Tumor cell-intrinsic Piezo2 drives radioresistance by impairing CD8+ T cell stemness maintenance

Abstract

Changes in mechanosensitive ion channels following radiation have seldom been linked to therapeutic sensitivity or specific factors involved in antitumor immunity. Here, in this study, we found that the mechanical force sensor, Piezo2, was significantly upregulated in tumor cells after radiation, and Piezo2 knockout in tumor cells enhanced tumor growth suppression by radiotherapy. Specifically, loss of Piezo2 in tumor cells induced their IL-15 expression via unleashing JAK2/STAT1/IRF-1 axis after radiation. This increase in IL-15 activates IL-15Rα on tumor-infiltrating CD8+ T cells, thereby leading to their augmented effector and stem cell-like properties, along with reduced terminal exhausted feature. Importantly, Piezo2 expression was negatively correlated with CD8 infiltration, as well as with radiosensitivity of patients with rectum adenocarcinoma receiving radiotherapy treatment. Together, our findings reveal that tumor cell-intrinsic Piezo2 induces radioresistance by dampening the IRF-1/IL-15 axis, thus leading to impaired CD8+ T cell-dependent antitumor responses, providing insights into the further development of combination strategies to treat radioresistant cancers.

Graphical abstract

Figure 1.

Effects of Piezo2 KO on tumor response to radiation. (A) Scanning electron microscope (SEM) showing the size of MC38 cells with one fraction of 30 Gy IR or without IR (left; scale bar = 10 µm), and the quantification of cell size (right). (B–D) MC38 cells were irradiated at a single fraction of 30 Gy. At 60 h after radiation, total RNA was extracted and further analyzed by RNA-seq. GO enrichment analysis of membrane potential signaling pathway (B), GSEA of cellular response to abiotic stimulus (C), and volcano plot exhibiting the DEGs of mechanosensitive ion channels from RNA-seq data (D). (E) The Piezo2 mRNA expression in MC38 and B16F1 cells after IR treatment was shown from three independent experiments. (F) Piezo2 expression was determined on the surface or in the cytosol of CD45⁻tdTomato⁺ tumor cells sorted from WT MC38 tumors on day 5 after IR treatment. Representative data were shown from one independent experiment using pooled tdTomato⁺ tumor cells from tumors (n = 6 mice per group). (G) Mice were subcutaneously inoculated with WT MC38 and Piezo2^−/− MC38 tumor cells, and then established tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from three independent experiments (n = 6–8 mice per group). (H) Mice were subcutaneously inoculated with WT B16F1 and Piezo2^−/− B16F1 tumor cells, and then established tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from two independent experiments (n = 3–4 mice per group). (I) The tumor growth curve of unirradiated secondary tumors (WT MC38, left flank) and irradiated primary tumors (WT MC38 or Piezo2^−/− MC38, right flank) in C57BL/6 mice was represented from two independent experiments (n = 5–6 mice per group). Data were represented as means ± SEM. The comparisons of two nonparametric datasets in A and E were calculated by the Mann–Whitney U test. G–I were analyzed by one-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001. Source data are available for this figure: SourceData F1.

Figure S1.

Cell size was changed after IR accompanied by the increase of Piezo2 expression and Ca²⁺ uptake. Related to Fig. 1. (A) The cytoskeleton staining in MC38 cells with or without radiation. Scale bar = 15 µm. (B) The cytoskeleton staining in B16F1 cells with or without radiation. Scale bar = 30 µm. (C and D) MFI of FSC-A tdTomato⁺ tumor cells from tumors on day 3 (C) and 6 (D) after IR were represented from three independent experiments (n = 4–6 mice per group). (E and F) The assessments of Piezo2 expression in MC38 (E) and B16F1 (F) tumor cells following radiation treatment were represented from three independent experiments. (G) Determination of Ca²⁺ concentration in tdTomato⁺ cells from irradiated tumors was shown from two independent experiments (n = 8–9 mice per group). (H) Determinations of Piezo2 expression in MC38 (E) and B16F1 (F) cells with or without gRNA were represented from three independent experiments. (I) Determinations of MC38 (left) and B16F1 (right) cell viability with or without Piezo2 by CCK-8 were shown from two independent experiments. Data were represented as means ± SEM. The comparisons of two nonparametric datasets were calculated by Mann–Whitney U test (A and B). C–G were analyzed by unpaired Student’s t test. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData FS1.

