Challenges and Opportunities Modeling the Dynamic Tumor Matrisome

We need novel strategies to target the complexity of cancer and, particularly, of metastatic disease. As an example of this complexity, certain tissues are particularly hospitable environments for metastases, whereas others do not contain fertile microenvironments to support cancer cell growth. Continuing evidence that the extracellular matrix (ECM) of tissues is one of a host of factors necessary to support cancer cell growth at both primary and secondary tissue sites is emerging. Research on cancer metastasis has largely been focused on the molecular adaptations of tumor cells in various cytokine and growth factor environments on 2-dimensional tissue culture polystyrene plates. Intravital imaging, conversely, has transformed our ability to watch, in real time, tumor cell invasion, intravasation, extravasation, and growth. Because the interstitial ECM that supports all cells in the tumor microenvironment changes over time scales outside the possible window of typical intravital imaging, bioengineers are continuously developing both simple and sophisticated in vitro controlled environments to study tumor (and other) cell interactions with this matrix. In this perspective, we focus on the cellular unit responsible for upholding the pathologic homeostasis of tumor-bearing organs, cancer-associated fibroblasts (CAFs), and their self-generated ECM. The latter, together with tumoral and other cell secreted factors, constitute the “tumor matrisome”. We share the challenges and opportunities for modeling this dynamic CAF/ECM unit, the tools and techniques available, and how the tumor matrisome is remodeled (e.g., via ECM proteases). We posit that increasing information on tumor matrisome dynamics may lead the field to alternative strategies for personalized medicine outside genomics

Co-Emergence of Specialized Endothelial Cells from Embryonic Stem Cells.

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad+/CD31+ cells, the tip/stalk-like EC contained predominantly Flt4+/Dll4+/CXCR4+/Flt-1- cells, while the phalanx-like EC was composed of higher numbers of Flt-1+ cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel

Genetic Mutations Associated with Hormone-Positive Breast Cancer in a Small Cohort of Ethiopian Women

In Ethiopia, a breast cancer diagnosis is associated with a prognosis significantly worse than that of Europe and the US. Further, patients presenting with breast cancer in Ethiopia are far younger, on average, and patients are typically diagnosed at very late stages, relative to breast cancer patients of European descent. Emerging data suggest that a large proportion of Ethiopian patients have hormone-positive (ER+) breast cancer. This is surprising given 1) that patients have late-stage breast cancer at the time of diagnosis, 2) that African Americans with breast cancer frequently have triple negative breast cancer (TNBC), and 3) these patients typically receive chemotherapy, not hormone-targeting drugs.To further examine the similarity of Ethiopian breast tumors to those of African Americans or of those of European descent, we sequenced matched tumor and normal adjacent tissue from Ethiopian patients from a small pilot collection. We identified mutations in 615 genes across all three patients, unique to the tumor tissue. Across this analysis, we found far more mutations shared between Ethiopian patient tissue and White patients (103) than we did comparing to African Americans (3). Several mutations were found in extracellular matrix encoding genes with known roles in tumor cell growth and metastasis. We suggest future mechanistic studies on this disease focus on these genes first, toward finding new treatment strategies for breast cancer patients in Ethiopia

Tumor cell–organized fibronectin maintenance of a dormant breast cancer population

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, nonproliferative state before reactivation and outgrowth. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system with carefully controlled ECM substrates to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle–arrested, and actively proliferating cells. Cell populations capable of entering dormancy formed an organized, fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation, and cancer cell outgrowth after dormancy required MMP-2–mediated fibronectin degradation. We propose this approach as a useful, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation

Multiplex Zymography Captures Stage-specific Activity Profiles of Cathepsins K, L, and S in Human Breast, Lung, and Cervical Cancer

<p>Abstract</p> <p>Background</p> <p>Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties.</p> <p>Methods</p> <p>Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity.</p> <p>Results</p> <p>Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p < .05), and stage II for breast; n = 6, p < .0001). Interestingly, cervical and breast tumor cathepsin activity was highest at the earliest stage we tested, stages I and II, respectively, and then were significantly lower at the latest stages tested (III and IV, respectively) (n = 6, p < 0.01 and p < 0.05), but lung cathepsin activity increased from one stage to the next (n = 6, p < .05). Using cathepsin K as a diagnostic biomarker for breast cancer detected with multiplex zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients.</p> <p>Conclusions</p> <p>To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.</p

Platt Lab Overview

Manu O. Platt is an assistant professor in the Wallace H. Coulter Department of Biomedical Engineering. He is the director of the Platt Lab for Repair Regeneration and Remodeling