Despite the implementation of multiple HER2-targeted therapies, patients with advanced HER2+breast cancer ultimately develop drug resistance. Stromal fibroblasts represent an abundant cell type in the tumor microenvironment and have been linked to poor outcomes and drug resistance. Here, we show that fibroblasts counteract the cytotoxic effects of HER2 kinase-targeted therapy in a subset of HER2+breast cancer cell lines and allow cancer cells to proliferate in the presence of the HER2 kinase inhibitor lapatinib. Fibroblasts from primary breast tumors, normal breast tissue, and lung tissue have similar protective effects on tumor cells via paracrine factors. This fibroblast-mediated reduction in drug sensitivity involves increased expression of antiapoptotic proteins and sustained activation of the PI3K/AKT/MTOR pathway, despite inhibition of the HER2 and the RAS-ERK pathways in tumor cells. HER2 therapy sensitivity is restored in the fibroblast cocultures by combination treatment with inhibitors of MTOR or the antiapoptotic proteins BCL-XL and MCL-1. Expression of activated AKT in tumor cells recapitulates the effects of fibroblasts resulting in sustained MTOR signaling and poor lapatinib response. Lapatinib sensitivity was not altered by fibroblasts in tumor cells that exhibited sustained MTOR signaling due to a strong gain-of-function PI3KCA mutation. These findings indicate that in addition to tumor cell-intrinsic mechanisms that cause constitutive PI3K/AKT/MTOR pathway activation, secreted factors from fibroblasts can maintain this pathway in the context of HER2 inhibition. Our integrated proteomic–phenotypic approach presents a strategy for the discovery of protective mechanisms in fibroblast-rich tumors and the design of rational combination therapies to restore drug sensitivity.
Soft lithography microfluidics offer many benefits over conventional biological assays; however, the impact this field is inhibited by the lack of widespread adoption of this technology in part due to prohibitive cost and fabrication time. Recent improvements in three-dimensional (3D) printing technologies such as digital light processing (DLP) printing offer a cost-effective and rapid prototyping solution to microfluidic fabrication. Limited information is available about how 3D printing parameters and resin cytocompatibility impact the performance of 3D printed molds for fabrication of polydimethylsiloxane (PDMS)-based microfluidics for cellular studies. Using a low-cost, commercially available DLP 3D printer, we assess the cytocompatibility of several resins, optimize printer settings and characterize minimum feature size of our system. We demonstrate the applications of DLP printing for soft lithography microfluidics by developing four assays to characterize cell viability, drug response, establish concentration gradients, and monitor live-cell 3D invasion into a hydrogel.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.