Mimicking the tumor microenvironment to regulate macrophage phenotype and assessing chemotherapeutic efficacy in embedded cancer cell/macrophage spheroid models

Abstract

Tumor associated macrophages (TAMs) are critical stromal components intimately involved with the progression, invasion, and metastasis of cancer cells. To address the need for an in vitro system that mimics the clinical observations of TAM localizations and subsequent functional performance, a cancer cell/macrophage spheroid model is described. The central component of the model is a triple negative breast cancer spheroid embedded in a three-dimensional collagen gel. Macrophages are incorporated in two different ways. The first is a heterospheroid, a spheroid containing both tumor cells and macrophages. The heterospheroid mimics the population of TAMs infiltrated into the tumor mass, thus being exposed to hypoxia and metabolic gradients. In the second model, macrophages are diffusely seeded in the collagen surrounding the spheroid, thus modeling TAMs in the cancer stroma. The inclusion of macrophages as a heterospheroid changes the metabolic profile, indicative of synergistic growth. In contrast, macrophages diffusely seeded in the collagen bear the same profile regardless of the presence of a tumor cell spheroid. The macrophages in the heterospheroid secrete EGF, a cytokine critical to tumor/macrophage co-migration, and an EGF inhibitor decreases the metabolic activity of the heterospheroid, which is not observed in the other systems. The increased secretion of IL-10 indicates that the heterospheroid macrophages follow an M2/TAM differentiation pathway. Lastly, the heterospheroid exhibits resistance to paclitaxel. In summary, the collagen embedded heterospheroid model promotes TAM-like characteristics, and will be of utility in cancer biology and drug discovery.

Embedded Spheroids as Models of the Cancer Microenvironment

“…Embedded spheroids possess two populations, a migratory or invasive population and a non‐migratory core. This duality in cell behavior is readily apparent in glioma, breast, and melanoma spheroids ( Figure 5 ). This is a consequence of the two distinct microenvironments present.…”

“…Tevis et al also report a heterospheroid and a spheroid/diffuse model with macrophages (RAW 264.7) and breast cancer cells (MDA‐MB‐231). These co‐culture model systems contain macrophages dispersed throughout the surrounding collagen gel containing a breast cancer spheroid (Figure B) or macrophages as a component of the heterospheroid (Figure C) . The macrostructure and interactions imparted by the heterospheroid model are sufficient to encourage EGF‐based interactions between the two cell types, increase system metabolism, and promote the cytokine‐based TAM phenotype in the macrophages contained within the heterospheroid ( Figure 4 ).…”

“…B) Cytokine based interaction of macrophages and tumor cells as monitored by EGF concentration detected via an ELISA from the four models (10 000 cancer cells (10C); a heterospheroid with 10 000 cancer cells and 5000 macrophages (10C5M); a 10 000 cancer cell spheroid surrounded by 5000 macrophages (10Cd5M); n = 4; (**p < 0.005, *p < 0.05). Reproduced with permission . Copyright 2017, Elsevier.…”

The role of collagen in cancer: from bench to bedside

“…Notably, in the context of an αvβ3 integrin-specific collagen hydrogel, M2-like immunosuppressive macrophages promoted angiogenesis in glioblastoma, while M1-like proinflammatory macrophages suppressed angiogenesis, which was regulated by Src-PI3K-YAP signaling associated with TGF-β1 [139]. IL-10 induces M2 differentiation in heterospheroid macrophages [140]. In contrast, collagen degradation is enhanced not by M2 macrophages but rather by M1 macrophages, as endogenous macrophages are recruited, modified and activated to produce MMPs and hepatic growth factors, thereby enhancing hepatocyte proliferation and the release of TNF-related apoptosis-inducing ligands by natural killer cells, leading to hepatic stellate cell apoptosis [141].…”

M2 macrophages contribute to cell proliferation and migration of breast cancer

“…It was accepted that the TAMs in cancer contribute to tumor progression by enhancing the capacity of tumor invasion, metastasis, drug-resistance, angiogenesis, as well as immune escape (17)(18)(19)(20) . MicroRNAs (miRNAs) are single-stranded RNA molecules that mediated gene expression at posttranscriptional level in various processes.…”