Integrating Macrophages into Organotypic Co-Cultures: A 3D In Vitro Model to Study Tumor-Associated Macrophages

Abstract

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…In collagen gels, monotypic or heterotypic cultures can be developed, thereby increasing the possibility of better mirroring the in vivo conditions by integrating tumor's neighboring cell types [122]. Culture of fibroblasts in collagen gels have allowed a better understanding of the reciprocal interactions between cells and ECM, and, namely, the regulation of cell behavior mediated by the mechanical properties of ECM [123].…”

Impact by pancreatic stellate cells on epithelial-mesenchymal transition and pancreatic cancer cell invasion: Adding a third dimension in vitro

“…In a study with squamous carcinoma cells, M2 macrophages together with fibroblasts enhance the invasion rate of cancer cells compared to fibroblasts alone (Linde et al, 2012). In a similar manner, the combined presence factors, which all are associated with invasion, metastasis and ECM degradation (Goswami et al, 2005, Baran et al, 2009, Kessenbrock et al, 2010, Kuwada et al, 2003, Lesina et al, 2011.…”

“…The organotypic model has previously displayed favourable results for visualisation of cancer cell invasion and interplay with the local microenvironment (Froeling et al, 2009, Linde et al, 2012. The desmoplastic stroma produced by pancreatic stellate cells has been shown to create a treatment barrier around the tumour, limiting bioavailability of drugs (Jaster and Emmrich, 2008, Erkan et al, 2012, Whatcott et al, 2015.…”

Bistability of the cytokine-immune cell network in a cancer microenvironment

“…More importantly, we can determine the cells/signals that are most responsible for biasing the immune cell population into the immune-suppressing state. From the work by Linde et al [49], cancer cells alone can bias macrophages into the M2-like state (not immune-promoting) and fibroblasts can further speed up the process. Introducing the T cells would then test whether CD4 + Th1 and CD8 + T cells can help the co-culture system to remain stable in the immune-promoting state and whether fibroblasts, together with cancer cells, may revert this effect and polarize the immune cell population into the immune-suppressing state.…”

“…Linde et al [49] showed that during three-week long organotypic co-culture of cancer cells and unpolarized macrophages, the percentage of macrophages with a protein maker, which is usually expressed on M 2 -like macrophage, gradually elevates. For the co-cultures with additional fibroblasts, the percentage of M 2 -like macrophages increases faster compared to the co-cultures with cancer cells and macrophages only.…”