Macrophage subtypes inhibit breast cancer proliferation in culture

Abstract

Macrophages are a highly plastic cell type that adopt distinct subtypes and functional states depending on environmental cues. These functional states can vary wildly, with distinct macrophages capable of displaying opposing functions. We sought to understand how macrophage subtypes that exist on two ends of a spectrum influence the function of other cells. We used a co-culture system with primary human macrophages to probe the effects of macrophage subtypes on breast cancer cell proliferation. Our studies revealed a surprising phenotype in which both macrophage subtypes inhibited cancer cell proliferation compared to cancer cells alone. Of particular interest, using two different proliferation assays with two different breast cancer cell lines, we showed that differentiating macrophages into a "pro-tumor" subtype inhibited breast cancer cell proliferation. These findings are inconsistent with the prevailing interpretation that "pro-tumor" macrophages promote cancer cell proliferation and suggest a re-evaluation of how these interpretations are made.

Figure 1.. MDA-MB-231 breast cancer cells do not exhibit increased proliferation when in culture with M2-like macrophages.

(A) Schematic outlining co-culture approaches. (B) Example gating of Ki67 cell cycle index flow cytometry assays. (C-F) Quantification of MDA-MB-231 cells in each stage of the cell cycle at 48 (C) and 72 (E) hours, following gating strategies outlined in (B) when MDA-MB-231 cells are plated alone (“231 mono”), when MDA-MB-231 cells are plated with M1-like macrophages (“231 + M1 MΦ”) and when MDA-MB-231 cells are plated with M2-like macrophages (“231 + M2 MΦ”) (n=4 independent donors). (D, F) Comparison of only the G2/M phase of the cell cycle from data in (C) at 48 hours and data in (E) at 72 hours (n=4; Tukey’s 1-way ANOVA). (G) Representative example of MDA-MB-231 cell confluence over 72 hours when MDA-MB-231 cells are plated under conditions described in (C), plus MDA-MB-231 cells plated alone at double density (“231 double density”). (H) Quantification of MDA-MB-231 cell confluence at 72 hours in (G), reported as the fold increase in cell area over time (n=4 independent donors; Tukey’s 1-way ANOVA).

Figure 2.. M2-like macrophages maintain differentiation state over the experimental time course and do not induce MDA-MB-231 cell death.

(A-C) Quantification of CD206+ macrophages across culture conditions. Quantification of the percent of CD206+ macrophages measured by surface CD206 expression at 24 hours (A), 48 hours (B), and 72 hours (C) of culture when M1-like macrophages are plated alone (“M1 MΦ”), when M1-like macrophages are plated with MDA-MB-231 cells (“231 + M1 MΦ”), when M2-like macrophages are plated alone (“M2 MΦ”), and when M2-like macrophages are plated with MDA-MB-231 cells (“231 + M2 MΦ”) (n=4 independent donors; Tukey’s 1-way ANOVA). (D-F) Quantification of Annexin V positive MDA-MB-231 cells at 24 hours (D), 48 hours (E), and 72 hours (F) when MDA-MB-231 cells are plated as described in (A-C). The positive control is heat-killed cells (see methods) (n=3 independent donors; Tukey’s 1-way ANOVA). Analysis was performed on the same day; thus, the same positive control was used for all time points.

Figure 3.. MDA-MB-468 cells do not show increased proliferation when in culture with M2-like macrophages.

(A-D) Quantification of MDA-MB-468 cells in each stage of the cell cycle at 48 (A) and 72 (C) hours when MDA-MB-468 cells are plated alone (“468 mono”), when MDA-MB-468 cells are plated with M1-like macrophages (“468 + M1 MΦ”) and when MDA-MB-468 cells are plated with M2-like macrophages (“468 + M2 MΦ”) (n=4 independent donors). (B, D) Comparison of only the G2/M phase of the cell cycle from data in (A) at 48 hours and data in (C) at 72 hours (n=4; Tukey’s 1-way ANOVA). (E) Representative example of MDA-MB-468 cell confluence over 72 hours when MDA-MB-468 cells are plated as in (A), plus MDA-MB-468 cells are plated alone at double density (“468 double density). (F) Quantification of MDA-MB-468 cell confluence at 72 hours in (E), reported as the fold increase in cell area over time (n=4 independent donors; Tukey’s 1-way ANOVA).

