The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer. To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyperplasia (MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L, reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity-based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

Breast cancer frequently metastasizes to lymphatics and the presence of breast cancer cells in regional lymph nodes is an important prognostic factor. Delineating the mechanisms by which breast cancer cells disseminate and spatiotemporal aspects of interactions between breast cancer cells and lymphatics is needed to design new therapies to prevent lymphatic metastases. As triple-negative breast cancer (TNBC) has a high incidence of lymphatic metastasis, we used a three-dimensional (3D) coculture model of human TNBC cells and human microvascular lymphatic endothelial cells (LECs) to analyze TNBC:LEC interactions. Non-invasive analyses such as live-cell imaging in real-time and collection of conditioned media for secretomic analysis were facilitated by our novel microfluidic chambers. The volumes of 3D structures formed in TNBC:LEC cocultures are greater than that of 3D structures formed by either LEC or TNBC monocultures. Over 4 days of culture there is an increase in multicellular invasive outgrowths from TNBC spheroids and an association of TNBC spheroids with LEC networks. The increase in invasive phenotype also occurred when TNBC spheroids were cultured in LEC-conditioned media and in wells linked to ones containing LEC networks. Our results suggest that modeling spatiotemporal interactions between TNBC and LECs may reveal paracrine signaling that could be targeted to reduce lymphatic metastasis.

This study evaluated the paracrine signaling between breast carcinoma-associated fibroblasts (CAFs) and breast cancer (BCa) cells. Resolving cell–cell communication in the BCa tumor microenvironment (TME) will aid the development of new therapeutics. Here, we utilized our patented TAME (tissue architecture and microenvironment engineering) 3D culture microphysiological system, which is a suitable pathomimetic avatar for the study of the BCa TME. We cultured in 3D BCa cells and CAFs either alone or together in cocultures and found that when cocultured, CAFs enhanced the invasive characteristics of tumor cells, as shown by increased proliferation and spread of tumor cells into the surrounding matrix. Secretome analysis from 3D cultures revealed a relatively high secretion of IL-6 by CAFs. A marked increase in the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) when carcinoma cells and CAFs were in coculture was also observed. We theorized that the CAF-secreted IL-6 functions in a paracrine manner to induce GM-CSF expression and secretion from carcinoma cells. This was confirmed by evaluating the activation of STAT3 and gene expression of GM-CSF in carcinoma cells exposed to CAF-conditioned media (CAF-CM). In addition, the treatment of CAFs with BCa cell-CM yielded a brief upregulation of GM-CSF followed by a marked decrease, indicating a tightly regulated control of GM-CSF in CAFs. Secretion of IL-6 from CAFs drives the activation of STAT3 in BCa cells, which in turn drives the expression and secretion of GM-CSF. As a result, CAFs exposed to BCa cell-secreted GM-CSF upregulate inflammation-associated genes such as IL-6, IL-6R and IL-8, thereby forming a positive feedback loop. We propose that the tight regulation of GM-CSF in CAFs may be a novel regulatory pathway to target for disrupting the CAF:BCa cell symbiotic relationship. These data provide yet another piece of the cell–cell communication network governing the BCa TME.

Background/Objectives: Carcinoma-associated fibroblasts (CAFs), a prominent cell type in the tumor microenvironment (TME), significantly contributes to cancer progression through interactions with cancer cells and other TME components. Consequently, targeting signaling pathways driven by CAFs has potential to yield new therapeutic approaches to inhibit cancer progression. However, the mechanisms underlying their long-term interactions with cancer cells in vivo remains poorly understood. Methods: To address this, we developed a three-dimensional (3D) parallel coculture model of human triple-negative breast cancer (TNBC) cells and CAFs using our innovative TAME devices. This model allowed for the analysis of TNBC paracrine interactions via their secretome over extended culture periods (at least 70 days). Results: Using TNBC cell lines (MDA-MB-231, MCF10.DCIS, and HCC70), we found that TNBC spheroids in 3D parallel cocultures with CAFs exhibited more pronounced invasive finger-like outgrowths than those in cocultures of TNBC cells and normal fibroblasts (NFs) over a period of 50–70 days. We also established that the CAF-derived secretome affects TNBC migration towards the CAF secretome region. Additionally, we observed a preferential migration of CAFs, but not NFs, toward TNBC spheroids. Conclusions: Overall, our results suggest that paracrine interactions between TNBC cells and CAFs enhance TNBC invasive phenotypes and promote reciprocal migration.