Figure 2.

Effects of Piezo2 KO on intratumoral CD8⁺ T cell function and stemness after radiation. (A–I) Mice were transplanted subcutaneously with 2 × 10⁶ MC38 cells and then established tumors were treated locally with one fraction of 18-Gy IR. (A) To deplete CD8⁺ T cells, mice were injected with anti-CD8 antibody (200 µg per mouse, intraperitoneally (i.p.) starting on the day receiving IR treatment, every 2 days for a total of three times), RIgG, rat IgG. The tumor growth curve of WT and Piezo2^−/− MC38 tumors in C57BL/6 mice with or without anti-CD8 treatment after IR was represented from two independent experiments (n = 4–6 mice per group). (B) Representative data and quantification of the percentage of CD8⁺ T cells in CD45⁺ cells from WT and Piezo2^−/− MC38 tumors on days 7 and 14 after IR treatment were shown from three independent experiments (n = 3–8 mice per group). (C) Representative data and quantification of the percentage of IFN-γ⁺TNF-α⁺CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 after IR were shown from three independent experiments (n = 3–6 mice per group). (D) Quantitation of the percentage of IFN-γ⁺CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on days 7 and 14 after IR was shown from three independent experiments (n = 3–6 mice per group). (E–H) Representative data and quantification of the percentage of Ki-67⁺ (E), TCF-1⁺ (F), CD62L⁺ (G), and TOX⁺ (H) in PD-1⁺CD44⁺TIM3^low CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 after IR are shown from three independent experiments (n = 3–6 mice per group). (I) Quantification of the percentage of PD-1⁺ in CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 after IR was shown from three independent experiments (n = 4–6 mice per group). (J and K) Mice were transplanted subcutaneously with 2 × 10⁶ MC38-OVA cells, and then established tumors were treated locally with one fraction of 18-Gy IR. On the next day of receiving radiation treatment, 2 × 10⁶ activated OT-I CD8⁺ T cells were adoptively transferred into mice via retroorbital intravenous injection. Representative data and quantification of the percentage of OT-I CD8⁺ T cells (J), and IFN-γ⁺TNF-α⁺ (K) in OT-I CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 11 after IR were shown from two independent experiments (J, n = 5–7 mice per group; K, n = 4–6 mice per group). (L) Quantification of the percentage of TCF-1⁺ in OT-I CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 11 after IR was shown from two independent experiments (n = 4–5 mice per group). (M) Mice were transplanted subcutaneously with 1 × 10⁶ cells WT, Piezo2^−/−, and Piezo2^−/−-RE B16F1 cells, and then established tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from two independent experiments (n = 4–6 mice per group). Data were represented as means ± SEM. A and M were analyzed by two-way ANOVA with multiple comparison tests; B–L were performed by one-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01, ***P < 0.001, ns, no significant difference.

Figure S2.

Piezo2 deficiency in tumor cells affected CD8⁺ T cell infiltration and differentiation within irradiated tumors. Related to Fig. 2. (A–D) Quantifications of the percentage of Ki-67⁺ (A), TCF-1⁺ (B), CD62L⁺ (C), and TOX⁺ (D) in exhausted CD8⁺ T cells from irradiated tumors on day 14 after IR were shown from two independent experiments (n = 3–9 mice per group). (E) Quantifications of PD-1⁺ in CD8⁺ T cells from irradiated tumors on day 14 after IR were shown from two independent experiments (n = 5–6 mice per group). (F and G) Quantifications of expression of Slamf6 on CD8⁺ T cells from irradiated tumors on day 7 (F) and 14 (G) after IR were shown from two independent experiments (n = 3–8 mice per group). (H–J) The percentage of CD8⁺ T cell (H), IFN-γ⁺TNF-α⁺CD8⁺ T (I), and IFN-γ⁺ CD8⁺ T (J) cells in DLNs from tumor-bearing mice was represented from two independent experiments (n = 4–6 mice per group). (K and L) MFI summary of Ki-67 (K) and the proportion of TCF-1⁺ (L) in CD8⁺ T cells in DLNs were shown from two independent experiments (n = 4–7 mice per group). (M) The determination of Piezo2 expression in MC38-OVA cells with or without gRNAs was represented by two independent experiments. (N) The assessment of Piezo2 expression in Piezo2^−/− B16F1 with or without mPiezo2-CMV-Sport6 plasmid was shown from two independent experiments. (O and P) Ca²⁺ concentration measurement in CD45⁻ cells (O) and CD8⁺ T cell infiltration (P) in CD45⁺ cells within irradiated WT, Piezo2^−/−, and Piezo2^−/−-RE tumors were shown from two independent experiments (n = 3–6 mice per group). (Q and R) The percentage of IFN-γ⁺CD8⁺ T cells (Q) and TOX⁺ among exhausted CD8⁺ T cells (R) from irradiated WT, Piezo2^−/−, and Piezo2^−/−-RE tumors was represented from two independent experiments (n = 3–6 mice per group). Data were represented as means ± SEM. Statistical analysis was performed by one-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01, ns, no significant difference. Source data are available for this figure: SourceData FS2.

