Tumor microenvironment profoundly modifies functional status of macrophages: peritoneal and tumor-associated macrophages are two very different subpopulations

Abstract

Macrophages are key players in the inflammatory response. In this study, we tested the hypothesis that although all macrophage subpopulations in tumor hosts are affected by the disease, it is the close proximity to the tumor that induces major alterations in these cells. We compared tumor-associated macrophages (TAMs) with peritoneal macrophages from mice bearing D1-DMBA-3 mammary tumors (T-PEMs). Our results show that TAMs downregulate IL-12p70 but upregulate IL-12p40, IL-23, IL-6 and IL-10. Some NFκB and C/EBP transcription factors family members are decreased in TAMs; however NFκBp50 homodimers, STAT1/pSTAT1 and STAT3/pSTAT3 are overexpressed. Furthermore, while TAMs block T-cell proliferation and are more prone to apoptosis compared to T-PEMs, both types of macrophages have an impaired phagocytic capacity. Moreover, TAMs constitutively express iNOS and produce nitric oxide but do not express arginase and are Gr-1(high) and CD11b(low). Collectively, our analysis of two spatially distinct macrophage subpopulations in tumor-bearing mice revealed that the tumor modulates them differently into two molecularly and functionally dissimilar macrophage subpopulations.

Keywords: Immunosuppression; Inflammation; MDSC; NPEM; Peritoneal and tumor-associated macrophages; TAM; TPEM; Treg; Tumor microenvironment; myeloid derived suppressor cell; normal peritoneal elicited macrophage; regulatory T cell; tumor associated macrophage; tumor peritoneal elicited macrophage.

Fig. 1

TAMs do not express IL-12p70 yet they constitutively upregulate IL-12p40, IL-23, IL-6 and IL-10 cytokines. ELISAs of IL-12p70 (A) IL-12p40 (B) IL-23 (C) IL-6 (D) TNFα (E) and IL-10 (F) measured protein concentration in 24-h supernatants of N-PEMs, T-PEMs and TAMs cultured with and without LPS (10 μg/mL). Columns: mean of four different experiments with similar results; bars, SEM.

Fig. 2

TAMs and T-PEMs differently regulate the expression of IL-12p70 and IL-23. IL-12p35, IL-12p40 and IL-23p19 mRNA levels were determined by qRT-PCR using GAPDH for normalization in constitutive and LPS-activated individual samples of N-PEMs, T-PEMs and TAMs. Mean reflects results of individual animals in one of three different experiments with similar outcomes.

Fig. 3

TAMs and T-PEMs differently express proinflammatory transcription factors and show different susceptibility to apoptosis. Western blots experiments comparing N-PEMs, T-PEMs and TAMs in different conditions are presented. (A) Constitutive expression patterns of several transcription factors in the three macrophage subgroups. (B) Activated levels of caspase 3 in N-PEMs, T-PEMs and TAMs; first three lanes represent N-PEMs cultured with 0, 10, and 100 nmol, respectively, of staurosporine for 2 h; lane 4, untreated T-PEMs and lane 5, untreated TAMs cultured for the same amount of time. (C) Constitutive Bcl-x and p53 expression in N-PEMs, T-PEMs and TAMs. Figures represent one of three different experiments with similar results.

Fig. 4

TAMs exhibit a more immature phenotype than T-PEMs and do not express the classical MDSCs phenotype. (A) Flow cytometric analysis of CD11b, F4/80, CD68, CD115, and Gr-1 in resting N-PEMs, T-PEMs and TAMs (MFI values). (B) Western blot analysis shows constitutive expression of iNOS, arginase and MMP9 by N-PEMs, T-PEMs and TAMs. (C) iNOS activity was detected as nitrite concentration levels in 48-h supernatants of constitutive or LPS-stimulated N-PEMs, T-PEMs and TAMs, and (D) Arginase activity measured as urea concentration levels in 48-h supernatants of constitutive and LPS-activated N-PEMs, T-PEMs and TAMs. Figures represent one of three different experiments with similar results. MFI, Mean fluorescence intensity.

Fig. 5

TAMs and T-PEMs differ in their regulation of T cell proliferation and their phagocytosis capabilities. (A) Analysis of ex vivo proliferation of αCD3/αCD28/IL-2-activated T splenocytes from normal mice in the absence or the presence of N-PEMs, T-PEMs and TAMs; columns represent mean of four different experiments with similar results. (B) Analysis of the ability of N-PEMs, T-PEMs and TAMs to internalize fluorescent Zymosan particles using fluorescence microscopy, with and without pre-treatment with phagocytosis-interfering Cytochalasin-D; columns show mean of four different experiments with similar results.

Fig. 6

TAMs coexist with CD3⁺ T cells, Tregs and IL-17-producing cells in the mammary tumor microenvironment. (A) Histologies (H&E) and IHC results showing F4/80⁺ macrophages and CD3⁺ lymphocytes colonizing D1-DMBA3 tumors with increasing degrees of progression. (B) One representative experiment showing flow cytometry analysis of CD4⁺CD25⁺Foxp3⁺ Tregs in spleens from normal mice and spleens and tumors from tumor-bearing mice. (C) Histogram showing percentages of Tregs in the three locations: data for the bar graphs was obtained from different experiments where a total of 18 normal and 18 tumor-bearing mice were used; NS (normal spleen), TS (tumor spleen), T (tumor). (D–E) Histograms corresponding to flow cytometry analysis (not shown) demonstrating presence of IL-17-producing cells (6D: percentages and 6E: MFI) in spleens from normal mice and spleens and tumors from tumor hosts (same numbers of mice as in C were used).