Establishment of ion channel and ABC transporter assays in 3D-cultured ReNcell VM on a 384-pillar plate for neurotoxicity potential.

Neurotoxicity potential of compounds by inhibition of ion channels and efflux transporters has been studied traditionally using two-dimensionally (2D) cultured cell lines such as CHO and HEK-293 overexpressing the protein of interest. However, these approaches are time consuming and do not recapitulate the activity of ion channels and efflux transporters indigenously expressed in neural stem cells (NSCs) in vivo. To overcome these issues, we established ion channel and transporter assays on a 384-pillar plate with three-dimensionally (3D) cultured ReNcell VM and demonstrated high-throughput measurement of ion channel and transporter activity. RNA sequencing analysis identified major ion channels and efflux transporters expressed in ReNcell VM, followed by validating 3D ReNcell-based ion channel and transporter assays with model compounds. Major ion channel activities were measured by specifically inhibiting potassium channels Kv 7.2 with XE-991 and Kv 4.3 with fluoxetine, and a calcium channel with 2-APB. Activities of major efflux transporters, MDR1, MRP1, and BCRP, were assessed using their respective blockers, verapamil, probenecid, and novobiocin. From this study, we demonstrated that 3D-cultured ReNcell VM on the 384-pillar plate could be a good alternative to rapidly identify environmental chemicals and therapeutic compounds for their role in modulating the activity of ion channels and efflux transporters, potentially leading to neurotoxicity.

Hepatobiliary malignancies have distinct peripheral myeloid-derived suppressor cell signatures and tumor myeloid cell profiles.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that are increased in patients with numerous malignancies including viral-derived hepatocellular carcinoma (HCC). Here, we report an elevation of MDSCs in the peripheral blood of patients with other hepatobiliary malignancies including non-viral HCC, neuroendocrine tumors (NET), and colorectal carcinoma with liver metastases (CRLM), but not cholangiocarcinoma (CCA). The investigation of myeloid cell infiltration in HCC, NET and intrahepatic CCA tumors further established that the frequency of antigen-presenting cells was limited compared to benign lesions, suggesting that primary and metastatic hepatobiliary cancers have distinct peripheral and tumoral myeloid signatures. Bioinformatics analysis of The Cancer Genome Atlas dataset demonstrated that a high MDSC score in HCC patients is associated with poor disease outcome. Given our observation that MDSCs are increased in non-CCA malignant liver cancers, these cells may represent suitable targets for effective immunotherapy approaches.

Development of a Cx46 Targeting Strategy for Cancer Stem Cells.

Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis.

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis.