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This paper describes a dielectrophoretic method for selection of circulating melanoma cells (CMCs), which lack reliable identifying surface antigens and are extremely rare in blood. This platform captures CMCs individually by dielectrophoresis (DEP) at an array of wireless bipolar electrodes (BPEs) aligned to overlying nanoliter-scale chambers, which isolate each cell for subsequent on-chip single-cell analysis. To determine the best conditions to employ for CMC isolation in this DEP-BPE platform, the static and dynamic dielectrophoretic response of established melanoma cell lines, melanoma cells from patient-derived xenografts (PDX) and peripheral blood mononuclear cells (PBMCs) were evaluated as a function of frequency using two established DEP platforms. Further, PBMCs derived from patients with advanced melanoma were compared with those from healthy controls. The results of this evaluation reveal that each DEP method requires a distinct frequency to achieve capture of melanoma cells and that the distribution of dielectric properties of PBMCs is more broadly varied in and among patients versus healthy controls. Based on this evaluation, we conclude that 50 kHz provides the highest capture efficiency on our DEP-BPE platform while maintaining a low rate of capture of unwanted PBMCs. We further quantified the efficiency of single-cell capture on the DEP-BPE platform and found that the efficiency diminished beyond around 25% chamber occupancy, thereby informing the minimum array size that is required. Importantly, the capture efficiency of the DEP-BPE platform for melanoma cells when using optimized conditions matched the performance predicted by our analysis. Finally, isolation of melanoma cells from contrived (spike-in) and clinical samples on our platform using optimized conditions was demonstrated. The capture and individual isolation of CMCs, confirmed by post-capture labeling, from patient-derived samples suggests the potential of this platform for clinical application.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of deadly primary brain tumors and brain metastasis. The phenothiazine (PTZ) class of anti-psychotic agents were originally developed to inhibit dopamine- and serotonin-receptors (DR/5-HTRs). PTZs have demonstrated anti-cancer properties in numerous studies through a variety of proposed mechanisms, including on-target (DR/5-HTR) and off-target (e.g. calmodulin (CaM) inhibition) effects. PTZs inhibit DR/5-HTRs in the low- to mid-nanomolar range whereas anti-cancer effects tend to be observed only at a higher (low micromolar) dose range. Thus, clinical efficacy of existing PTZs may be limited by toxicity of on-target effects (sedation) of these drugs as DR/5-HTR antagonists. However, it has been unclear whether anti-cancer effects of PTZs are entirely separate from their activity as DR/5-HTR antagonists. Here we evaluate the in vitro anti-cancer efficacy of a novel PTZ analog (CWHM-974, a N-benzyl-linked fluphenazine (FLU) derivative), originally developed to treat brain-resident fungal infections, that is a 100-1000-fold less potent DR/5-HTR antagonist than FLU. Experimental procedures: To evaluate the anti-cancer efficacy of FLU and CWHM-974, we assessed cytotoxicity following 72h exposure against a broad panel of cancer cell lines. To investigate CaM as a target for CWHM-974, we evaluated the correlation between the sensitivity to CMW-974 and sensitivity to the CaM inhibitor W-7 as well as CaM gene expression levels in a panel of cancer cell lines. We also tested the anti-cancer efficacy of a sulfoxide analog of CWHM-974 (SLU-0010894) which lacks CaM binding activity. Results: We confirmed the anti-cancer efficacy of FLU and demonstrated that CWHM-974 is consistently 2-10-fold more potent than FLU in a broad panel of cancer cell lines. Among the most sensitve cancer cell lines are melanoma, which is a cancer that frequently metastasizes to the brain. Consistent with the hypothesis that CaM is a relevant target for CWHM-974, we found that: 1) CaM mRNA (CALM1) levels are correlated with sensitivity to CWHM-974; 2) the activities of CWHM-974 and W7 are correlated, and 3) most importantly, SLU-0010894 does not exhibit anti-cancer activity. Conclusions: The anti-cancer efficacy of the PTZ derivative CWHM-974 is seperable from DR/5-HTR antagonism. Our data suggests that further optimization of this compound may preserve the desirable pharmacologic properties for targeting brain cancers while reducing toxicity due to DR/5-HTR antagonism. Moreover, we provide a variety of evidence suggesting that anti-cancer efficacy of CWHM-974 is associated with CaM binding. Ongoing efforts will characterize the activity of CWHM-974 in mouse models of brain metastasis. Citation Format: Marion Vanneste, Anita Venzke, Soumitra Guin, Damian Krysan, Marvin Meyers, Michael Henry. Anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4881.

