The epithelial-mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem-like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal and EMT tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre+/−;PtenL/L;KrasG12D/+ prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT and mesenchymal cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT (EpCAM+/Vim-GFP+) and mesenchymal (EpCAM−/Vim-GFP+) characteristics at the primary tumor site and in circulation. These EMT and mesenchymal tumor cells displayed enhanced stemness and invasive character compared to epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia-mesenchyme plasticity. Interestingly, while mesenchymal tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade.

PI3K/AKT and RAS/MAPK pathway co-activation in the prostate epithelium promotes both epithelial-mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically-defined cellular and in vivo model systems from which epithelial, EMT, and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial-mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase (HDAC) inhibitor LBH589, inhibits epithelial-mesenchymal plasticity and stemness activities in vitro and dramatically reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and AR acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to ADT. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.

Introduction: A majority of residents provide care for critically ill patients, yet only a minority of medical schools require ICU rotations. Therefore, many medical students enter residency without prior ICU experience. The third-year internal medicine (IM) clerkship at our institution's Veterans Affairs Medical Center (VAMC) provided an opportunity for medical students to rotate through an open ICU as part of their inpatient ward rotation. Prior to March 2019, no structured critical care curriculum existed within the IM clerkship to prepare students for this experience. Methods: We created a seven-session ICU curriculum integrated within the VAMC IM clerkship addressing core critical care topics and skills including bedside presentations, shock, and respiratory failure. IM residents facilitated the curriculum's case-based, small-group discussions. We assessed curricular efficacy and impact with a pre-and posttest and end-of-curriculum survey. Results: Forty-one students participated in the curriculum from March to November 2019. As a result, students agreed that their overall clerkship experience improved (73% strongly agree, 24% agree). Students also reported increased comfort in their ability to participate in the management of critically ill patients (44% strongly agree, 51% agree). Objectively, student performance on a 15-question pre-and posttest improved from a precurricular average of 7.5 (50%) questions correct to a postcurricular average of 10.7 (71%) questions correct (p <.0001; CI 2.2-4.4). Discussion: Following implementation of our ICU curriculum, medical student attitudes regarding overall IM clerkship experience, self-perceived confidence in critically ill patient management, and medical knowledge all improved.

<p>Supplementary Figure S4. Characterization of CTCs and iCTCs by FACS analysis.</p>

<p>Supplementary Figure S2. The anterior portion of the urethra and proximal region of the anterior lobes regenerate tumors containing all three prostate epithelial lineages.</p>