Hedgehog signaling promotes tumor-associated macrophage polarization to suppress intratumoral CD8+ T cell recruitment

Abstract

Tumor-associated macrophages (TAMs) usually display an antiinflammatory M2-like phenotype to facilitate tumor growth. However, what drives M2 polarization of TAMs and how TAMs suppress antitumor immunity within the tumor microenvironment (TME) remain largely undefined. Using several murine tumor models, we showed that hedgehog (Hh) signaling in myeloid cells is critical for TAM M2 polarization and tumor growth. We also found that tumor cells secrete sonic hedgehog (SHH), an Hh ligand, and that tumor-derived SHH drives TAM M2 polarization. Furthermore, Hh-induced functional polarization in TAMs suppresses CD8+ T cell recruitment to the TME through the inhibition of CXCL9 and CXCL10 production by TAMs. Last, we demonstrated that Krüppel-like factor 4 (Klf4) mediates Hh-dependent TAM M2 polarization and the immunosuppressive function. Collectively, these findings highlight a critical role for tumor-derived SHH in promoting TAM M2 polarization, a mechanism for TAM-mediated immunosuppression, and may provide insights into the design of new cancer immunotherapeutic strategies.

Keywords: Cancer; Cancer immunotherapy; Immunology; Macrophages.

Figure 1. Loss of Smo in myeloid cells interferes with tumor growth and M2 polarization of TAMs in vivo.

(A) Tumor growth of Hepa1-6 mouse hepatoma cells inoculated s.c. in Smo^fl/fl and Smo^ΔM mice. Tumor volumes on day 15 at sacrifice are shown. (B) Arg1, Mrc1, Il10, and Tnf mRNA levels in Smo^fl/fl and Smo^ΔM TAMs were measured by qRT-PCR. Expression of mRNAs was normalized to Actb and compared with that of Smo^fl/fl. (C) Surface CD206 expression and intracellular Arg-1 production in Smo^fl/fl and Smo^ΔM TAMs by FACS. (D) TNF-α and IL-10 secretion from Smo^fl/fl and Smo^ΔM TAMs was measured by ELISA. Values are mean ± SEM of a minimum of 3 independent experiments. **P < 0.005; ***P < 0.0005. n = 15 mice per group (A); n = 5 biological replicates per group (B–D). Wilcoxon-Mann-Whitney U test (A); 2-tailed Student’s t test (B–D).

Figure 2. Loss of Hh signaling in myeloid cells suppresses M2 polarization of TAMs and promotes survival in autochthonous HCC.

(A) Representative gross image of Smo^fl/flMdr2^–/– and Smo^ΔMMdr2^–/– mice at 12 months after birth. Tumor number and tumor area were quantified by counting and measuring of neoplastic nodules per H&E-stained liver sample. (B) Kaplan-Meier survival curve for the 2 mouse cohorts, Smo^fl/flMdr2^–/– and Smo^ΔMMdr2^–/–. The median survival times were 11.9 months for the Smo^fl/flMdr2^–/– cohort (black line) and 20 months for the Smo^ΔMMdr2^–/– cohort (red line). (C–E) Expression of Arg-1, CD206 (Mrc1), IL-10, and TNF-α in Smo^fl/flMdr2^–/– and Smo^ΔMMdr2^–/– TAMs was measured by qRT-PCR (C), FACS (D), and ELISA (E). Expression of mRNAs was normalized to Actb and compared with that of Smo^fl/flMdr2^–/– TAMs. Values are the mean ± SEM of a minimum of 3 independent experiments. *P < 0.05; **P < 0.005; ***P < 0.0005. n = 8 biological replicates per group (A); n = 15 mice per group (B); n = 5 biological replicates per group (C–E). Two-tailed Student’s t test (A and C–E); log-rank test (B).

Figure 3. SHH is sufficient to induce M2 polarization of macrophages in vitro.

(A) Shh, Ihh, and Dhh mRNA levels in Hepa1-6 hepatoma cells were measured by qRT-PCR. Expression was normalized to reference gene Actb. Concentration of SHH ligands in Hepa1-6 supernatants and control DMEM was assayed using ELISA. (B–D) Macrophages (Mφ) from C57BL/6 mice were treated with 100 pg/mL SHH or 100 pg/mL IL-4 for 3 days or left untreated (control). Expression of Arg-1, CD206 (Mrc1), IL-10, and TNF-α in control macrophages and SHH- or IL-4–treated macrophages was measured by qRT-PCR (B), FACS (C), and ELISA (D). Expression of mRNAs was normalized to Actb and compared with that of control macrophages (B). Values are the mean ± SEM of a minimum of 2 independent experiments. **P < 0.005; ***P < 0.0005. n = 3 technical replicates per group (A); n = 5 biological replicates per group (B–D). One-way ANOVA (B–D).

Figure 4. Tumor-derived SHH promotes TAM M2 polarization in vivo.

(A) Tumor growth of Hepa1-6 Shh-WT and Hepa1-6 Shh-KO cells inoculated s.c. in C57BL/6 mice. (B–D) Expression of Arg-1, CD206 (Mrc1), IL-10, and TNF-α in TAMs isolated from Hepa1-6 Shh-WT or Hepa1-6 Shh-KO tumors was measured by qRT-PCR (B), FACS (C), and ELISA (D). Expression of mRNAs was normalized to Actb and compared with that of Hepa1-6 Shh-WT TAMs (B). (E) Tumor growth of LLC1 Shh-WT and LLC1 Shh-KO cells inoculated s.c. in C57BL/6 mice. (F–H) Expression of Arg-1, CD206 (Mrc1), IL-10, and TNF-α in TAMs isolated from LLC1 Shh-WT or Shh-KO tumors was measured by qRT-PCR (F), FACS (G), and ELISA (H). Expression of mRNAs was normalized to Actb and compared with that of LLC1 Shh-WT TAMs (F). Values are the mean ± SEM of a minimum of 3 independent experiments. *P < 0.05; **P < 0.005; ***P < 0.0005. n = 5 mice per group (A–H). Two-tailed Student’s t test (B–D and F–H); Wilcoxon-Mann-Whitney U test (A and E).

