Selective ROCK Inhibitor Enhances Blood Flow Recovery after Hindlimb Ischemia.

The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.

Development of a cancer metastasis-on-chip assay for high throughput drug screening.

Metastasis is the cause of most triple-negative breast cancer deaths, yet anti-metastatic therapeutics remain limited. To develop new therapeutics to prevent metastasis, pathophysiologically relevant assays that recapitulate tumor microenvironment is essential for disease modeling and drug discovery. Here, we have developed a microfluidic metastasis-on-chip assay of the early stages of cancer metastasis integrated with the triple-negative breast cancer cell line (MDA-MB-231), stromal fibroblasts and a perfused microvessel. High-content imaging with automated quantification methods was optimized to assess the tumor cell invasion and intravasation within the model. Cell invasion and intravasation were enhanced when fibroblasts co-cultured with a breast cancer cell line (MDA-MB-231). However, the non-invasive breast cancer cell line, MCF7, remained non-invasive in our model, even in the presence of fibroblasts. High-content screening of a targeted anti-cancer therapy drug library was conducted to evaluate the drug response sensitivity of the optimized model. Through this screening, we identified 30 compounds that reduced the tumor intravasation by 60% compared to controls. Multi-parametric phenotypic analysis was applied by combining the data from the metastasis-on-chip, cell proliferation and 2D cell migration screens, revealing that the drug library was clustered into eight distinct groups with similar drug responses. Notably, MEK inhibitors were enriched in cluster cell invasion and intravasation. In contrast, drugs with molecular targets: ABL, KIT, PDGF, SRC, and VEGFR were enriched in the drug clusters showing a strong effect on tumor cell intravasation with less impact on cell invasion or cell proliferation, of which, Imatinib, a multi-kinase inhibitor targeting BCR-ABL/PDGFR/KIT. Further experimental analysis showed that Imatinib enhanced endothelial barrier stability as measured by trans-endothelial electrical resistance and significantly reduced the trans-endothelial invasion activity of tumor cells. Our findings demonstrate the potential of our metastasis-on-chip assay as a powerful tool for studying cancer metastasis biology, drug discovery aims, and assessing drug responses, offering prospects for personalized anti-metastatic therapies for triple-negative breast cancer patients.