Figure 4.. Conditioned media from M2-like macrophages increases MDA-MB-468 proliferation compared to conditioned media from M1-like macrophages.

(A) Representative example of proliferation of MDA-MB-231 cell confluence over 72 hours when naïve MDA-MB-231 cells are treated with M2-like macrophage conditioned media (“M2 mono CM”), when naive MDA-MB-231 cells are treated with MDA-MB-231/M2-like macrophage co-culture conditioned media (“M2 + 231 CM”), when naïve MDA-MB-231 cells are treated with M1-like macrophage conditioned media (M1 mono CM), and when naïve MDA-MB-231 cells are treated with MDA-MB-231/M1-like macrophage co-culture conditioned media (“M1 + 231 CM”). (B) Quantification of MDA-MB-231 cell confluence at 48 hours when treated with M1-like or M2-like macrophage monoculture CM in (A) (n=4 independent donors; Mann-Whitney test). (C) Quantification of MDA-MB-231 cell confluence at 72 hours when treated with MDA-MB-231/M1-like or MDA-MB-231/M2-like macrophage co-culture conditioned media in (A) (n=5 independent donors; Mann-Whitney test). (D) Representative example of proliferation of MDA-MB-468 cell numbers over 72 hours when naïve MDA-MB-468 cells are treated as described in (A). (E) Quantification of MDA-MB-468 cell confluence at 48 hours when treated with M1-like or M2-like macrophage monoculture CM in (D) (n=5 independent donors; Mann-Whitney test). (F) Quantification of MDA-MB-468 cell confluence at 72 hours when treated with MDA-MB-468/M1-like or MDA-MB-468/M2-like macrophage co-culture conditioned media in (D) (n=5 independent donors; Mann-Whitney test).

Figure 5.. Conditioned media from M2-like macrophages inhibit breast cancer cell proliferation compared to cancer cells alone.

(A) Representative example of proliferation of MDA-MB-231 cell numbers over 72 hours when naïve MDA-MB-231 cells are treated with MDA-MB-231 monoculture conditioned media (“231 mono”), when MDA-MB-231 cells are in direct co-culture with M1-like macrophages (“231 + M1 MΦ”), when naïve MDA-MB-231 cells are treated with MDA-MB-231/M1-like macrophage co-culture conditioned media (“M1 mono CM”), and when naïve MDA-MB-231 cells are treated with MDA-MB-231/M1-like macrophage co-culture conditioned media (“M1 + 231 CM”). (B) Quantification of MDA-MB-231 cell confluence at 72 hours when plated in conditions listed in (A) (n=4 independent donors; Tukey’s 1-way ANOVA). (C) Representative example of proliferation of MDA-MB-231 cell numbers over 72 hours when naïve MDA-MB-231 cells are treated with conditions described in (A), but with M2 macrophages. (D) Quantification of MDA-MB-231 cell confluence at 72 hours when plated in conditions listed in (C) (n=4 independent donors; Tukey’s 1-way ANOVA). (E) Representative example of proliferation of MDA-MB-468 cell numbers over 72 hours when naïve MDA-MB-468 cells are treated with conditions described in (A). (F) Quantification of MDA-MB-468 cell confluence at 72 hours when plated in conditions listed in (E) (n=5 independent donors; Tukey’s 1-way ANOVA). (G) Representative example of proliferation of MDA-MB-468 cell numbers over 72 hours when naïve MDA-MB-468 cells are treated with conditions described in (B). (H) Quantification of MDA-MB-468 cell confluence at 72 hours when plated in conditions listed in (G) (n=5 independent donors; Tukey’s 1-way ANOVA).