Figure 5. Loss of Hh signaling in TAMs promotes CD8⁺ T cell infiltration via CXCL9 and CXCL10.

(A and B) Quantification of tumor-infiltrating CD8⁺ T cells by immunofluorescent staining in Hepa1-6 cells implanted s.c. into Smo^fl/fl and Smo^ΔM mice (A) and in the autochthonous HCC model (B). (C) Chemotaxis of CD8⁺ T cells toward macrophages treated with IFN-γ (control) and IFN-γ plus SHH. (D) Ccl3, Ccl4, Ccl5, Cxcl9, and Cxcl10 mRNA levels in macrophages treated with IFN-γ or with IFN-γ plus SHH were measured by qRT-PCR. Expression was normalized to Actb and compared with untreated. (E) Chemotaxis of CD8⁺ T cells toward macrophages treated with IFN-γ alone, IFN-γ plus CXCL9 and/or CXCL10-neutralizing antibodies, and IFN-γ plus SHH. (F and G) Expression of Cxcl9 and Cxcl10 in Smo^fl/fl or Smo^ΔM TAMs was measured by qRT-PCR (F) and ELISA (G). Expression of mRNAs was normalized to Actb and compared with that of Smo^fl/fl TAMs (F). (H) Tumor growth of Hepa1-6 in Smo^fl/fl and Smo^ΔM mice injected with CXCR3-blocking antibody or isotype control. (I) Percentage of tumor CD8⁺ T cell infiltration quantified by FACS. (J) Frozen tissue sections were stained for CD8⁺ T cells and quantified under high-power field (hpf). Values are the mean ± SEM of a minimum of 3 independent experiments. **P < 0.005; ***P < 0.0005. n = 8 biological replicates per group (A and B); n = 5 technical replicates per group (C and E); n = 5 biological replicates per group (D, F–J). Two-tailed Student’s t test (A–D, F, and G); 1-way ANOVA (E); Kruskal-Wallis test (H); 2-way ANOVA (I and J). α, anti.

Figure 6. Hh-induced M2 TAM polarization and function are mediated by Klf4.

(A) Klf2, Klf4, Stat6, Pparg, and Cebpb mRNA levels in control and SHH-treated macrophages were measured by qRT-PCR. Expression was normalized to Actb and compared with control. (B) Klf4 mRNA levels in Smo^fl/fl and Smo^ΔM TAMs were measured by qRT-PCR. Expression was normalized to reference gene Actb and compared with that of Smo^fl/fl. (C) Gli1 transcription factor binds to the Klf4 promoter region as demonstrated by ChIP. Gli1 activity was inhibited using 5 μM GANT61 or constitutively activated using Smo^CM macrophages. (D) Tumor volumes of Hepa1-6 hepatoma cells inoculated s.c. in Klf4^fl/fl and Klf4^ΔM mice on day 18 at sacrifice. (E) Expression of Arg1, Mrc1, Il10, and Tnf mRNAs in Klf4^fl/fl and Klf4^ΔM TAMs was quantified by qRT-PCR. Expression was normalized to reference gene Actb and compared with that of Klf4^fl/fl TAMs. (F) Percentages of tumor CD8⁺ T cell infiltration into tumors from Klf4^fl/fl and Klf4^ΔM mice. (G) CXCL9 and CXCL10 production by Smo^CM and Smo^CMKlf4^ΔM TAMs was measured by ELISA. (H) Tumor volumes of Hepa1-6 hepatoma cells inoculated s.c. in Smo^CM and Smo^CMKlf4^ΔM mice on day 18 at sacrifice. (I) Expression of Arg1, Mrc1, Il10, and Tnf mRNAs in Smo^CM and Smo^CMKlf4^ΔM TAMs was quantified by qRT-PCR. Expression was normalized to reference gene Actb and compared with that of Smo^CM TAMs. (J) Percentages of tumor CD8⁺ T cell infiltration in tumors from Smo^CM and Smo^CMKlf4^ΔM mice. Values are the mean ± SEM of a minimum of 3 independent experiments. *P < 0.05; **P < 0.005; ***P < 0.0005. n = 5 biological replicates per group (A and B); n = 3 technical replicates per group (C); n = 6 biological replicates per group (D–J). Two-tailed Student’s t test (A, B, D, and J).

Figure 7. Hh inhibition and immune checkpoint blockade have synergistic antitumorigenic effects.

(A) Tumor-bearing mice inoculated with 1 × 10⁶ Hepa1-6 cells were treated with vehicle only (DMSO), vismodegib (VMD) (2 mg/mouse), anti–PD-1 antibody (200 μg/mouse), and a combination of VMD and anti–PD-1 antibody 3 times weekly for 3 weeks. (B) Tumor-bearing mice inoculated with 0.5 × 10⁶ LLC1 cells were treated with vehicle only (DMSO), VMD (2 mg/mouse), anti–PD-1 antibody (200 μg/mouse), and a combination of VMD and anti–PD-1 antibody 3 times weekly for 3 weeks. **P < 0.005; ***P < 0.0005. n = 5 biological replicates per group (A and B). Kruskal-Wallis test (A and B).