Circulating tumor cells (CTCs) face challenges to their survival, including mechanical and oxidative stresses that are different from cancer cells in solid primary and metastatic tumors. The impact of adaptations to the fluid microenvironment of the circulation on the outcome of the metastatic cascade is not well understood. Here, we find that cancer cells exposed to brief pulses of high-level fluid shear stress (FSS) exhibit enhanced invasiveness and anchorage-independent proliferation in vitro and enhanced metastatic colonization/tumor formation in vivo. Cancer cells exposed to FSS rapidly alter their metabolism in a manner that promotes survival by providing energy for cytoskeletal remodeling and contractility as well as reducing equivalents to counter oxidative stress associated with cell detachment. Thus, exposure to FSS may provide CTCs with an unexpected survival benefit that promotes metastatic colonization.

Abstract Background and Purpose: Circulating tumor cells (CTCs) are exposed to mechanical and biochemical stresses including fluid shear stress (FSS) and oxidative distress. 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; herein, 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 and MDA-MB-231 breast cancer cells to FSS prior to injection into the tail-vein of NCG mice. We then monitored metastatic colonization by weekly bioluminescence imaging. Further, we examined if FSS exposure altered the invasive potential of PC-3 and MDA-MB-231 cells, as well as their ability to survive/proliferate under anchorage-independent conditions. We conducted GC/LC-MS metabolomic profiling and measured lactate production following FSS exposure. To investigate oxidative burden on FSS exposed cells, we used dihydroethidium to measure general ROS production and a lipid peroxide sensor. Results: We found that FSS exposure shortens the time for metastatic colonization for both cell lines. We found that FSS exposure leads to approximately two-fold increase in invasion through collagen matrix, which, for PC-3 cells, is RhoA dependent. 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 increased lactate levels. We blocked glycolysis in PC-3 and MDA-MB-231 cells with 2-deoxyglucose 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. The rapid changes in metabolism 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. Citation Format: Devon L. Moose, Amanda N. Pope, Marion Dykstra, Eric B. Taylor, Patrick Breheny, Michael D. Henry. Fluid shear stress enhances metastatic potential and rapidly alters metabolism of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 199.

IntroductionAbl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in many solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation and motility and tumor growth in vivo in some settings.MethodsTo investigate the role of Abl kinases in tumor progression, we used RNAi to generate Abl-deficient cells in a model of androgen receptor-indifferent, metastatic prostate cancer. The effect of Abl kinase depletion on tumor progression and metastasis was studied in an in vivo orthotopic model, and tumor cell motility, 3D growth, and signaling was studied in vitro.ResultsReduced Abl family kinase expression resulted in a highly aggressive, metastatic phenotype in vivo that was associated with AKT pathway activation, increased growth on 3D collagen matrix, and enhanced cell motility in vitro. Inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells, while treatment with the Abl kinase inhibitor, imatinib, promoted 3D growth of multiple additional tumor cell types. Moreover, Abl kinase inhibition also promoted soft-agar colony formation by pre-malignant fibroblasts.ConclusionsCollectively, our data reveal that Abl family kinases can function to suppress malignant cell phenotypes in vitro, and tumor progression and metastasis in vivo.

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.

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.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (<1 dyne/cm(2)) and the highest levels present briefly as cells pass through the heart and around heart valves (>500 dynes/cm(2)). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Pancreatic neuroendocrine neoplasms (pNENs) are slow growing cancers of increasing incidence that lack effective treatments once they become metastatic. Unfortunately, nearly half of pNEN patients present with metastatic liver tumors at diagnosis and current therapies fail to improve overall survival. Pre-clinical models of pNEN metastasis are needed to advance our understanding of the mechanisms driving the metastatic process and for the development of novel, targeted therapeutic interventions. To model metastatic dissemination of tumor cells, human pNEN cell lines (BON1 and Qgp1) stably expressing firefly luciferase (luc) were generated and introduced into NSG immunodeficient mice by intracardiac (IC) or intravenous (IV) injection. The efficiency, kinetics and distribution of tumor growth was evaluated weekly by non-invasive bioluminescent imaging (BLI). Tumors formed in all animals in both the IC and IV models. Bioluminescent Qgp1.luc cells preferentially metastasized to the liver regardless of delivery route, mimicking the predominant site of pNEN metastasis in patients. By comparison, BON1.luc cells most commonly formed lung tumors following either IV or IC administration and colonized a wider variety of tissues than Qgp1.luc cells. These models provide a unique platform for testing candidate metastasis genes and anti-metastatic therapies for pNENs.

During metastasis, cancer cells traverse the circulation to reach distant organs. Conventionally, this journey has been regarded as mechanically destructive to circulating tumor cells from solid tissues. We have recently shown that cancer cells from diverse tissues actively resist destruction by fluid shear stress through a mechano-adaptive RhoA-actomyosin mechanism.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

Pharmacological ascorbate (P-AscH−, high-dose, intravenous vitamin C) is cytotoxic to tumor cells in doses achievable in humans. Phase I studies in pancreatic cancer (PDAC) utilizing P-AscH− have demonstrated increases in progression free survival, suggesting a reduction in metastatic disease burden. The purpose of this study was to determine the effects of P-AscH− on metastatic PDAC. Several in vitro and in vivo mechanisms involved in PDAC metastases were investigated following treatment with P-AscH−. Serum from PDAC patients in clinical trials with P-AscH− were tested for the presence and quantity of circulating tumor cell-derived nucleases. P-AscH− inhibited invasion, basement membrane degradation, decreased matrix metalloproteinase expression, as well as clonogenic survival and viability during exposure to fluid shear stress. In vivo, P-AscH− significantly decreased formation of ascites, tumor burden over time, circulating tumor cells, and hepatic metastases. Both in vitro and in vivo findings were reversed with the addition of catalase suggesting that the effect of P-AscH− on metastatic disease is mediated by hydrogen peroxide. Finally, P-AscH− decreased CTC-derived nucleases in subjects with stage IV PDAC in a phase I clinical trial. We conclude that P-AscH− attenuates the metastatic potential of PDAC and may prove to be effective for treating advanced disease.

During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.

Abl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation, motility, and in vivo tumor growth in some settings. To investigate the role of Abl kinases in prostate cancer, we generated Abl-deficient cells in a pre-clinical model of spontaneously metastatic, androgen-indifferent prostate cancer. Loss of Abl family kinase expression resulted in a highly aggressive, metastatic phenotype in vivo that was associated with AKT pathway activation, increased growth on 3D collagen matrix, and enhanced cell motility in vitro . Treatment of Abl kinase-expressing cells with the Abl kinase inhibitor imatinib phenocopied the malignant phenotypes observed in Abl-deficient tumor cells. In addition, inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells. Our data reveal that Abl family kinases can function as suppressors of prostate cancer progression and metastasis by restraining AKT signaling, a signaling pathway known to be associated with emergence of metastatic castration-resistant prostate cancer.### Competing Interest StatementThe authors have declared no competing interest.

Epithelial-to-mesenchymal transition (EMT) is implicated in cancer metastasis and drug resistance. Specifically targeting cancer cells in an EMT-like state may have therapeutic value. In this study, we developed a cell imaging-based high-content screening protocol to identify EMT-selective cytotoxic compounds. Among the 2,640 compounds tested, salinomycin and monensin, both monovalent cation ionophores, displayed a potent and selective cytotoxic effect against EMT-like cells. The mechanism of action of monensin was further evaluated. Monensin (10 nM) induced apoptosis, cell cycle arrest, and an increase in reactive oxygen species (ROS) production in TEM 4-18 cells. In addition, monensin rapidly induced swelling of Golgi apparatus and perturbed mitochondrial function. These are previously known effects of monensin, albeit occurring at much higher concentrations in the micromolar range. The cytotoxic effect of monensin was not blocked by inhibitors of ferroptosis. To explore the generality of our findings, we evaluated the toxicity of monensin in 24 human cancer cell lines and classified them as resistant or sensitive based on IC50 cutoff of 100 nM. Gene Set Enrichment Analysis identified EMT as the top enriched gene set in the sensitive group. Importantly, increased monensin sensitivity in EMT-like cells is associated with elevated uptake of 3H-monensin compared to resistant cells.

