Understanding the role of ECM in Tumor Progression using Synthetic Microenvironments
Within the tumor microenvironment, changes in cancer cell-extracellular matrix (ECM) interactions influence each stage of the metastatic cascade, from the loss of basement membrane adhesion to colonization of distant sites.55,56 Multiple techniques exist for studying microenvironmental interactions, yet it has been challenging to systematically interrogate the functional implications of specific cell–ECM interactions. Based on techniques first developed in our study of liver microenvironments, we have synthesized 2D and 3D tumor microenvironments in vitro to query their interactions with cells derived from mouse models of lung tumors developed in the laboratory of Tyler Jacks (MIT). To synthesize 2D microenvironments, we utilized robotic spotting technologies to create non-covalent arrays of 768 unique pairwise ECM combinations in 4000 spots. The ECM molecules that made up this library included many glycosaminoglycans, which are difficult to study through integrin manipulation alone. Based on their adhesion patterns, we discovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3 or galectin-8. These molecules correlate with human disease and their interactions are mediated in part by a3ß1 integrin.
An ECM microarray identifies galectin-3 as a mediator of lung cancer metastasis.
(A) Schematic depicting the ECM microarray platform. (B) Kras;p53 mouse model of lung adenocarcinoma metastasis. Cell lines were derived from primary tumors that did not give rise to metastases (TnonMet), primary tumors
that did give rise to metastases (TMet), and lymphatic and distant metastases (N and M). (C)Fluorescence immunostaining of the ECM combinations on the printed microarrays. (D) M lines display preferential adhesion to combinations of fibronectin with galectins. (E)Representative galectin-3 staining of primary lung tumors and lymph node metastases on human tissue microarrays. (F) Flow cytometric analysis of surface expression of integrin a3 and the T-Antigen on TMet and M lines. (G) shRNA knockdown of a3 reduces experimental metastasis in vivo. (Flaim et al., 2008; Reticker-Flynn et al., 2012)
In addition, by building on recent advances in microfluidic technology, we developed a platform for high-throughput fabrication of tunable cellular microniches ‘‘microtissues’’) that allow us to probe tumor cell response to a range of microenvironmental cues, including ECM, soluble factors, and stromal cells, all in 3D. We further combined this tunable microniche platform with rapid, flow-based population level analysis, which permits analysis and sorting of microtissue populations both pre- and post-culture based on many criteria, including cell growth and population patterns. We used this platform to examine responses to a selection of ECM molecules and soluble factors, and also to study patterns of drug responsiveness and toxicity. We found that tumor cells grown in microtissues behaved differently than when grown in more traditional 2D cultures. Our findings highlight the importance of the microenvironmental context in therapeutic development and that the platform we present here allows the high-throughput study of tumor response to drugs as well as basic tumor biology in well-defined microenvironmental niches. (Li et al., 2013)