Fig 2: CTHRC1 + CAFs promote EGFR-TKI resistance via metabolic reprogramming in cancer cells.A KEGG enrichment analysis shows the top upregulated pathways in PC9 co-cultured with CTHRC1+ CAFs (n = 3). B Schematic show change of metabolites involved glycolysis pathway in PC9 co-cultured with CTHRC1+ CAFs (n = 4). Cell glycolytic profile of indicated lung cancer cells co-cultured with CTHRC1+ CAFs was evaluated by measuring glycolytic proton efflux rate (GlycoPER, C), 2-NBDG incorporation (D) and lactate production (E, n = 4). F Volcano plot of differently expressed genes in PC9 co-cultured with CTHRC1+ CAFs. G Expression of the key glycolytic enzymes in PC9 co-cultured with CTHRC1+ CAFs. PC9 cells with or without shHK2 and CTHRC1+ CAFs were co-injected subcutaneously into nude mice (n = 5). Treating with Osi (5 mg/kg). The digital photographs of tumors dissected from mice in different groups (H). Tumor growth curves (I) and tumor weight (J) were monitored. Representative immunofluorescent images for TUNEL staining in xenografts (K). Scale bar, 50 μm. L–O Cell glycolytic profile of PC9 co-cocultured with CTHRC1+ CAFs after Cthrc1 knockdown, or treating with recombinant CTHRC1 (rCTHRC1, 500 ng/mL). P HK2 expression in PC9 co-cultured with CTHRC1+ CAFs after Cthrc1 knockdown (top) or treating with rCTHRC1 (500 ng/mL) at 0, 2, 4 and 8 h (bottom). Q PC9 cells and CTHRC1+ CAFs with or without shCthrc1 were co-injected subcutaneously into nude mice (n = 5). Treating with Osi (5 mg/kg). Tumor growth curves were monitored (left). Representative immunofluorescent images for TUNEL staining in xenografts (right). R PC9 cells were injected into nude mice (n = 5) and treated with Osi (5 mg/kg) or rCTHRC1 (100 μg per mice) combined with Osi. Tumor growth curves were monitored (left). Representative immunofluorescent images for TUNEL staining in xenografts (right). Scale bar, 50 μm. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Lactate drives CTHRC1 + CAFs phenotype via histone lactylation.A Cthrc1 expression in primary CAFs isolated from PDX-S co-cultured with primary cancer cells from PDX-S (CC-S) and PDX-R (CC-R). B Lactate levels in tumor tissues derived from PDXs (n = 4). Cthrc1 expression in primary CAFs isolated from PDX-S treating with lactate at different concentrations (C) or timepoints (D). RNA-seq of primary CAFs from PDX-S after lactate treatment (20 mM) (n = 3). Volcano plot of differently expressed genes (E). Venn diagram shows significant upregulated overlap genes between in vivo scRNA-seq and in vitro RNA-seq (F). GSEA shows CTHRC1 + CAF signature expression after lactate treatment (G). H Representative immunofluorescent images of CTHRC1 + CAF marker genes in primary CAFs after lactate treatment (20 mM). Scale bar, 20 μm. I Representative snapshots of CUT&Tag tracks for H3K18la on Cthrc1 promoter. J Levels of pan-Kla, H3K18la, and CTHRC1 in primary CAFs isolated from PDXs. K CUT&RUN-qPCR testing of H3K18la enrichment on Cthrc1 promoter in primary CAFs isolated from PDXs. L Luciferase reporter assays for Cthrc1 promoter activity in primary CAFs isolated from PDXs. M Levels of pan-Kla, H3K18la, and CTHRC1 in primary CAFs isolated from PDX-S co-cultured with CC-R alone or with C646 treatment (100 μM). Cthrc1 expression (N) and promoter activity (O) in primary CAFs isolated from PDX-S co-cultured with CC-R alone or with C646 treatment (100 μM). P Representative immunofluorescent images of CTHRC1+ CAFs and H3K18la in TN and PD lung cancer samples. Scale bar, 50 μm Q Quantification of H3K18la intensity in TN (n = 22) and PD (n = 22) lung cancer samples from clinical cohort 1. R Kaplan-Meier survival curves of lung cancer patients from clinical cohort 2 who received EGFR-TKI treatment with low and high H3K18la intensity (n = 32). S Schematic proposing a positive feedback loop between CTHRC1+ CAFs and EGFR-TKI resistant cancer cells. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: CTHRC1 + CAFs promote acquired resistance to EGFR-TKI treatment in lung cancer.A Schematic illustration of PDX models establishment from EGFR-TKI (osimertinib, Osi) sensitive and resistant patients. B Tumor growth curves of tumor xenografts from PDXs that were treated with Osi (5 mg/kg, n = 5 mice per group). C Representative images of cytokeratin (pan-CK), COL1A1, and CTHRC1 in situ immunofluorescent staining in tumor tissues from PDXs. Scale bar, 50 μm. D Flow cytometric analysis of CTHRC1 expression in CAFs in tumor tissues from PDXs. E Representative immunofluorescent staining images of CTHRC1 + CAF marker genes in primary CAFs derived from PDXs. Scale bar, 20 μm. F Cell viability of indicated lung cancer cell lines after Osi treatment or co-cultured with indicated CAFs and treating with Osi. Representative images of the colony formation (G) and the percentage of dead cells in indicated lung cancer cell lines (H) after being cultured alone, treated with Osi (0.01 μM) alone, or co-cultured with indicated CAFs and treating with Osi. PC9 cells with or without indicated CAFs were injected subcutaneously into nude mice (n = 6). Treating with Osi (5 mg/kg) or vehicle. The digital photographs of tumors dissected from mice in different groups (I). Tumor growth curves (J) and tumor weight at the therapeutic endpoint were monitored (K). Representative immunofluorescent images for TUNEL staining apoptotic cells in xenografts at the therapeutic endpoint (L). Scale bar, 50 μm. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: CTHRC1/Glycolysis/H3K18la forms a positive feedback loop in EGFR-TKI-resistant tumor.A Lactate production in PC9 cocultured with primary CAFs isolated from PDX-S after rCTHRC1 treatment (500 ng/mL, n = 4). B Levels of pan-Kla, H3K18la, and CTHRC1 in primary CAFs isolated from PDX-S after rCTHRC1 treatment (500 ng/mL). C Lactate production in PC9 cocultured with CTHRC1+ CAFs after Cthrc1 knockdown (n = 4). D Levels of pan-Kla, H3K18la, and CTHRC1 in Cthrc1-knockdown CAFs after lactate treatment. Representative images of the colony formation (E) and the percentage of dead cells (F) in indicated lung cancer cells co-cultured with Cthrc1-knockdown CAFs and treating with Osi (0.01 μM) alone or Osi combined with lactate (20 mM). PC9 cells and CTHRC1+ CAFs with or without shCthrc1 were co-injected into nude mice (n = 5). Treating with Osi (5 mg/kg) or Osi combined with lactate (1 g/kg). The digital photographs of tumors dissected from mice (left). Tumor growth curves (middle) and tumor weight (right) were monitored (G). Representative immunofluorescent images for TUNEL staining in xenografts (H). I Molecular docking analysis of binding between CTHRC1 and TGFBR2. J Docking of Gambogenic Acid (GA) into the crystal structures of CTHRC1. K Coculture IP detected the CTHRC1-TGFBR2 and TGF-β1-TGFBR2 interactions after GA treatment (0.5 μM). Representative images of the colony formation (L) and the percentage of dead cells (M) in indicated lung cancer cells co-cultured with CTHRC1+ CAFs and treating with Osi (0.01 μM) alone or Osi combined with GA (0.5 μM). PC9 cells and CTHRC1+ CAFs were co-injected into nude mice (n = 5). Treating with Osi (5 mg/kg) alone or Osi combined with GA (10 mg/kg). The digital photographs of tumors dissected from mice (left). Tumor growth curves (middle) and tumor weight (right) were monitored (N). Representative immunofluorescent images for TUNEL staining in xenografts (O). Scale bar, 50 μm. P Representative immunofluorescent images of CTHRC1+ CAFs and H3K18la in xenografts after Osi combined with GA treatment. Scale bar, 50 μm. Q A proposed working model for how a CTHRC1/glycolysis/H3K18la positive feedback loop in lung cancer tumor microenvironment drives EGFR-TKI resistance, and interruption of this feedback loop by blocking CTHRC1 overcomes drug resistance. ns not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.