Canine mammary cancer cells direct macrophages toward an intermediate activation state between M1/M2

Abstract

Background: Canine mammary carcinoma is the most common cancer in female dogs and is often fatal due to the development of distance metastasis. The microenvironment of a tumour often contains abundant infiltrates of macrophages called tumour-associated macrophages (TAMs). TAMs express an activated phenotype, termed M2, which sustains proliferation of cancer cells, and has been correlated with poor clinical outcomes in human cancer patients. Cancer cells themselves have been implicated in stimulating the conversion of macrophages to a TAM with an M2 phenotype. This process has yet to be fully elucidated. Here we investigate the interplay between cancer cells and macrophages in the context of canine mammary carcinoma.

Results: We show that cancer cells inhibit lipopolysaccharide (LPS)-induced macrophage activation. Further, we show that macrophage associated proteins, colony-stimulating factor (CSF)-1 and C-C motif ligand (CCL)-2, stimulate macrophages and are responsible for the effects of cancer cells on macrophages. We suggest the existence of a feedback loop between macrophages and cancer cells; while cancer cells influence the phenotype of the TAMs through CSF-1 and CCL2, the macrophages induce canine mammary cancer cells to upregulate their own expression of the receptors for CSF-1 and CCL2 and increase the cancer cellular metabolic activity. However, these cytokines in isolation induce a phenotypic state in macrophages that is between M1 and M2 phenotypes.

Conclusions: Overall, our results demonstrate the extent to which canine mammary carcinoma cells influence the macrophage phenotype and the relevance of a feedback loop between these cells, involving CSF-1 and CCL2 as important mediators.

Fig. 1

Cancer cells inhibit LPS induced activation of macrophages. Macrophages (RAW) were co-cultured with either canine mammary carcinoma (REM), Human mammary cancer cells (MCF7) or non-cancerous cells (HEK293T), stimulated with 1 μg/ml LPS for 48 h. LPS-induced activation of macrophages was measured by changes in granularity (a (i)), percentage of MHC II+ cells (a (ii)) or mean fluorescence intensity of MHC II (a (iii)), and expression of the M2 marker, CD301 (b). Macrophages were distinguished from cancer cells in flow cytometry by CSFE staining. All experiments are replicates of 4. Asterisks indicate statistically significant differences in relation to LPS-treated cells, where P < 0.05 (unless where indicated) by Kruskal-Wallis test; “ns” indicates non-significant differences

Fig. 2

M1/M2 polarization of macrophages involves CCR2 signalling. RAW cells were pre-treated for 1 h with 10 μM RS102895 hydrochloride, a small molecule inhibitor of CCR2 (CCR2i) prior to stimulation with 1 μg/ml LPS for 48 h. An equivalent volume of DMSO was used as a vehicle control. LPS-induced activation of macrophages was measured by changes in the percentage of high granularity cells (a (i)) before (a (ii)) and after LPS-treatment (a (iii)), expression of MHC II before (b (i)) or after LPS stimulation (b (ii)), and expression of the M2 marker, CD301 (c). All experiments are replicates of 3. Asterisks indicate statistically significant differences in relation to the CCR2i treated cells, where P < 0.05 by Kruskal-Wallis (Figure A) or ANOVA (Figures B and C); “ns” indicates non-significant differences

Fig. 3

Cancer cell effects on macrophages are CSF-1 dependent. Canine recombinant CSF-1R binds to CSF-1 as detected by ELISA. BSA was used as a control protein for coating the plate (a). REM134 cells, RAW cells and soluble canine recombinant CSF1R (1 μg/ml every 24 h) were co-cultured for 3 days prior to LPS-induced activation of macrophages. Activation of macrophages was measured by granularity (b) and expression of MHC II (c). All experiments are replicates of 3. Asterisks indicate statistically significant differences in relation to the LPS treated cells, where P < 0.05 by ANOVA; “ns” indicates non-significant differences

Fig. 4

CSF-1 and CCL2 contribute to cancer cell proliferation and activity. Macrophages (a (i)) and REM134 (a (ii)) cells were incubated with increasing doses of CSF-1. Cellular proliferation was measured after 48 h. PBS + 0.1 % BSA was used a vehicle control at 0 ng/ml. REM134 cells were incubated with increasing concentrations of CCL2 (a (iii)) or conditioned media (CM) from CCL2 treated macrophages (a (iv)). Cellular proliferation was measured after 48 h. PBS + 0.1 % BSA was used a vehicle control at 0 ng/ml. Experiments are replicates of 6. Statistical analysis considered each group against the control group. Expression of CSF-1R was assayed in Lilly cancer cells, after exposure to macrophage conditioned media, by immunofluorescence (b (i)). Expression of CCR2 was measured in REM134 cells, after exposure to macrophage-conditioned media, by qRT-PCR (b (ii)). Cellular activity was measured by glucose uptake. REM134 cells were pre-incubated with macrophage-conditioned media for 72 h prior to analysis. Ethanol was used as a vehicle control (VC). Cells were incubated at 4 °C as a negative control (c). Figures B and C are replicates of 3. Statistical analysis considered each group against the REM group. Asterisks or horizontal bars indicate statistically significant differences to the 0 ng/ml or REM group, where P < 0.05 by Kruskal-Wallis (Figure A) or Mann–Whitney (Figures B and C); “ns” indicates non-significant differences

Fig. 5

Proliferation of canine mammary carcinoma cells is CSF-1R dependent. Canine mammary carcinoma cell lines, REM134 and Lilly were treated with increasing doses of either CCR2 small-molecule inhibitor (i-iv), toceranib (ii-v), or CSF-1R inhibitor  (iii-vi) for 48 h before assaying for cell viability. All experiments are replicates of 3. Asterisks indicate statistically significant differences to the DMSO group, where P < 0.05 by ANOVA

Fig. 6

The dual role of CSF-1 in macrophage activation. Macrophages were incubated with media containing increasing concentrations of either recombinant CSF-1 (a (i)) or recombinant CCL2 (a (ii)) for 2 days prior to analysis of MHC II expression by flow cytometry. Protein expression levels of COX-2 and Twist were determined by Western blotting. β-actin was used as a loading control. 20 μg protein loaded (b). Macrophages were incubated with media containing increasing concentrations of either recombinant CSF-1 (c (i)) or recombinant CCL2 (c (ii)) for 2 days prior LPS-induced activation (300 μg/ml LPS for 48 h). MHC II expression levels were determined by flow cytometry. Figures (i) are replicates of 6, Figures (ii) are replicates of 3. Asterisks indicate statistically significant differences to the RAW or RAW + LPS group, where P < 0.05 by Kruskal-Wallis (Figures A(i) and C(ii)) or ANOVA, unless where indicated; “ns” indicates non-significant differences

Fig. 7

CSF-1 and Twist-1 are key mediators of macrophage activation. Twist-1 protein levels were depleted by treating with siRNA against Twist-1 for 24 h. Immunofluorescence was carried out using anti-Twist-1 antibody (a). The effects of the reduction of Twist-1 levels on LPS-induced activation of macrophages were assayed by expression of MHC II (b (i)); granularity (b (ii)); and phagocytosis (c). All experiments are replicates of 3. Horizontal bars indicate statistically significant differences on a Student t-test, where P < 0.05, unless where indicated; “ns” indicates non-significant differences

Fig. 8

Schematic representation of the molecular interactions following macrophage and cancer cell activation. Arrows indicate that the pathway is increased; blunt-ended lines represent a negative effect on another receptor/pathway