5b). cell debris could affect the polarization of macrophages to the M2-like phenotype, we examined the expression of the M2-like macrophage marker, CD206 in Rabbit Polyclonal to PIK3R5 macrophages co-cultured with tumor cell debris or live breast cancer cells. The CD206 expression levels of macrophages were higher in macrophages treated with tumor cell debris compared to those co-cultured with live breast cancer cells AZ876 (Fig. 2f). Likewise, BMDMs co-incubated with breast tumor cell debris showed elevated mRNA levels of which is an immunosuppressive M2-like marker (Supplemental Fig. S1a). These results suggest that engulfment of tumor cell debris causes an immunosuppression, which can be an important aspect of the TME changes seen in the wake of anti-cancer therapy. Engulfment of tumor cell debris induces HO-1 expression in macrophages Chemotherapy used for tumor eradication in breast cancer patients has been reported to upregulate HO-1 expression, which is associated with the low survival rate [23], [26]). Moreover, TAMs represent the major tumoral source of HO-1 in breast cancer [26]. To determine whether HO-1 expression of TAMs was enhanced after chemotherapy, the HO-1 expression in macrophages in breast cancer patient AZ876 samples collected before and after chemotherapy was measured. In macrophages of breast cancer patients following chemotherapy, overexpression of HO-1 was confirmed (Fig. 3a). The elevated expression of HO-1 in TAMs derived from 4T1 tumor-bearing mice injected with PTX was confirmed by immunofluorescence staining (Fig. 3b). Open in a separate window Fig. 3 Phagocytosis of tumor cell debris induces HO-1 expression in macrophages. (a) Representative images of CD11b and HO-1 expressed in tumor tissues from breast cancer patients before and after chemotherapy. (b) Representative immunofluorescence images of HO-1 expression in TAMs from 4T1 breast cancer-bearing mice. (c) The protein expression levels of HO-1 in macrophages co-incubated for 8?h with tumor cell debris generated from murine 4T1 measured by Western blot analysis. (d) The HO-1 expression levels of macrophages (F4/80+) cultured with live cancer cells or tumor cell debris analyzed by flow cytometry. Comparisons between the control and experimental groups were performed with the Students and in M1-like polarized macrophages co-cultured with 4T1 tumor cell debris (Supplemental Fig. S1b, c). Open in a separate window Fig. 4 HO-1 expression induced by is involved in the modulation of macrophage polarization. (a, b) BMDMs from WT or HO-1 KO mice were treated with LPS (100?ng/ml) for 24?h. The resulting M1-like polarized macrophages were co-cultured with 4T1 tumor cell debris for 30?h. The proportion of macrophages expressing TNF- (a) and IL-12p40 (b) was determined by flow cytometry. (c) BMDMs from WT or HO-1 KO mice were co-treated with 4T1 tumor AZ876 cell debris for 24?h, and the expression of CD206 in macrophages was analyzed by flow cytometry. (d) BMDMs from WT or HO-1 KO mice were co-cultured with or without 4T1 tumor cell debris for 8?h, and the mRNA levels of were analyzed by qPCR. *compared to the WT macrophages (Fig. 4d). Taken together, these results suggest that HO-1 signaling induced by tumor cell debris represses the M1-like polarization of macrophages in the TME following chemotherapy. HO-1 inactivation amplifies the therapeutic efficacy of PTX To determine whether HO-1 inactivation could enhance the therapeutic efficiency of PTX, we used HO-1 AZ876 KO mice implanted with 4T1 breast cancer cells. We injected PTX into the mice when tumors were grown for 10?days. Notably, the therapeutic efficacy of PTX was increased in HO-1 KO mice compared to the WT mice (Fig. 5a). However, the tumor weight was not that much different between HO-1 WT and KO mice treated with PTX due to the relatively larger size of initial tumors at the time point of PTX injection (Supplemental Fig. S2a and Fig. S2b)..