Macrophage depletion reduces postsurgical tumor recurrence and metastatic growth in a spontaneous murine model of melanoma

Abstract

Surgical resection of tumors is often followed by regrowth at the primary site and metastases may emerge rapidly following removal of the primary tumor. Macrophages are important drivers of tumor growth, and here we investigated their involvement in postoperative relapse as well as explore macrophage depletion as an adjuvant to surgical resection. RETAAD mice develop spontaneous metastatic melanoma that begins in the eye. Removal of the eyes as early as 1 week of age did not prevent the development of metastases; rather, surgery led to increased proliferation of tumor cells locally and in distant metastases. Surgery-induced increase in tumor cell proliferation correlated with increased macrophage density within the tumor. Moreover, macrophages stimulate tumor sphere formation from tumor cells of post-surgical but not control mice. Macrophage depletion with a diet containing the CSF-1R specific kinase inhibitor Ki20227 following surgery significantly reduced postoperative tumor recurrence and abrogated enhanced metastatic outgrowth. Our results confirm that tumor cells disseminate early, and show that macrophages contribute both to post-surgical tumor relapse and growth of metastases, likely through stimulating a population of tumor-initiating cells. Thus macrophage depletion warrants exploration as an adjuvant to surgical resection.

Keywords: CSF1R inhibition; macrophages; postsurgical relapse; tumor-initiating cell.

Figure 1. Surgical resection enhances primary tumor regrowth

(A) IHC labeling of eye sections from non-tumor-bearing (ret^−/−) and tumor-bearing (Ret^+/−) mice, with S100B (red) and Ki67 (blue). At the time of surgery (3wk after birth) Ret^+/− eyes show signs of hyperplasia (arrow). 4 weeks after surgery (VE), tumor regrowth has occurred and tumors were comparable to those in orbits of control mice. (B) Quantification of the number of tumor nodules relapsing within the orbit of the eye 4 and 6wk after surgery. Each point represent one mouse; 1-way ANOVA, N.S. not significant (n = 12–14 mice). (C) The percentage of Ki67⁺ cells in the relapsed tumors 4 and 6wks after surgery. Each point represents one tumor nodule; 1-way ANOVA, *P < 0.05 (n = 4–6 mice). (D) Quantification of the area of the relapsed tumor nodules 4 and 6wk after surgery. Each point represents one tumor nodule; 1-way ANOVA, **P < 0.01 (n = 3–5 mice).

Figure 2. Surgical resection enhances the growth of metastases

(A) Number of metastases present in mice 4 and 6wk after surgery. Each point represents one mouse; 1-way ANOVA, N.S. not significant (n = 8–10 mice). (B) The percentage of Ki67⁺ tumor cells present in the metastases 4 and 6wks after surgery. Each point represents one tumor nodule; 1-way ANOVA, *P < 0.05 (n = 3–5 mice). (C) Quantification of the area of the metastases 4 and 6wk after surgery. Each point represents one tumor nodule; 1-way ANOVA, *P < 0.05 (n = 3–7 mice).

Figure 3. Surgery increases macrophage infiltration which correlates with tumor growth

(A) Representative immunofluorescence image of tumors from control and VE animals labeled with antibodies against CD45 (green) and F4/80 (red). (B) Density of F4/80⁺ TAMs in relapsed eye tumors and metastases at 4 and 6wkPO. Each point represents one tumor nodule; 1-way ANOVA, ****P < 0.0001 (n = 3–7). (C) Graph showing the correlation (Pearson) of TAM numbers with tumor area at 4wkPO and 6wkPO at the primary site. (D) Graph showing the correlation (Pearson) of TAM density and percentage of Ki67⁺ cells within the tumors at 4wkPO at the primary site.

Figure 4. Surgery accelerates the emergence of macrophage-responsive tumor-initiating cells

(A) Graph showing the fold-change in percentage of cells forming tumor spheres in the presence of TAMs, relative to without TAMs. Tumor cells from young mice (age 10–15 weeks) that underwent VE followed by recovery for 4–6wks were stimulated with TAMs derived from non-VE old mice (> 30wk). Bars represent mean ± SE; 2-way ANOVA, ***P < 0.001, ****P < 0.0001 (n = 3–4 mice). (B) Density of F4/80⁺ TAMs in primary relapsed tumors from mice that underwent VE followed by 4wks of recovery on a normal (4wkPO) or Ki20227-supplemented diet (4wkPO + Ki), and non-VE age-matched control mice. Each point represents one tumor nodule; 1-way ANOVA, ****P < 0.0001 (n = 3–7 mice). (C) Graph showing the fold-change in percentage of cells forming tumor spheres in the presence of TAMs, relative to without TAMs. Tumor cells from VE mice on normal (VE) or Ki20227 diet (VE+Ki) were stimulated with TAMs derived from non-VE old mice. Bars represent mean ± SE; 2-way ANOVA, ****P < 0.0001, N.S. not significant (n = 3–4 mice). (D) Graph showing the gene expression level of Sms (left panel) and APAO (right panel) in CD34⁻ tumor cells derived from VE mice on normal (VE) or Ki20227 diet (VE+Ki) and non-VE age-matched control mice. Each point represents one mouse; 1-way ANOVA, *P < 0.05 (n = 6–7 mice).

Figure 5. Macrophage depletion reduces tumor growth after surgery

(A) Percentage of Ki67⁺ cells in relapsed eye tumors from VE mice on normal (VE) or Ki20227 diet (VE+Ki) compared with non-VE age-matched control mice. Each point represents one tumor nodule; 1-way ANOVA, *P < 0.05 (n = 4–6 mice). (B) Area of relapsed eye tumors from VE mice on normal (VE) or Ki20227 diet (VE+Ki) compared with non-VE age-matched control mice. Each point represents one tumor nodule; 1-way ANOVA, **P < 0.01 (n = 3–5 mice). (C) Percentage of Ki67⁺ cells in metastases from VE mice on normal (VE) or Ki20227 diet (VE+Ki) compared with non-VE age-matched control mice. Each point represents one tumor nodule; 1-way ANOVA, *P < 0.05 (n = 3–5 mice). (D) Area of metastases from VE mice on normal (VE) or Ki20227 diet (VE+Ki) compared with non-surgical age-matched control mice. Each point represents one tumor nodule; 1-way ANOVA, *P < 0.05, **P < 0.01 (n = 3–7 mice).