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. 2023 Jun 21;10(24):2300881. doi: 10.1002/advs.202300881

Figure 8.

Figure 8

ANO1‐mediated ferroptosis inhibition provokes TGF‐β release and recruits cancer‐associated fibroblasts into TIME, generating resistance to immunotherapy. A) Correlation between ANO1 and all key microenvironmental cells in TCGA‐STAD/ESCA/COAD/READ datasets. B) The IHC‐based expression of infiltrated α‐SMA in prospective GI cancer patient‐cohort was stratified by the positivity of ANO1. C) The representative staining for CAFs and CD8+ T cells and D) their distribution ratio in tumor/stroma regions were assessed by mIHC analysis. The E) expression and F) secretion of TGF‐β by GC cell HGC27 were assessed after ANO1 knockdown. For MC38 and MC38‐R CDXs, the T‐distributed stochastic neighbor embedding (t‐SNE) maps of G) major cell components in TIME, H) CAF proportions, and I) intensity of ANO1 were assessed by single‐cell RNA sequencing. J) Expression of CAF markers in MC38/MC38‐R CDXs after acquired resistance to immunotherapy. K) Expression of CAF markers in CT26 CDX after ANO1 overexpression. L) Expression and M) fluorescent staining of CAF markers in CT26 CDX after ANO1 knockdown and anti‐PD‐1 antibody treatment. N) PI3K‐Akt signaling inhibitor Dactolisib (Dacto) repressed the TGF‐β upregulation induced by ANO1 overexpression. The O) expression and P) secretion of TGF‐β by GC cells were reduced by ANO1 knockdown and rescued by ferroptosis inhibitor Fer‐1. For CT26 CDX, changes of Q) IHC‐based expression of infiltrated CD8/PD‐L1/α‐SMA in xenograft tissue, and R) ELISA‐based expression of IFN‐γ/granzyme B/TGF‐β in CDX tissues were measured after combining ANO1 knockdown, anti‐PD‐1 antibody and liproxstatin treatment. S) The effect of talabostat mesylate on inducing granzyme B expression in CT26 CDX tissues. T) A schematic model summarizing ANO1's mechanism in mediating TIME remodeling and immunotherapeutic resistance. U) The tumor growth inhibition for the triple combination of ANO1 knockdown, anti‐PD‐1 antibody, and CAF inhibitor talabostat mesylate (talabostat) treatment. STAD, stomach adenocarcinoma. ESCA, esophageal carcinoma. COAD, colon cancer. READ, rectum cancer. CAF, cancer‐associated fibroblast. NAF, normal tissue‐associated fibroblast. *p < 0.05. ns, not significant. Error bars, mean ± SEM.