Figure 3.

Increase of tumor cell–derived IL-15 is responsible for the enhanced antitumor immunity after radiation by Piezo2 deficiency. (A and B) WT and Piezo2^−/− MC38 cells were irradiated at a single fraction of 30 Gy. At 60 h after radiation, total RNA was extracted and further analyzed. (A) RNA-seq analysis of indicated cytokine mRNA expression. Bar plots from RNA-seq reveal the genes coding T cell–related cytokines. (B) Il-15 mRNA expression in WT and Piezo2^−/− MC38 or WT and Piezo2^−/− B16F1 with or without radiation was shown from two independent experiments. (C) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT MC38-tdTomato and Piezo2^−/− MC38-tdTomato cells. Tumors were treated locally with one fraction of 18-Gy IR. On days 7 and 14 after radiation, IL-15 expression in MC38-tdTomato⁺ cells was represented from three independent experiments (n = 5–9 mice per group). (D) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT MC38 and Piezo2^−/− MC38 cells. Tumors were treated locally with one fraction of 18-Gy IR. Anti-IL-15 was administered intratumor at 100 µg per mouse to mice every 2 days for a total of four times from the day receiving radiotherapy. Tumor growth was measured twice a week. The tumor growth curve was represented from two independent experiments (n = 6 mice per group). (E) Representative data and quantification of the percentage of IFN-γ⁺ in CD8⁺ T cells from irradiated WT and Piezo2^−/− MC38 tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–5 mice per group). (F and G) Representative data and quantification of the percentage of TOX⁺ (F) and TCF-1⁺ (G) in PD-1⁺CD44⁺TIM3^lowCD8⁺ T cells from tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–6 mice per group). (H) Representative data and quantification of the percentage of Ki-67⁺ in CD8⁺ T cells from irradiated WT and Piezo2^−/− MC38 tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–5 mice per group). (I) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT, IL-15^−/−, Piezo2^−/−, and Piezo2^−/−IL-15^−/− MC38 cells. Tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from two independent experiments (n = 4–6 mice per group). (J) Representative data and quantification of the frequency of IFN-γ⁺TNF-α⁺CD8⁺ T cells from irradiated WT, IL-15^–/–, Piezo2^−/−, and Piezo2^–/–IL-15^−/− MC38 tumors on day 16 after IR were shown from two independent experiments (n = 4–6 mice per group). Data were represented as means ± SEM. B, C, E–H, and J were calculated by one-way ANOVA with multiple comparison tests. D and I were analyzed by two-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant difference.

Figure S3.