Introduction: Epithelial to Mesenchymal Transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therefore, therapeutic targeting of EMT-like phenotypes in cancer cells may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Recently, GPX4 inhibitors (GPX4i) became of interest in this field as they were identified as potent and selective compounds for cells in a mesenchymal state through induction of lipid peroxidation induced cell death also known as ferroptosis. The sensitivity to these inhibitors was also shown to be dependent on the master EMT regulator, ZEB1. However, the link between ZEB1 expression and ferroptosis susceptibility remains unclear. Methodology: We used PC-3E and TEM 4-18 cell lines, originally isolated from the PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer, as our models of epithelial and EMT-like phenotypes respectively. To better understand the role of ZEB1 in ferroptosis susceptibility, we used ZEB1 KO TEM 4-18 cells as well as a panel of cancer cell lines that cover a broad spectrum of epithelial and EMT-like phenotypes. After evaluating the sensitivity to GPX4i in these cell lines, we examined different parameters affecting the lipid peroxidation pathway. We particularly focused on the regulation of iron metabolism by ZEB1. Results: The three GPX4i tested (ML210, ML162 and RSL3) displayed a greater toxicity against TEM 4-18 cells. Inhibitors of ferroptosis (ferrostatin-1, liproxstatin-1) completely reversed the effect of GPX4i indicating that ferroptosis was indeed triggered by GPX4i and responsible for their toxicity. Sensitivity to GPX4i correlated with ZEB1 expression in our panel of cancer cell lines and ZEB1 KO reversed toxicity of GPX4i in TEM 4-18 cells. Sensitivity to GPX4i correlated with a slightly higher labile iron pool in ZEB1 expressing cells. Expression of proteins with a previously established connection to iron metabolism such as Ferritin Light Chain (FtL), Ferritin Heavy Chain (FtH), Transferrin Receptor (TfR) and Prominin-2 (PROM2) were also dysregulated between resistant and sensitive cells. In particular, the ratio FtH:FtL and expression of PROM2 was positively correlated with resistance to GPX4i. In addition, using the Cancer Cell Line Encyclopedia (CCLE) database, we found that ZEB1 expression negatively correlated with PROM2 and Iron Responsive Element Binding Protein-2 (IREB2) suggesting these genes could be transcriptional targets of ZEB1. Conclusions: We validated GPX4i as potent EMT selective cytotoxic compounds and confirmed that the sensitivity to these inhibitors depends on ZEB1 expression. However, the mechanisms leading to sensitivity to GPX4i and how they connect to the EMT program have yet to be identified. As these mechanisms might be diverse, we hypothesize that ZEB1 regulates iron metabolism which is critical for ferroptosis induction. Citation Format: Marion Vanneste, Michael Darrin Henry, Akansha Jain. ZEB1 confers sensitivity to ferroptosis through regulation of iron metabolism [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A028.

Background and Purpose: Circulating tumor cells (CTCs) are exposed to mechanical and biochemical stresses including fluid shear stress (FSS), due to blood flow, and oxidative distress, from a lack of matrix attachment. Recently we have shown that viable CTCs actively resist destruction when exposed to FSS by a mechano-adaptive mechanism that depends on RhoA-actomyosin activity (PMID: 32187555). Since the RhoA-actomyosin axis is implicated in other cellular behaviors that might contribute to metastasis, here we explored the hypothesis that exposure to FSS alters the biology of viable CTCs to promote metastatic colonization. Methodology: To determine if exposure to FSS alters metastatic potential, we exposed PC-3 prostate cells to FSS prior to injection into the tail-vein of NCG mice. We then monitored metastasis formation by weekly bioluminescence imaging. We also examined whether FSS exposure altered the ability of PC-3 and MDA-MB-231 cells to invade through a collagen I matrix, as well as their ability to survive/proliferate under anchorage-independent conditions. We conducted GC/LC-MS metabolomic profiling immediately after FSS exposure. To investigate oxidative burden on FSS exposed cells, we used dihydroethidium (10µM, 30 minutes) to measure general ROS production and a lipid peroxide sensor (5µM, 20 minutes). Results: We found that FSS exposure shortens the time for metastasis formation by 20% (median of 42 vs 52.5 days metastasis free; p=0.0222, log-rank). For both PC-3 and MDA-MB-231 cells we found that FSS exposure leads to ~2x increase in invasion through collagen matrix, and that increase depends on RhoA. Moreover, FSS exposure resulted in increased proliferation (~25%) under anchorage independent conditions in both cell lines. Metabolomic profiling revealed that FSS exposure resulted increased glycolysis, reduced entry into the TCA cycle, and a decrease in the metabolites that feed the folate cycle. We blocked glycolysis in PC-3 and MDA-MB-231 cells with 2-deoxyglucose (25mM, 2 hours) and found that this increased the fraction of cells destroyed by FSS ~20-30%. We also investigated redox stress and found that compared to cells held in suspension, those exposed to FSS demonstrate lower ROS burden. Interestingly, cells exposed to FSS exhibit lower levels of lipid peroxides in a manner that depends on RhoA. Conclusion: Our findings demonstrate that brief pulses of FSS exposure can enhance metastatic potential, increase invasive and anchorage-independent proliferative capacity and rapidly alter metabolism. The phenotypic changes driven by exposure to FSS depend in part on RhoA activation but may also reflect other mechanisms by which cancer cells sense and respond to FSS. FSS rapidly alters cellular metabolism in a manner that may act to reduce oxidative distress while CTCs are in suspension. In summary, our data indicate that FSS exposure in the circulation not only represents a physical force that CTCs must overcome to survive, but it also provides instructive cues that may enhance the metastatic behavior of some CTCs. This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR006) of the Conference Proceedings. Citation Format: Devon L. Moose, Amanda N. Pope, Marion Vanneste, Patrick Breheny, Eric B. Taylor, Michael D. HenryDevon L. Moose, Amanda N. Pope, Marion Vanneste, Patrick Breheny, Eric B. Taylor, Michael D. Henry. Fluid shear stress enhances the metastatic potential and rapidly alters metabolism of circulating tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B047.