Cancer-associated fibroblasts impair the cytotoxic function of NK cells in gastric cancer by inducing ferroptosis via iron regulation

Abstract

As the predominant immunosuppressive component within the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) inhibit Natural Killer cell (NK cell) activity to promote tumor progression and immune escape; however, the mechanisms of cross-talk between CAFs and NK cells in gastric cancer (GC) remain poorly understood. In this study, we demonstrate that NK cell levels are inversely correlated with CAFs abundance in human GC. CAFs impair the anti-tumor capacity of NK cells by inducing ferroptosis, a cell death process characterized by the accumulation of iron-dependent lipid peroxides. CAFs induce ferroptosis in NK cells by promoting iron overload; conversely, decreased intracellular iron levels protect NK cells against CAF-induced ferroptosis. Mechanistically, CAFs increase the labile iron pool within NK cells via iron export into the TME, which is mediated by the upregulated expression of iron regulatory genes ferroportin1 and hephaestin in CAFs. Moreover, CAF-derived follistatin like protein 1(FSTL1) upregulates NCOA4 expression in NK cells via the DIP2A-P38 pathway, and NCOA4-mediated ferritinophagy is required for CAF-induced NK cell ferroptosis. In a human patient-derived organoid model, functional targeting of CAFs using a combination of deferoxamine and FSTL1-neutralizing antibody significantly alleviate CAF-induced NK cell ferroptosis and boost the cytotoxicity of NK cells against GC. This study demonstrates a novel mechanism of suppression of NK cell activity by CAFs in the TME and presents a potential therapeutic approach to augment the immune response against GC mediated by NK cells.

Keywords: Cancer-associated fibroblasts; Ferritinophagy; Ferroptosis; Iron regulation; NK cells.

Fig. 1

CAFs levels are inversely correlated with NK cells levels in human gastric cancer and inhibit NK cells viability and cytotoxicity in vitro. A, The correlation of immune cells and CAFs in GC tissues according to the TCGA database. B–C, Representative immunofluorescence staining of α-SMA and CD56 in GC tumor tissues and the correlation of α-SMA and CD56 in 21 GC tumor tissues Arrows indicate the expression of CD56 (scale bar = 50um). D, Chemotaxis of pb-NK cells (labeled with Calcein-AM) induced by NFs and CAFs was determined by fluorescence intensity assay. E, Flow cytometry assessment of cell death for NK92 cells cultured alone (BLANK) or co-cultured with CAFs. F-G, The proliferation of NK92/pb-NK cells co-cultured with CAFs was measured by CCK8 assay. H–I, Protein levels of TNF-a and IFN-y secreted by NK92/pb-NK cells co-cultured with CAFs were measured by ELISA. J-M, The effects of CAFs on NK92 and pb-NK cytotoxicity against SNU16 and MKN45 GC cells were measured by DELFIA EuTDA cell cytotoxicity assay. Data are representative of at least three independent experiments. Student t-test and pearson correlation analysis were used to analyze the data (mean ± SD; *P < 0.05, **P < 0.01).

Fig. 2

CAFs induce ferroptosis by promoting intracellular iron overload in NK cells. A, The viability of NK92 cells co-cultured with CAFs was measured by CCK8 assay in the presence of specific inhibitors Z-VAD (caspase-3 inhibitor), Nec1 (necroptosis inhibitor), or Fer1 (ferroptosis inhibitor) for 48 h. B–C, The effects of CAFs on NK92 and pb-NK cytotoxicity against MKN45 and SNU16 GC cells were measured by DELFIA EuTDA cell cytotoxicity assay (Fer1 5 μM, Lip1 50 nM). D-E, Lipid ROS in NK92 cells co-cultured with CAFs was detected using C11 BODYPI 581/591 probe. NK92 cells treated with 0.5 μM RSL3 for 48 h served as a positive control (scale bar = 20um). F, MDA in NK92 cells co-cultured with CAFs was detected using an MDA assay kit. NK92 cells treated with 0.5 μM RSL3 for 48 h served as a positive control. G, The mitochondria of pb-NK cells were analyzed by transmission electron microscopy (scale bar = 1μm/500 nm). H–I, Ferrous iron in pb-NK cells was detected by ferroorange assay in untreated cells or co-culture with CAFs (+CAFs) (scale bar = 20um). J, Ferrous iron in NK92 cells was detected by ferroorange assay in untreated cells after co-culture with CAFs in the absence or presence of DFO (10 μM, 48 h). K, Lipid ROS in NK92 cells co-cultured with CAFs only or CAFs plus DFO (10 μM) was detected by C11 BODYPI 581/591 probe. L, MDA in NK92 cells co-cultured with CAFs only or CAFs plus DFO (10 μM) was detected by MDA assay. M, 4-HNE in NK92 cells co-cultured with CAFs only or CAFs plus DFO (10 μM) was detected by ELISA. Data are representative of at least three independent experiments. Student t-test was used to analyze the data (mean ± SD; *P < 0.05, **P < 0.01).

Fig. 3

CAFs elevate NK cell labile iron pool levels via iron export into the TME. A-B, The mRNA levels of hephaestin (HEPH) and ferroportin1 (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 versus CAFs was analyzed by western blot assay. D-E, Iron ions in NFs and CAFs were detected by Calcein-AM staining (scale bar = 100um). F-G, Iron ions in CAFs were detected by Calcein-AM staining after treatment with indicted doses of DFP (0–100 μM, 48 h) (scale bar = 100um). H, Changes in iron ions within CAFs after removal of DFP were detected by Calcein-AM. I, Iron ions in CAFs only or CAFs plus DFP co-cultured with NK92 was detected by Calcein-AM. J, The viability of NK92 cells co-cultured with CAFs only or CAFs plus DFP was measured by CCK8 assay (Fer1:5 μM). K, Ferrous iron in NK92 cells co-cultured with CAFs only or CAFs plus DFP (100 μM) was detected by ferroorange assay. The fluorescence was measured on a fluorescence microplate reader. L, Lipid ROS in NK92 cells co-cultured with CAFs only or CAFs plus DFP (100 μM) was detected by C11 BODYPI 581/591 probe. M, MDA in NK92 cells co-cultured with CAFs only or CAFs plus DFP (100 μM) was detected by MDA assay. N, 4-HNE in NK92 cells co-cultured with CAFs only or CAFs plus DFP (100 μM) was detected by ELISA. Data are representative of at least three independent experiments. Student t-test was used to analyze the data (mean ± SD; *P < 0.05, **P < 0.01).

Fig. 4

Ferritinophagy via NCOA4 is involved in CAF-induced intracellular iron overload in NK cells. A, Protein expression of TfR, FPN1, FTL and FTH in pb-NK and NK92 cells after incubation with FAS (100 μM) was analyzed by western blotting. B–C, Western blotting analysis of TfR, FPN1, FTL and FTH in pb-NK and NK92 cells co-cultured with CAFs, with CAFs and DFO (10 μM), or with DFP (100 μM)-pre-treated CAFs. D, The mRNA levels of genes associated with ferritin regulation in NK92 cells after co-culture with CAFs were analyzed by QRT-PCR. E, Western blotting of NCOA4 and FTH in NK92 cells co-cultured with CAFs. F, Western blotting 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 measured by CCK8 assay (Fer1 5 μM). H, Ferrous iron in NK92siNC or NK92siNCOA4 cultured with or without CAFs was detected by ferroorange assay. I, Lipid ROS was detected by C11 BODYPI 581/591 probe. J, MDA was detected by MDA assay. Data are representative of at least three independent experiments. Student t-test was used to analyze the data (mean ± SD; *P < 0.05, **P < 0.01).

Fig. 5

CAF-derived FSTL1 upregulates NCOA4 expression in NK cells via the DIP2A-P38 pathway. A, The correlation between NCOA4 and FSTL1 expression in GC from TCGA database. B, The FSTL1 concentration in cell supernatants from NK cells (pb-NK, NK92), GC cell lines (MKN-45, N87, SNU5, SNU16, and AGS) and paired NF and CAFs from GC patients was analyzed by ELISA. C, The mRNA level of NCOA4 in NK92 cells treated with rhFSTL1 (20 ng/ml) for 48 h were analyzed by QRT-PCR. D, Protein expression of NCOA4 and FTH in NK92 cells treated with rhFSTL1 (20 ng/ml; 48 h) was analyzed by western blotting. E, Ferrous iron in NK92 cells treated with or without rhFSTL1 (20 ng/ml; 48 h) was detected by ferroorange assay. F, Protein expression of NCOA4 and FTH in NK92 cells co-cultured with CAFs-shFSTL1 or CAFs-shNC for 48 h was analyzed by western blotting. G, Ferrous iron in NK92 cells co-cultured with CAFs-shFSTL1 or CAFs-shNC for 48 h was detected by ferroorange assay. H, Lipid ROS in NK92 cells co-cultured with CAFs-shFSTL1 or CAFs-shNC was detected using C11 BODYPI 581/591 probe. I, MDA in NK92 cells co-cultured with CAFs-shFSTL1 or CAFs-shNC was detected by MDA assay. J, 4-HNE in NK92 cells co-cultured with CAFs-shFSTL1 or CAFs-shNC was detected by ELISA. K, Protein expression of NCOA4 in NK92siDIP2A or control cells treated with rhFSTL1 (20 ng/ml; 48 h) was analyzed by western blotting. L, Protein expression of p-p38 and p38 in NK92 cells co-cultured with CAFs-shFSTL1 or CAFs-shNC was analyzed by western blotting. M, Protein expression of p-p38 and p38 in NK92siDIP2A or control cells treated with rhFSTL1 (20 ng/ml; 48 h) was analyzed by western blotting. 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 μM) 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 was analyzed by western blotting. Data are representative of at least three independent experiments. Student t-test and pearson correlation analysis were used to analyze the data (mean ± SD; *P < 0.05, **P < 0.01).

Fig. 6

Combined application of DFO and FSTL1-neutralizing antibody alleviates CAF-induced NK cell ferroptosis and boosts the cytotoxicity of NK cells against GC. A, Ferrous iron in NK92 cells co-cultured with CAFs with or without FSTL1-neutralizing antibody (FnAB, 1 μg/ml) and/or DFO (10 μM) was detected by ferroorange assay. B, Lipid ROS in NK92 cells co-cultured with CAFs with or without FnAB (1 μg/ml) and DFO (10 μM) was detected by C11 BODYPI 581/591 probe. C, MDA in NK92 cells co-cultured with CAFs with or without FSTL1-neutralizing antibody (FnAB, 1 μg/ml) and DFO (10 μM) was detected by MDA assay. D, 4-HNE in NK92 cells co-cultured with CAFs with or without FSTL1-neutralizing antibody (FnAB, 1 μg/ml) and DFO (10 μM) was detected by ELISA.E, The cell death of NK92 cells co-cultured with CAFs with or without FnAB (1 μg/ml) and DFO (10 μM) was analyzed by flow cytometry. F-G, The cytotoxicity of NK92 cells and pb-NK against MKN45 and SNU16 GC cells was measured by DELFIA EuTDA cell cytotoxicity assay after co-culture with CAFs with or without FnAB (1 μg/ml) and DFO (10 μM). H, Protein expression of MSLN in GC organoids (PDO1T and PDO3T) and GC cells (MKN45 and SNU16) was analyzed by western blotting. I, The cell death of PDO1T co-cultured with NK92 or CAR-NK cells (PDO1T: NK92 or CAR-NK = 1:5) was analyzed by flow cytometry. J-K, The cell death of PDO1T co-cultured with CAFs in the presence of DFO/FnAB and/or CAR-NK cells (PDO1T: CAFs: NK = 1:2:5, DFO: 10 μM, FnAB: 1 μg/ml) was analyzed by flow cytometry. L-M The cell death of PDO3T co-cultured with CAFs in the presence of DFO/FnAB and/or CAR-NK cells (PDO1T: CAFs: NK = 1:2:5, DFO: 10 μM, FnAB: 1 μg/ml) was analyzed by Calcein-blue-AM(scale bar = 20um). The data are presented as the mean ± SD from three independent experiments. Student t-test was used to analyze the data (*P < 0.05; **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Schematic representation of the putative mechanism by which CAFs induce NK cell ferroptosis in GC. CAFs increase labile iron within NK cells to induce ferroptosis by exporting iron into TME and also induce FSTL1-NCOA4-mediated ferritinophagy in GC. Therefore, incorporating FSTL1-neutralizing antibody and DFO treatment may present a promising therapeutic strategy to treat GC by overcoming the immune suppression from the tumor stroma.