Spheroid formation and invasion capacity are differentially influenced by co-cultures of fibroblast and macrophage cells in breast cancer

Abstract

Interactions with stromal components influence the growth, survival, spread, and colonization capacities of tumor cells. Fibroblasts and macrophages which are responsible for the stroma production and maintenance are of the basic elements found in tumor microenvironment. Cellular density and ratio of stromal cells to tumor cells can also have modulatory effects in cancer. Here, the contribution of fibroblast and/or macrophage cells on the malignant behavior of breast cancer cells was modeled in co-culture systems. Co-cultures were established at different cell densities and ratios with 4T1 breast cancer, NIH/3T3 or 3T3-L1 fibroblast, and J774A.1 monocyte/macrophage cell lines. Flow cytometry-based proliferation, 3D growth on alginate matrix, and matrigel invasion assays were performed to determine the change in the malignant assets of tumor cells. The data were also supported by immunocytochemical and morphological analyses. Co-culturing with fibroblasts (especially, NIH/3T3 cells) significantly supported the proliferation, scattering, and invasiveness of 4T1 cells whereas inclusion of macrophages disrupted this positive influence. On the other hand, the invasion capacity of 4T1 cells was not enhanced in the co-cultures with fibroblasts whose motility were inhibited with pertussis toxin pretreatment. Particularly at low-density seeding in 3D cultures, 4T1 cells could form substantially more spheroids than that of in the co-cultures with fibroblasts. Only, increasing the amount of fibroblasts could restore the 3D-growth. Intriguingly, co-existence of macrophage, fibroblast, and tumor cells in 3D cultures provided a convenient stroma sustaining the spheroid formation and growth. In conclusion, fibroblasts can form a favorable environment for tumor cells' spread and motility whereas restricting their 3D-growth capacity. On the other hand, presence of macrophages may disrupt the influence of fibroblasts and enhance the spheroid formation by the tumor cells.

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…Similarly cells in the TME play a critical role in cancer progression. Co-culturing MCTS with other cells types, such as immune cells and fibroblasts, can provide insights into host tumor cell interactions (Hauptmann et al , 1993; Esendagli, 2014). Microfluidic devices are suitable for such studies as tumor cells can be co-cultured with multiple cell types in presence of the ECM components.…”

Matrigel: From discovery and ECM mimicry to assays and models for cancer research

“…Prostate MCTS have been combined with osteoblasts and endothelial cells to recapitulate the metastatic niche [110]. Bladder cancer MCTS were evaluated for their ability to induce differentiation of monocytes into tumorassociated macrophages [138], and breast MCTS have been combined with fibroblasts and macrophages to evaluate the impact on spheroid formation and invasion [139] and with embryonic stem (ES) cells to evaluate the lineage-specific differentiation in the tumor microenvironment [140]. From these studies, it is becoming apparent that the tumor interactions with all facets of its environment, cellular and extracellular, are critical for developing physiologically relevant models.…”

Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

“…To test this hypothesis, an adjacent culture system and/or assay on differential cell populations may be the next approach to establish a further improved 3D co-culture system that may mimic the increased resistance as well as the EMT state at the same time. Incorporation of other TME factors such as macrophages or endothelial cells could also be considered to develop a more clinically relevant in vitro model to study the effects of TME on EMT as well as chemoresistance [38][39][40][41][42].…”