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Subject: tissue engineering

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    Dynamic 3D In Vitro Bone Metastatic Testbeds for Prostate and Breast Cancer
    (North Dakota State University, 2022) Jasuja, Haneesh
    Metastatic prostate cancer spreads preferentially to the bone, causing skeletal complications associated with significant morbidity and a poor prognosis, despite current therapeutic approaches. Increasing evidence suggests the synergistic role of biochemical and biophysical cues in cancer progression at metastases. However, the mechanism underlying the crosstalk between interstitial flow-induced mechanical stimuli and prostate cancer progression in the bone microenvironment remains poorly understood. To this end, we have developed 3D in vitro dynamic models of prostate cancer bone metastasis using perfusion bioreactor and horizontal flow bioreactor to delineate the role of flow-induced shear stress on prostate cancer progression and migration, respectively at metastases. Using a perfusion bioreactor, we observed changes in the expressions of MET biomarkers and the tumoroid morphologies of prostate cancer cells under dynamic culture. Evaluation of cell adhesion proteins indicated that the altered cancer cell morphologies resulted from the constant force pulling due to increased E-cadherin and FAK proteins under shear stress. Using a horizontal flow bioreactor, we demonstrated that the percent cell migration rate of prostate cancer cells was increased in the presence of bone under dynamic conditions. The results showed that interstitial fluid flow did not alter the CXCR4 level, but bone upregulated CXCR4 levels, leading to increased MMP-9 levels. In addition, both αvβ3 integrins and MMP-9 levels were upregulated by fluid flow conditions, contributing to an increased migration rate under dynamic conditions. Breast cancer cells also tend to preferentially disseminate to bone and colonize within the remodeling bone site to cause bone metastases. We have previously developed a 3D in vitro breast cancer bone metastasis model using hMSCs and commercial breast cancer cells (MCF-7 and MDAMB231), recapitulating late-stage breast cancer metastasis to bone. In the present study, we have validated our model using patient-derived breast cancer cell lines- NT013 and NT023, exhibiting hormone-positive and triple-negative characteristics, respectively that showed MET and formed tumors in the presence of bone. In addition, the results showed ET-1 (NT013) and DKK-1 (NT023) mediated stimulation and abrogation of the osteogenesis via Wnt/β catenin pathway, in line with our previous results with MCF-7 and MDAMB231 cell lines.
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    Tissue-Engineered Nanoclay-Based Bone-Mimetic 3D In Vitro Testbed for Studying Breast Cancer Metastasis to Bone
    (North Dakota State University, 2020) Kar, Sumanta
    Breast cancer shows a high affinity towards the bone, causing bone-related complications leading to poor clinical prognosis. Approximately 80% of breast cancer patients die within five years after primary cancer has metastasized to the bones. The tumor stage strongly influences the survival rates of patients with breast cancer that has spread to bone at the time of diagnosis. There are currently no effective therapeutics available for bone metastases due to the failure of animal models and the scarcity of human bone metastasized samples, as most patients with advance stages of cancer are already in palliative care. Therefore, it is imperative to develop translational models to elucidate disease mechanisms at the cellular and molecular level. Here, we report the development of tissue-engineered nanoclay-based bone-mimetic three-dimensional (3D) in vitro model for studying later stages of cancer pathogenesis at the metastatic bone site using osteogenically-differentiated human mesenchymal stem cells (MSCs) and human breast cancer cells (MDA-MB-231 and MCF-7). This 3D model provides an ideal microenvironment suitable for cell-cell and cell-matrix interactions while retaining the behavior of breast cancer cells with different metastatic potential along with mimicking mesenchymal to epithelial transition (MET) of breast cancer cells. Sequential cultures of MSCs with MCF-7 gave rise to tumoroids, while sequential cultures of MSCs with MDA-MB-231 formed disorganized clusters of cells with poor cell-cell adhesion. We further evaluated how cancer-derived factors and cytokines affect bone leading to up to metastasis and conferring drug resistance, respectively. Results showed that Wnt/β-catenin and interleukin-6 (IL-6) mediated IL-6/STAT3 pathways are responsible for bone-related complications and conferring drug resistance, respectively. Furthermore, we have utilized the 3D in vitro model to develop methods for non-invasive and rapid prediction of cancer progression using various biophysical techniques such as spectroscopy and nanoindentation. Spectroscopy methods showed significant contributions of proteins, lipids, and nucleic acids, while the nanoindentation method showed F-actin mediated softening of cancer cells during cancer progression at the metastatic bone site, respectively. Collectively, 3D in vitro model provides an ideal platform for studying the molecular mechanism of breast cancer progression at the metastatic bone site, drug development, and discovery of biomarkers for cancer progression.