Calcitriol in the Presence of Conditioned Media from Metastatic Breast Cancer Cells Enhances Ex Vivo Polarization of M2 Alternative Murine Bone Marrow-Derived Macrophages.

Joanna Wietrzyk,Natalia Łabędź,Artur Anisiewicz,Izabela Krauze

doi:10.3390/cancers12113485

Joanna Wietrzyk, Natalia Łabędź

Open Access

https://doi.org/10.3390/cancers12113485

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Abstract

Simple SummaryIn this study, we stimulated bone marrow-derived macrophages to M0, M1, and M2 subtypes, with or without calcitriol, or with or without 4T1 (metastatic), 67NR (non-metastatic), and Eph4-Ev (normal) cell culture supernatants (CMs) to test their effect on polarization. We showed that calcitriol increased the expression of Cd206 and Spp1 mRNA and CD36, CCL2, and arginase levels for M2 macrophages and decreased Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS for M1 macrophages. 4T1 CM influenced the expression of the studied genes and proteins to a greater extent than 67NR and Eph4; the strongest effect was noted for M2 macrophages. We show that calcitriol and 4T1 CM enhance the polarization of M2 macrophages and M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells. We indicate that the immunosuppressive properties of calcitriol may unfavorably affect the tumor microenvironment, and supplementation with vitamin D in oncological patients may not always bring benefits.In this study, we differentiated murine bone marrow-derived macrophages (BMDMs) into M0, M1, and M2 in the presence or absence of calcitriol. Real-time PCR analysis of gene expression, FACS analysis of surface markers, and chemokine/cytokine production assays were performed. In addition, the effect of the conditioned media (CM) from murine breast cancer 4T1 (metastatic) and 67NR (non-metastatic) and Eph4-Ev (normal) cells with and without calcitriol on the polarization of M1/M2 cells was determined. We found that calcitriol enhanced the differentiation of M2 macrophages, which was manifested by increased expression of Cd206 and Spp1 mRNA and CD36, Arg, and CCL2 in M2 BMDMs and by decreased expression of Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS in M1 BMDMs. 4T1 CM showed a higher effect on the gene and protein expression in macrophages than 67NR and Eph4-Ev, with the greatest effect observed on M2 macrophages which increased their differentiation and properties characteristic of alternative macrophages. Moreover, M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells through fibronectin and collagen type IV, respectively. Overall, our results indicated that vitamin D supplementation may not always be beneficial, especially in relation to cancers causing excessive, pathological activation of the immune system.

Highlights

Macrophages are a key element of innate immunity
Under the influence of factors released by cancer cells, such as chemokine CCL2, peripheral monocytes and local macrophages are recruited to the primary tumor and transformed into tumor-associated macrophages (TAMs) [2,3]
A tendency of enhanced proliferation was observed for bone marrow-derived macrophages (BMDMs) polarized in the presence of calcitriol; the differences were not statistically significant (Figure S1B)

Summary

Introduction

Macrophages are a key element of innate immunity Due to their numerous functions in all tissues, these cells are essential for the maintenance of local and overall homeostasis [1]. M1 classical macrophages exhibit antitumor activity, during cancer progression, a predominance of immunosuppressive macrophages is generally observed. These macrophages, which are phenotypically similar to M2 alternative macrophages, support the growth of primary tumors, increase the metastatic potential of tumor cells, and promote vascularization and remodeling of tumor stroma, accelerating cancer progression [4,5,6]. It was reported that overall survival or disease-free survival mostly correlated with the expression of the M2 phenotype: CD163+, CD204+, or CD206+ [8,9]

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