Cross-talk between tumors can affect responses to therapy

Abstract

Advanced stages of cancer often involve multiple tumors in different locations in the body. These tumors are associated with a microenvironment that can influence tumor responses to immunotherapy. Whether tumors and their disparate microenvironment can interact together at distance in a multiple tumor setting, through a form of cross-talk, and affect their responses to immunotherapy has never been described. Our study investigated the cross-talk between two tumors with disparate microenvironments in a mouse model. We demonstrated that immunosuppressive visceral tumors could influence distant subcutaneous (SC) tumors to render them resistant to immunotherapy. We observed distinct modifications in the SC tumor microenvironment following cross-talk with kidney tumors that exhibit a type-2 macrophage-related immunosuppressive microenvironment. Indeed, when a concomitant kidney tumor was present in the mouse, the SC tumors were highly infiltrated with M2 macrophages and had a reduced T cell and NK cell effector immune profile. Finally, blocking the M2-associated chemokine CCL2 or depleting macrophages, significantly improved the effect of immunotherapy on growth of SC tumors in the presence of concomitant kidney tumors. This work emphasizes the potential negative influence that a tumor, with a strong immunosuppressive microenvironment, can exert on distant tumors that would normally be treatment-responsive. This report may lead to a new vision of the prioritization in the treatment of advanced metastatic cancer.

Keywords: C-C motif ligand-2; CD; CCL2; CD137; CD40; DR5; T cell receptor.; clodronate; cluster of differentiation; IFNγ; immunosuppression; immunotherapy; interferon gamma; IH; interleukin; M2; intrahepatic; IK; intrakidney; IL; natural killer; SC; subcutaneous; TCR; tumor immunology; tumor microenvironment; type 2 macrophages; NK; type-2 macrophages.

Figure 1.

The presence of IK tumors can modify the response to Tri-mAb of distant SC tumors. (A) SC tumor growth and (B) survival of mice injected with Renca tumor cells in one location subcutaneously (SC) or intrakidney (IK) or in two locations simultaneously SC and SC (SC + SC) or SC and IK (SC + IK) and treated with Tri-mAb or PBS (Ctl). (A) The SC tumor growth averages are represented ±SEM, tumors were measured using a caliper. Statistics were calculated between SC Tri-mAb and SC + IK Tri-mAb, * p < 0.05, ** p < 0.005. (n = 6–9 mice per group, 1 representative experiment of 2 is shown), (B) * p < 0.05, ** p < 0.005. (n = 12–19 mice per group, two experiments pooled) (C) Survival of mice injected with two tumors SC and IK and treated with Tri-mAb or PBS (Ctl). Seven days after tumor inoculation some mice bearing kidney tumors were resected (res.) or not (non res.) (n = 7 mice per group) * p < 0.05 Fisher's Exact Test. (D) SC tumors growth in mice injected with Renca tumor cells in two locations: SC + SC or SC and intrahepatic (IH) (SC + IH) and treated with Tri-mAb or PBS. Bar-graph represents the average ±SEM of the SC tumor growth from the two groups that have been treated with Tri-mAb. n = 5–6 mice per group, * p < 0.05, *** p < 0.0005.

Figure 2.

Serum-soluble factors are unlikely responsible for the cross-talk between IK and SC tumors. (A) Cytometric bead array analysis of serum from mice injected with one tumor SC and two tumors SC + IK. Serum was harvested at day 12 after tumor injection. (n = 6-7 per group). (B) SC tumor growth and (C) survival of mice injected SC with Renca cells and treated with three doses of Tri-mAb alone (Tri-mAb), PBS or Tri-mAb in combination with serum (serum + Tri-mAb). In the serum transferred group, each mouse received five injections of 300 μL of serum intravenously (IV), harvested from a pool of 33 IK-tumor-bearing mice at day 13. (n = 6-8 mice per group).

Figure 3.

The potential cross-talk between SC and IK tumors downregulates the antitumor immune response and increases the M2 suppressive response in SC tumors. (A–D) To simplify the main finding from Table 1, gene expression determined, using RNA sequencing of various immune cells: (A) NK cells, (B) B cells, (C) T cells, and (D) type-2 macrophages (M2), was represented as histograms. Gene expression (A–C) downregulation or (D) upregulation is represented in fold change of expression in SC tumor when a concomitant IK tumor was present in the mice. (n = 6 mice per group, one experiment). Proteins corresponding to gene names are represented in Table 1. (E) Representative flow cytometry dot plots and (F) quantitative data of total macrophages representing F4/80^int/CD11b^hi and F4/80^hi/CD11^int populations that infiltrate some SC tumors when mice injected with two SC tumors (SC + SC) or one SC and one IK tumors (SC + IK). Tumors were taken for analysis at day 14 after inoculation. (n = 4 mice per group, one representative experiment of 3). *** p < 0.0005.

Figure 4.

A lower percentage of NK and T cells infiltrate SC tumors when a concomitant IK tumor is present. (A and B) Representative flow cytometry dot plots and (C and D) quantitative data for the percentage of (A, C) NK cells and (B, D) CD4⁺ and CD8⁺ T cells of total leukocytes that infiltrate SC tumors from mice injected with two SC tumors (SC + SC) or one SC and one IK tumors (SC + IK). (C) n = 12 mice per group, three experiments pooled. (D) n = 11 mice per group, three experiments pooled. * p < 0.05, ** p < 0.005, **** p < 0.0001.

Figure 5.

A dampened NK and T cell effector immune response occurs in SC tumors when a concomitant IK tumor is present. (A–C) Representative flow cytometry dot plots and histograms (D–F), quantitative data for (A and D) interferon-γ (IFNγ), (B and E) granzyme B (GzmB) and (C and F) CD69 in NK cells, CD4⁺ and CD8⁺ T cells as depicted. (A) Representative FACS data from one experiment n = 4 mice and (B, C) from three experiments, n = 11–12 mice. (A, B) the rectangular gate represents the isotype control. (C) Black line represents anti-CD69 antibody and gray line represents isotype control antibody. (D–F) percentages (%) of positive cells were calculated of total leukocytes (CD45.2⁺ cells). Graphs (D) are from one experiment (n = 4), (E) left panel and (F) are from three experiments pooled (n = 11–12 mice) and (E right panel) is from one representative experiment of two (n = 4) * p < 0.05, ** p < 0.005

Figure 6.

CCL2 is involved in the resistance of SC tumors to treatment when a concomitant IK tumor is present. (A and C) SC tumor growth and (B and D) survival of mice injected with Renca cells SC + IK and treated with Tri-mAb alone (Tri-mAb) or Tri-mAb and anti-CCL2 antibody (A, B) systemically with 200 μL (0.2 mg total per dose) intraperitoneally or (C, D) systemically with 170 μL and locally (intra-tumor) with 30 μL (0.3 mg total per dose). (A, B) n = 15–16 mice/group, two experiments pooled, (C, D) n = 7–9 mice/group, one experiment (A) * p < 0.05, **** p < 0.0001 at day 24 and day 27. (C) ** p < 0.005 at day 19 and *** p < 0.0005 at day 21. (D) * p < 0.05. (E) SC tumor growth and (F) survival of mice injected with Renca cells SC + IK and treated with either PBS (Ctl), Clodrolip (Clodro), Tri-mAb alone (Tri-mAb) or Clodrolip and Tri-mAb (Clodro + TrimAb). n = 5–8 mice/group, one experiment. * p < 0.05, **** p < 0.0001. The average of SC tumor growth is represented (±SEM).