During metastasis cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level fluid shear stress (FSS) in vitro relative to non-transformed epithelial cells. Herein we identify a mechanism of FSS resistance in cancer cells, and extend these findings to mouse models of circulating tumor cells (CTCs). We show that cancer cells acutely isolated from primary tumors are resistant to FSS. Our findings demonstrate that cancer cells activate the RhoA-myosin II axis in response to FSS, which protects them from FSS-induced plasma membrane damage. Moreover, we show that the myosin II activity is protective to CTCs in mouse models. Collectively our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III (MP81)1 Apr 2019MP81-02 LARGE, LOCALLY ADVANCED TUMOR PROGRESSION IN TWO PTEN/TRP53 DOUBLE KNOCKOUT (DOKO) MOUSE MODELS MONITORED WITH BIOLUMINESCENCE IMAGING (BLI) Courtney Yong*, Devon Moose, Nadine Bannick, Marion Vanneste, James A. Brown, Michael B. Cohen, and Michael D. Henry Courtney Yong*Courtney Yong* More articles by this author , Devon MooseDevon Moose More articles by this author , Nadine BannickNadine Bannick More articles by this author , Marion VannesteMarion Vanneste More articles by this author , James A. BrownJames A. Brown More articles by this author , Michael B. CohenMichael B. Cohen More articles by this author , and Michael D. HenryMichael D. Henry More articles by this author View All Author Informationhttps://doi.org/10.1097/01.JU.0000557421.72582.aeAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVES: We previously described a prostate-specific Pten knockout mouse with a genetically engineered luciferase reporter to monitor disease progression with BLI (Svensson et al, 2011, Am J Path). Here, we describe two Pten/Trp53 DOKO models of prostate cancer with genetically engineered luciferase reporters to monitor disease progression with BLI. METHODS: We generated 3 cohorts of Probasin (Pb)-Cre mediated Pten -/- combined with either Trp53 +/+ (WT), Trp53 ± (HET), or Trp53 -/- (DOKO) mice on an albino C57BL/6 background with a Cre-activatable luciferase reporter allele. An additional inducible DOKO model was created by intraprostatic injection of CMV-Cre or Keratin-5 (K5)-Cre adeno-associated virus. The virus with the CMV promoter will induce broad Cre expression in the prostate, while the K5 will be targeted to the basal cells. BLI was measured biweekly starting at 5 weeks of age for the Pb-Cre mice and after injections for the viral Cre mice. We analyzed BLI data and histopathology for each of the cohorts. RESULTS: The Pb-Cre DOKO mice develop large, locally invasive tumors that grow more quickly than the HET or WT genotypes. This difference is evident on BLI at 21 weeks of age (p=0.0009). The DOKO mice also have shorter survival compared to the HET or WT, with a median survival of 26.5 weeks compared to 50 and 41 weeks for WT and HET mice (p<0.0001). Pathologic analysis of the 3 groups showed sarcomatoid carcinoma in all DOKO mice, 41% of HET mice, and none of WT mice; high grade murine prostatic intraepithelial neoplasia (mPIN) in 35% of HET and 73% of WT mice; and low grade mPIN in 11% of HET and 13% of WT mice. There was invasion around pelvic lymphatics, but there was no pathologic confirmation of metastasis. To test androgen sensitivity, we castrated a separate cohort of Pb-Cre DOKO mice at age 10 weeks. BLI signal reached a nadir at 14 weeks but increased steadily to levels comparable to their non-castrate, age-matched counterparts. The Pten/Trp53 DOKO mouse cohorts with virally induced Cre are still active. Three out of five CMV-Cre mice have developed tumors evident on BLI at week 9; 2 of these were fatal at week 11. Of the K5-Cre mice, 2 out of 7 have tumors evident on BLI at week 11, and all are alive at present. CONCLUSIONS: The Pten/Trp53 DOKO mouse model of prostate cancer develops large, locally advanced, and fatal prostate tumors of high-grade pathology that are initially castrate sensitive but develop castrate resistance. Our model also demonstrates the use of noninvasive BLI to monitor tumor progression. Source of Funding: NIH Grant R21CA137490 Iowa City, IA; Winston-Salem, NC; Iowa City, IA© 2019 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 201Issue Supplement 4April 2019Page: e1171-e1171 Advertisement Copyright & Permissions© 2019 by American Urological Association Education and Research, Inc.MetricsAuthor Information Courtney Yong* More articles by this author Devon Moose More articles by this author Nadine Bannick More articles by this author Marion Vanneste More articles by this author James A. Brown More articles by this author Michael B. Cohen More articles by this author Michael D. Henry More articles by this author Expand All Advertisement PDF downloadLoading ...