Tumor cell–derived IL-15 was required for tumor growth suppression enhancement and CD8⁺ T cell regulation mediated by Piezo2 deficiency after IR treatment. Related to Fig. 3. (A) MFI summary of IL-15 expression in CD45⁺ or CD45⁻ cells in indicated MC38 tumors was represented from three independent experiments (n = 5–6 mice per group). (B) MFI summary of IL-15 expression in indicated immune cells was shown from two independent experiments (n = 6–9 mice per group). (C) The knockdown efficiency of IL-15 expression in MC38 cells was shown in two independent experiments. (D) Representative data and quantification of the percentage of TCF-1⁺ in exhausted CD8⁺ T cells within irradiated WT, IL-15^−/−, Piezo2^−/−, and Piezo2^−/−IL-15^−/− tumors were shown from two independent experiments (n = 3–5 mice per group). (E) Representative data and quantification of the percentage of IL-15Rα⁺ in CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 and 14 after IR treatment were shown from three independent experiments (n = 4–5 mice per group). (F) Quantification of the percentage of IL-15Rα⁺ in CD8⁺ T cells from DLNs of WT and Piezo2^−/− MC38 tumor–established mice on day 5 after IR treatment was shown from two independent experiments (n = 5–6 mice per group). (G–I) The positive relevance of IL-15Rα with IFN-γ (G), Ki-67 (H), and TCF-1 (I) in tumor-infiltrating CD8⁺ T cells was shown by two independent experiments (n = 4–6 mice per group). (J) MFI summary of IL-15Rα expression in indicated immune cells within tumors was shown by independent three experiments (n = 5–6 mice per group). (K) The expression of p-STAT5 in CD8⁺ T cells from irradiated tumors on days 7 and 14 upon radiation treatment was represented by two independent experiments (n = 3–7 mice per group). Data were represented as means ± SEM. The comparisons of two datasets were calculated by paired Student’s t test (A and G–I). Statistical analysis was performed by one-way ANOVA with multiple comparison tests (B, D–F, J, and K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns, no significant difference. Source data are available for this figure: SourceData FS3.

Figure 4.

IRF-1 is essential for IL-15 production after radiation in Piezo2 KO tumors. (A) The expression of IRF-1 from nuclear and cytoplasm of WT and Piezo2^−/− MC38 by 60 h after IR treatment was shown from three independent experiments. (B) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT and Piezo2^−/− MC38-tdTomato cells. Tumors were treated locally with one fraction of 18-Gy IR. On day 5 after radiation, IRF-1 expression in MC38-tdTomato⁺ cells was analyzed by flow cytometry. Representative data of IRF-1 expression (left) and quantification analysis (right) in irradiated WT and Piezo2^−/− MC38 tumors were shown from two independent experiments (n = 3–5 mice per group). (C) At 60 h after IR, IL-15 expression in WT, Piezo2^−/−, and Piezo2^−/−IRF-1^−/− B16F1 tumor cells was shown from three independent experiments. (D) The IL-15 expression in WT, Piezo2^−/−, and Piezo2^−/−IRF-1^−/− MC38 tumor cells by 60 h upon radiation treatment was shown from three independent experiments. (E) The IL-15 expression in CD45⁻ cells from irradiated WT, Piezo2^−/−, and Piezo2^−/−IRF-1^−/− MC38 tumors on day 14 after IR was represented from two independent experiments (n = 4–5 mice per group). (F) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT, Piezo2^−/−, and Piezo2^−/−IRF-1^−/− B16F1 cells. Tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was shown from two independent experiments (n = 4–6 mice per group). (G) Representative data and quantification of the percentage of PD-1⁺ in CD8⁺ T cells from irradiated WT or Piezo2^−/− MC38 tumors with or without IRF-1 on day 14 after IR were shown from two independent experiments (n = 4 mice per group). (H) Representative data and quantification of the percentage of TCF-1⁺ in CD8⁺ T cells from irradiated WT or Piezo2^−/− MC38 tumors with or without IRF-1 on day 14 after IR were shown from two independent experiments (n = 5–6 mice per group). (I) Representative data and quantification of the percentage of CD62L⁺ in CD8⁺ T cells from irradiated WT or Piezo2^−/− MC38 tumors with or without IRF-1 were shown from two independent experiments (n = 3–4 mice per group). Data were represented as means ± SEM. Statistical analysis was performed by one-way ANOVA with multiple comparison tests (A–E and G–I), and statistical analysis was calculated by two-way ANOVA with multiple comparison tests (F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData F4.

Figure S4.

IRF-1 was increased in irradiated cells and the KO efficiency determination for IRF-1 in Piezo2^−/− cells. Related to Fig. 4. (A) The assessment of IRF-1 expression in nuclear and cytoplasm in irradiated B16F1 cells was represented from two independent experiments. (B) The determination of IRF-1 expression by immunofluorescence was shown by two independent experiments. Scale bar = 40 µm. (C and D) The KO efficiency determination of shRNA silencing for IRF-1 in Piezo2^−/− MC38 and Piezo2^−/− B16F1 cells was shown by two independent experiments. Data were represented as means ± SEM. Statistical analysis was performed by one-way ANOVA with multiple comparison tests. *P < 0.05; ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData FS4.

