK = 1:2:5, DFO: 10 M, FNaFs: 1 G ML. Data are presented as the mean SD of the three independent experiments. Data analysis was performed using the Student's t-test (*p < 0.05；**p < 0.01）。(For an explanation of the color references in this legend, please refer to the online version of this article).
Figure 7 Schematic diagram of the possible mechanism by which CAFS induces ferroptosis in NK cells in GC.
CAFS increases ferritosis within NK cells, induces ferroptosis by deriving iron to the TME, and also induces FSTL1-NCOA4-mediated GC ferritin autophagy. Therefore, combining FSTL1 neutralizing antibodies and DFOs by overcoming immunosuppression of tumor stroma may be a promising strategy for GC.
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Summary. suggestion
This study provides valuable insights into the interactions between CAFS and NK cells in GC. CAFS increases iron instability within NK cells, induces iron eosinophilia by exporting iron to TME and promoting FSTL1-NCOA4-mediated ferrophage. The findings highlight the potential of targeting these pathways as a strategy to enhance the immune response of NK cells to GC. However, further research is needed to understand the intricate interactions between CAFS and immune cells in TME, and to have clinical implications for GC of these findings.
References. cancer-associated fibroblasts impair the cytotoxic function of nk cells in gastric cancer by inducing ferroptosis via iron regulation.redox biol. 2023 nov:67:102923. doi: 10.1016/j.redox.2023.102923.pmid: 37832398 pmcid: pmc10582581.
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