Metastatic, drug-resistant cancer is lethal. Epithelial-to-mesenchymal transition (EMT) is a type of phenotypic plasticity that has been implicated in cancer metastasis and drug resistance. One potential therapeutic approach is to specifically target cancer cells in an EMT-like state. In this study, we have developed a cell-based high-throughput screening (HTS) protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on epithelial (PC-3E) and mesenchymal-like (TEM 4-18) cell lines, both isolated from the parental PC-3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds was screened on co-cultured PC-3E GFP cells and TEM 4-18 mCherry cells. After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The mechanisms of action of one of the most potent and selective compound against EMT-like cells, monensin, were further evaluated. Among the 2,640 compounds tested, salinomycin and monensin, both monovalent cation ionophores, displayed a potent and selective cytotoxic effect against EMT-like cells. A closely related compound absent from the compound library, nigericin, was also evaluated and appeared to be the most potent of the three compounds tested. Monensin (10nM) induced apoptosis, cell cycle arrest, and an increase in ROS production in TEM 4-18 cells. In addition, monensin rapidly induced swelling of Golgi apparatus, a previously known effect of monensin, in multiple cell lines most likely resulting in a blockage of intracellular protein trafficking and cell death. We then evaluated the toxicity of monensin in 24 human cancer cell lines and classified them as resistant or sensitive based on IC50 cutoff of 100nM. Supporting these findings, Gene Set Enrichment Analysis identified EMT as the top of the gene sets enriched in the sensitive group. Increased monensin sensitivity in EMT-like cells is associated with elevated uptake of H3-monensin compared to resistant cells. In conclusion, using a high-throughput screening approach, we identified monensin as a potent and specific inhibitor of cancer cells in an EMT-like state where it rapidly disrupts Golgi function. At least part of selectivity of monensin for EMT-like cells is due to increased uptake of this compound. Citation Format: Marion Vanneste, Huang Qin, Devon Moose, Mengshi Li, Michael Schultz, Meng Wu, Michael Henry. Monensin is selectively cytotoxic to cancer cells in an EMT-like state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4186.

Metastatic disease is the leading cause of pancreatic ductal adenocarcinoma (PDAC) associated death. PDAC cells invade and enter the bloodstream early, before frank malignancy can be detected. Our objective was to develop an in vivo assay enabling the identification and quantification of circulating tumor cells (CTCs) from primary orthotopic PDAC tumors. Human PDAC cells expressing luciferase and green fluorescent protein were orthotopically injected into the pancreas of mice utilizing ultrasound guidance. Bioluminescent imaging was conducted to identify and track tumor growth. CTCs were then isolated and analyzed by flow cytometry to detect GFP-expressing cancer cells. Tumor growth as measured by bioluminescent imaging increased over time. The concentration of CTCs correlated with the strength of bioluminescent imaging signal. In addition, livers bearing macroscopic disease were harvested for further imaging under fluorescence stereomicroscopy and confocal microscopy, which confirmed the presence of metastases. This study represents an orthotopic animal model that reliably detects the presence of CTCs from PDAC. There is a positive correlation between the concentrations of CTCs with overall tumor burden.