Figure 5.

Increase of IRF-1 and IL-15 expression in Piezo2 KO tumor cells upon radiation depended on JAK2/STAT1 pathway. (A) The expression of p-JAK2 and p-STAT1 shown by western blot (left) and quantification (right) in WT and Piezo2^−/− MC38 with or without IR treatment for 15 min after IR were represented from three independent experiments. (B) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT and Piezo2^−/− MC38-tdTomato⁺ cells. Tumors were treated locally with one fraction of 18-Gy IR. On day 5 after radiation, p-STAT1 expression in MC38-tdTomato⁺ cells was analyzed by flow cytometry. Representative data and quantification of p-STAT1 expression in MC38-tdTomato⁺ cells from irradiated tumors were shown from two independent experiments (n = 5–6 mice per group). (C and D) Representative data and quantification of IL-15 expression in WT and Piezo2^−/− MC38 (C) and B16F1 (D) cells at 15 min after radiation with or without JAK inhibitor (1 µM) 1 h ahead of IR were shown from three independent experiments. (E and F) Representative data and quantification of IRF-1 expression in WT and Piezo2^−/− MC38 (E) and B16F1 (F) cells at 15 min after radiation with or without JAK inhibitor (1 µM) 1 h ahead of IR were shown from three independent experiments. (G) Representative data and quantification of IFN-γ expression in WT and Piezo2^−/− MC38-tdTomato⁺ cells from tumors on day 5 upon radiation were shown from two independent experiments (n = 4–6 mice per groups). (H) Representative data and quantification of IL-15 expression in irradiated MC38 cells with or without anti-IFN-γ treatment at 100 µg/ml for 60 h are shown from two independent experiments. Data were represented as means ± SEM. Statistical analysis was performed by one-way ANOVA with multiple comparison tests (A–H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001; ns, no significant difference. Source data are available for this figure: SourceData F5.

Figure S5.

The measurement of JAK2/STAT1 activation in B16F1 cells and suppression of phosphor-JAK2/STAT1 expression by JAK inhibitor in MC38 cells. Related to Fig. 5. (A) The expression of p-JAK2 and -STAT1 expression in WT and Piezo2^−/− B16F1 cells with or without IR treatment was represented by three independent experiments. (B) The expression of p-JAK2 and -STAT1 in WT and Piezo2^−/− MC38 cells with or without JAK inhibitor following IR treatment was shown from three independent experiments. Data were represented as means ± SEM. Statistical analysis was performed by one-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01; ns, no significant difference. Source data are available for this figure: SourceData FS5.

Figure 6.

Correlation analysis between Piezo2 expression and radiosensitivity in the clinic. (A) The expression of Piezo2 in tumor samples from patients with rectum adenocarcinoma (READ) post-radiotherapy stratified by responders (n = 15) and non-responders (n = 20). Scale bar = 50 µm. The blue and red arrows show the expression of Piezo2 detected by immunohistochemistry in non-responder and responder, respectively. (B) The infiltration of CD8⁺ T cells in tumor samples from patients with READ after radiotherapy grouped by responders (n = 15) and non-responders (n = 18). Scale bar = 50 µm. The blue and red arrows show the expression of CD8 detected by immunohistochemistry in non-responder and responder, respectively. (C) The correlation between Piezo2 expression and CD8⁺ T cell infiltration in READ tumor tissue samples following radiotherapy (n = 28). (D and E) Normalized Piezo2 (D) and IL-15 (E) expression in PDAC tumor tissue samples collected after radiotherapy stratified by responders (n = 24) and non-responders (n = 7) in the public database. Data were represented as means ± SEM. Statistical analysis was performed by unpaired Student’s t test (A, B, and E). The correlation between Piezo2 and CD8 infiltration was analyzed by Linear regression of Correlation built in GraphPad Prism 8.0 (C). The comparisons of two nonparametric datasets were calculated by the Mann–Whitney U test (D). *P < 0.05.