Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments

Abstract

Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.

Integrated cancer tissue engineering models for precision medicine

“…For example, Regier et al developed a compartmentalized system that allowed for tri-culture of breast cancer cells with stromal and immune components keeping each cell type physically separated while sharing the same culture medium. This allowed for direct analysis of paracrine signaling between co- and tri-cultures[229]. A similar model developed by Szot et al was able to evaluate the role of paracrine signaling from breast cancer cells in causing angiogenesis of endothelial cells through culture of cancer cells and endothelial cells separated by an acellular type I collagen matrix[230].…”

Microfluidic modelling of the tumor microenvironment for anti-cancer drug development

“…Instead of simultaneously culturing various cell types in the same chamber, a separate coculture system grows each cell type in a separate chamber and all the cell-cell interaction is achieved by substance diffusion through the culture media between individual culture chambers [131,[139][140][141][142][143][144][145][146][147][148] (Figure 4b). For example, cancer-associated fibroblasts (CAFs) and NSCLC cells were co-cultured in separate chambers connecting by a fluid channel and cell behavior was observed under the microscope [143].…”

The Tumor-on-Chip: Recent Advances in the Development of Microfluidic Systems to Recapitulate the Physiology of Solid Tumors

“…Modified from Han et al, 2016 [19]. ( c ) Different types of tumor-on-chip systems: from simple spheroids in a microflow to vascularized and perfusable tumor microtissues [20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72].…”