Fig 2: S100A4 increases ZIP1 expression in fibroblasts.a, b ZIP1 expression in mCAF treated with DOX at the indicated doses (a) or for the indicated times (b). Representative results from three (a, b) independent experiments are shown. c S100A4 levels in the same volume of LLC-GFP-luc cell culture mediums (CMs) with or without DOX treatment determined by western blotting. The same number of tumour cells were seeded and pre-treated with DOX (1 µM) for 6 h. Twenty-four hours after changing the fresh medium, CMs were collected. The same volume of CM from different treatment groups was analysed by Western blotting. A representative result from three independent experiments is shown. d ZIP1 expression in mCAF treated with indicated CM. For S100A4 neutralisation, CM was pre-incubated with a-S100A4 antibody 3B11 (6 µg/mL) for 1 h. A representative result from four independent experiments is shown. e, f ZIP1 expression in mCAF treated with S100A4 at the indicated doses (e) or for the indicated times (f). ß-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three (e, f) independent experiments are shown. g, h Expression of phosphorylated p65 (pp65), p65, phosphorylated ERK (pERK) (g), phosphorylated AKT (pAKT), AKT, phosphorylated p38 (pp38), and p38 (h) in mCAF treated with S100A4 for the indicated time periods. ß-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three (g, h) independent experiments are shown. i Expression of ZIP1 in mCAF treated with S100A4, SB202190 (p38 inhibitor, 50 µM), U0126 (ERK inhibitor, 10 µM), SC75741 (p65 inhibitor, 8 µM), and LY294002 (AKT inhibitor, 25 µM) as indicated. A representative result from three independent experiments is shown. j Expression of ZIP1 in mCAF treated with S100A4, TLR4-IN-C34 (TLR4 inhibitor, 10 µM), and FPS-ZM1 (RAGE inhibitor, 1 µM), as indicated. A representative result from three independent experiments is shown. k Diagram showing the signalling pathways for the regulation of ZIP1 by S100A4. l Extracellular S100A4 in LLC-GFP-luc tumours, with or without DOX treatment. n = 6 for the PBS group and n = 7 for the DOX group. Data are presented as mean ± SEM, n = 6 for PBS, n = 7 for DOX. Two-tailed t-test. Source data are provided as a Source Data file (a–j, l).

Fig 3: ZIP1+ fibroblasts interconnect lung cancer cells via upregulating CX43 and promote chemoresistance by Zn2+ transfer.Chemotherapy treatment induces Zn2+ released from dying cancer cells and the enrichment of ZIP1+ fibroblasts. ZIP1+ fibroblasts interconnect cancer cells with gap junctions by upregulating CX43. Upon treatment, the ability of Zn2+ uptake by cancer cells is suppressed. ZIP1+ fibroblasts act as a reservoir to absorb and transfer Zn2+ to lung cancer cells via gap junctions. In ZIP1+ fibroblasts, imported Zn2+ stimulates degradation of PTEN and activation of AKT to upregulate the expression of gap junction protein CX43. In lung cancer cells, the elevation of Zn2+ enhances the expression of ABCB1 and reduces the accumulation of drugs, resulting in chemoresistance.

Fig 4: ZIP1high fibroblasts are associated with poor survival and chemoresistance in patients with lung cancer.a Representative ZIP1 staining in lung adenocarcinoma (LA) tissues. A human liver section was used as a positive control. Arrow, ZIP1+ fibroblasts. Representative staining results from 68 lung cancer samples are shown. b Survival curve of patients with lung adenocarcinoma with stroma ZIP1high or stroma ZIP1low status. c Double-staining of ZIP1 and a-SMA in lung cancer tissues including tumour and paratumour tissue. Representative staining results from 90 tumour and 90 corresponding paratumour samples are shown. d ZIP1 expression levels in fibroblasts and percentage of ZIP1+ fibroblasts in tumour and paratumour tissues (n = 90). Data are presented as median with interquartile range. Two-tailed t-test. e, f Overall survival (e) and progression-free survival (f) of patients with lung cancer with ZIP1high or ZIP1low status. g, h Overall survival of patients with no observed relapse (g) or with pathological grade 2 lung adenocarcinoma (h). i, j Overall survival (i) or progression-free survival (j) of patients with ZIP1high and ZIP1low status in response to chemotherapy with paclitaxel/docetaxel-containing regimens (Taxane). k Correlation of ABCB1 expression in tumours with ZIP1 expression in cancer-associated fibroblasts (CAF) (n = 19). Representative co-staining of ABCB1, ZIP1 and a-SMA is shown. Arrowhead, ZIP1+ fibroblasts. Asterisk, cancer cell. Log-rank test for survival curve comparison. Mann–Whitney test for two-group comparison. Source data are provided as a Source Data file (b, d–k).

Fig 5: ZIP1+ fibroblasts promote chemoresistance of lung cancer cells in vitro and in vivo.a Experimental design (left) and results of DNA damage (?-H2AX expression) in LLC-GFP-luc cells co-cultured with Zip1+/+ or Zip1-/- MEFs with DOX (3 µM) treatment. Mean ± SEM, n = 4. Two-tailed t-test. b DOX accumulation in LLC-GFP-luc tumour cells co-cultured with Zip1+/+ or Zip1-/- MEFs with DOX (3 µM) treatment. Mean ± SEM, n = 4. Two-tailed t-test. c ABCB1 expression in LLC-GFP-luc tumour cells co-cultured with Zip1+/+ or Zip1-/- MEFs for 24 h. Mean ± SEM. Ctl, Zip1+/+: n = 3; Zip1-/-: n = 6. Two-tailed t-test. d DOX accumulation in LLC-GFP-luc tumour cells 6 h after DOX injection in tumour-bearing Zip1+/+, Zip1+/- and Zip1-/- mice. DOX (10 mg/kg) was intravenously injected into the mice at day 10 post-transplantation. Mean ± SEM. Zip1+/+: n = 4; Zip1+/-, Zip1-/-: n = 5. One-tailed Kruskal–Wallis test. e ABCB1 expression in LLC-GFP-luc cells transplanted into Zip1+/+ and Zip1-/- mice at day 10 post-transplantation. Mean ± SEM, n = 5. Two-tailed t-test. f Tumour growth curves for Zip1+/+ and Zip1-/- mice treated with DOX or PBS. Arrows, DOX injection. Mean ± SEM. P values are for indicated time points. Zip1+/+ PBS (n = 11), Zip1-/- PBS (n = 12), Zip1+/+ DOX (n = 8), Zip1-/- MEFs DOX (n = 10). g Tumour growth curves for co-injection of Zip1+/+ or Zip1-/- MEFs with LLC-GFP-luc cells, with DOX or PBS treatment. Arrows indicate DOX injection. Zip1+/+, Zip1-/- MEFs PBS, Zip1-/- MEFs DOX: n = 9; Zip1+/+ MEFs DOX: n = 6. Mean ± SEM. h Tumour growth curves for mice inoculated with LLC cells and mCAFs with a tet-off system-controlled expression of Zip1 (TET-Zip1). Mice were administered PTX, with or without doxycycline (Dc) treatment. Mean ± SEM. Arrow, PTX (10 mg/kg) treatment. TET-mock, TET-Zip1 PBS: n = 6; TET-Zip1 PTX, TET-Zip1 PTX + Dc: n = 5. Two-way ANOVA test for tumour growth curve comparison. Source data are provided as a Source Data file (a–h).