In vitro 3D spheroid model preserves tumor microenvironment of hot and cold breast cancer subtypes.

Dynamic interaction of cancer, immune and stromal cells with extracellular matrix components modulates and resists the response of standard care therapies. To mimic this, we designed an in vitro three-dimensional (3D) spheroid model using liquid overlay method to simulate hot (MDA-MB-231) and cold (MCF-7) breast tumor microenvironment (TME). Our study shows increased mesenchymal phenotype, stemness and suppressive microenvironment in MDA-MB-231-spheroids upon exposure to doxorubicin. Intriguingly, presence of human dermal fibroblasts enhances cancer-associated fibroblast (CAF) phenotype in MDA-MB-231-spheroids through increased expression of CXCL12 and FSP-1, leading to higher infiltration of immune cells (THP-1 monocytes). However, a suppressive TME was observed in both subtypes, as seen by upregulation of M2-macrophage specific CD68 and CD206 markers. Specifically, increased PDL-1 expressing tumor associated macrophages (TAMs) along with FoxP3 expressing T regulatory (T-regs) cells were found in MDA-MB-231-spheroids when cultured with peripheral blood mononuclear cells (PBMCs). Further, we found that addition of 1-methyl-tryptophan, a potent indoleamine-2,3-dioxygenase-1 (IDO-1) inhibitor, subsided the suppressive phenotype by decreasing the M2 polarisation via downregulation of tryptophan metabolism and IL10 expression, particularly in MCF-7 triculture spheroids. Thus, our in vitro 3D spheroid model of TME can be utilized in therapeutics to validate immunomodulatory drugs for various breast cancer subtypes. This article is protected by copyright. All rights reserved.