Circulating Tumor Cells Lines Research Articles

Analysis of the Plasticity of Circulating Tumor Cells Reveals Differentially Regulated Kinases During the Suspension-to-Adherent Transition.

Research on circulating tumor cells (CTCs) offers the opportunity to better understand the initial steps of blood-borne metastasis as main cause of cancer-related deaths. Here, we have used the colon cancer CTC-MCC-41 and breast cancer CTC-ITB-01 lines, which were both established from human CTCs as permanent cell lines as models to further study CTC biology with special emphasis on anchorage-independent survival and growth. Both cell lines showed a marked intrinsic plasticity to switch between suspension and adherent invitro growth, in 2D adherent culture conditions, and established an equilibrium of both growth patterns with predominant adherent cells in the CTC-MCC-41 line (77%) and suspension cells in the CTC-ITB-01 line (85%). Western blot analysis revealed a higher expression of pERK1/2 in CTC-ITB-01 adherent cells compared to the suspension counterpart that suggested the involvement of kinases in this process. Subsequent functional kinome profiling identified several serine/threonine as well as tyrosine kinases that were differentially regulated in adherent and suspension CTCs. In the adherent cells of the breast cancer line CTC-ITB-01 the activity of MSK1, Src family kinases and the PKG family was increased compared to the suspension counterpart. In adherent cells of the colorectal CTC-MCC-41 line, an increased activity of TYRO3 and JAK2 was detected, whereas p38 MAPK was strongly impaired in the suspension CTC-MCC-41 cells. Some of the regulated kinases, which include the Src family, TYRO3, MSK1, JAK2 and p38 MAPK, have been associated with crucial cellular processes including proliferation, migration and dormancy in the past. The investigated CTC lines exhibit a high plasticity, similar to the concept of 'adherent-to-suspension transition (AST)' that was recently suggested as a new hallmark of tumor biology by Huh etal. Moreover, we identified differentially regulated kinome profiles that may represent potential targets for future studies on therapeutic interventions.

Remembering Hypoxia: Uncovering the Long-Term Effects of Transient Oxygen Deprivation on IFN Signaling and Progression of Breast Cancer.

Hypoxia occurs in 90% of solid tumors and is strongly associated with an increased propensity for metastasis. Hypoxia induces tumor progression largely through inducing HIF-mediated transcription, resulting in alterations to tumor cell metabolism, as well as increases in migration and invasion. Hypoxia also results in a myriad of changes to the tumor microenvironment (TME). While many studies have examined the immediate effects of hypoxia on tumor cells and the associated TME, far fewer have focused on the long-term consequences of transient reductions in oxygen. In this issue of Cancer Research, Iriondo and colleagues examined whether short-term exposure to hypoxia leads to a "hypoxic memory" in the context of breast cancer. The authors used established cell lines and circulating tumor cell lines to demonstrate that these cells harbor a hypoxic memory that sustains downregulation of IFN signaling and antigen presentation (AP) pathways that contribute to tumor progression via alterations to tumor cells and the TME. The authors further showed that cells that have experienced hypoxia maintain the reduction in IFN signaling in vivo and are more aggressive. They determined that the hypoxic memory and reduction of IFN signaling can be reversed with a histone deacetylase inhibitor, entinostat, providing a potential means to reverse hypoxia-induced suppression of IFN signaling. As suppression of IFN signaling has the potential to influence both tumor cells and the TME, the identification of a strategy to inhibit long-term suppression of IFN signaling downstream of hypoxia could prove to be an effective means to target tumor progression. See related article by Iriondo et al., p. 3141.

Hypoxic Memory Mediates Prolonged Tumor-Intrinsic Type I Interferon Suppression to Promote Breast Cancer Progression.

Hypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis that is associated with tumor progression and metastasis. Most of the well-known hypoxia effects are mediated through hypoxia-inducible factors (HIF). Identification of the long-lasting effects of hypoxia beyond the immediate HIF-induced alterations could provide a better understanding of hypoxia-driven metastasis and potential strategies to circumvent it. Here, we uncovered a hypoxia-induced mechanism that exerts a prolonged effect to promote metastasis. In breast cancer patient-derived circulating tumor cell lines and common breast cancer cell lines, hypoxia downregulated tumor-intrinsic type I IFN signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells, via both HIF-dependent and HIF-independent mechanisms. Hypoxia induced durable IFN/AP suppression in certain cell types that was sustained after returning to normoxic conditions, presenting a "hypoxic memory" phenotype. Hypoxic memory of IFN/AP downregulation was established by specific hypoxic priming, and cells with hypoxic memory had an enhanced ability for tumorigenesis and metastasis. Overexpression of IRF3 enhanced IFN signaling and reduced tumor growth in normoxic, but not hypoxic, conditions. The histone deacetylase inhibitor entinostat upregulated IFN targets and erased the hypoxic memory. These results point to a mechanism by which hypoxia facilitates tumor progression through a long-lasting memory that provides advantages for circulating tumor cells during the metastatic cascade. Significance: Long-term cellular memory of hypoxia leads to sustained suppression of tumor-intrinsic type I IFN signaling and the antigen presentation pathway that facilitates tumorigenesis and metastasis. See related commentary by Purdy and Ford, p. 3125.

Establishing Single-Cell Clones from In Vitro-Cultured Circulating Tumor Cells.

Cancer is a common health problem with more than 90% of deaths due to metastases. Circulating tumor cells (CTCs) contain precursors that can initiate metastases. However, CTCs are rare, heterogeneous, and difficult to expand in culture. We have previously created CTC-derived cell lines from stage IV breast cancer patients. These CTC lines were used to establish single-cell CTC clones using flow cytometry cell sorting.

In vitro cross-talk between metastasis-competent circulating tumor cells and platelets in colon cancer: a malicious association during the harsh journey in the blood.

Background: Platelets are active players in hemostasis, coagulation and also tumorigenesis. The cross-talk between platelets and circulating tumor cells (CTCs) may have various pro-cancer effects, including promoting tumor growth, epithelial-mesenchymal transition (EMT), metastatic cell survival, adhesion, arrest and also pre-metastatic niche and metastasis formation. Interaction with CTCs might alter the platelet transcriptome. However, as CTCs are rare events, the cross-talk between CTCs and platelets is poorly understood. Here, we used our established colon CTC lines to investigate the colon CTC-platelet cross-talk in vitro and its impact on the behavior/phenotype of both cell types. Methods: We exposed platelets isolated from healthy donors to thrombin (positive control) or to conditioned medium from three CTC lines from one patient with colon cancer and then we monitored the morphological and protein expression changes by microscopy and flow cytometry. We then analyzed the transcriptome by RNA-sequencing of platelets indirectly (presence of a Transwell insert) co-cultured with the three CTC lines. We also quantified by reverse transcription-quantitative PCR the expression of genes related to EMT and cancer development in CTCs after direct co-culture (no Transwell insert) with platelets. Results: We observed morphological and transcriptomic changes in platelets upon exposure to CTC conditioned medium and indirect co-culture (secretome). Moreover, the expression levels of genes involved in EMT (p < 0.05) were decreased in CTCs co-cultured with platelets, but not of genes encoding mesenchymal markers (FN1 and SNAI2). The expression levels of genes involved in cancer invasiveness (MYC, VEGFB, IL33, PTGS2, and PTGER2) were increased. Conclusion: For the first time, we studied the CTC-platelet cross-talk using our unique colon CTC lines. Incubation with CTC conditioned medium led to platelet aggregation and activation, supporting the hypothesis that their interaction may contribute to preserve CTC integrity during their journey in the bloodstream. Moreover, co-culture with platelets influenced the expression of several genes involved in invasiveness and EMT maintenance in CTCs.

Small cell lung cancer: circulating tumor cell lines and expression of mediators of angiogenesis and coagulation.

Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a putative release of tissue factor (TF) by circulating tumor cells (CTCs) represents a target to impair the dissemination of small cell lung cancer (SCLC), the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines that have been established at the Medical University of Vienna. Five CTC and SCLC lines were analyzed using a TF enzyme-linked immunosorbent assay (ELISA) tests, RNA sequencing, and western blot arrays covering 55 angiogenic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators was investigated. The results demonstrate that the SCLC CTC cell lines express no significant amounts of active TF but thrombospondin-1 (TSP-1), urokinase-type plasminogen activator receptor (uPAR), vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 in two cases. The major difference between the SCLC and SCLC CTC cell lines found was the loss of the expression of angiogenin in the blood-derived CTC lines. Topotecan and epirubicin decreased the expression of VEGF, whereas hypoxia-like conditions upregulated VEGF. Active TF capable of triggering coagulation seems not to be expressed in SCLC CTC cell lines in significant levels and, thus, CTC-derived TF seems dispensable for dissemination. Nevertheless, all CTC lines form large spheroids, termed tumorospheres, which may become trapped in clots of the microvasculature and extravasate in this supportive microenvironment. The contribution of clotting to the protection and dissemination of CTCs in SCLC may be different from other solid tumors such as breast cancer.

Abstract IA008: Biology of metastasis-competent circulating tumor cells

Abstract The characterization of circulating tumor cells (CTCs) holds promises for precision medicine because these cells are an important clinical indicator of treatment efficacy. We established the first and still only nine permanent colon CTC lines from peripheral blood samples of a patient with metastatic colon cancer collected at different time points during treatment and cancer progression. The study objectives were (i) to compare the gene expression profiles of these CTC lines, and (ii) to determine the main features acquired during treatment. The number of upregulated genes was higher in the CTC lines obtained after treatment, indicating that they acquired properties to escape treatment pressure. Among these upregulated genes, some are involved in the mTOR and PI3K/AKT signaling pathways. Moreover, cytidine deaminase expression was significantly increased in the CTC lines obtained after failure of the first- and second-line 5-fluorouracile-based treatments, suggesting that these CTCs can eliminate this specific drug and resist to therapy. Several enzymes involved in xenobiotic metabolism also were upregulated after treatment, suggesting the activation of detoxification mechanisms in response to chemotherapy. Finally, the significant higher expression of aldolase B in four of the six CTC lines obtained after treatment withdrawal and cancer progression indicated that these clones originated from liver metastases. In conclusion, these CTC lines generated at different time points during treatment of metastatic colon cancer in a single patient are characterized by the deregulation of different genes that promote (i) drug resistance, (ii) xenobiotic and energy metabolism, and (iii) stem cell properties and plasticity. Citation Format: Catherine Alix-Panabières. Biology of metastasis-competent 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 IA008.

Changes of protein expression during tumorosphere formation of small cell lung cancer circulating tumor cells.

Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times of approximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs are distinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTC lines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition (EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion is markedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showed variable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres.

Abstract 611: Assessment of tumor mutation burden and microsatellite instability by single-cell circulating tumor cell genomic profiling

Abstract The success of immune checkpoint inhibitors rests on biomarkers such as tumor mutation burden (TMB) and microsatellite instability (MSI), both FDA-approved predictors of anti PD-1/L1 therapy benefit. Tissue biopsies often collected once in the metastatic setting through an invasive procedure, or archived primary tumor tissue often collected much prior to treatment consideration, are the specimen types of choice for biomarker identification. The tissue sample originates from a limited region of one disease site, which may limit its usefulness given intra-patient tumor heterogeneity. TMB and MSI measurement by liquid biopsy, including proteins, circulating tumor cells (CTC), and cell-free circulating tumor DNA (ctDNA), is an attractive, minimally-invasive way to obtain a real-time picture of the entire disease. While TMB and MSI assessment from ctDNA have been reported, their measurement can be limited by low ctDNA tumor fraction. Single-cell next generation sequencing of CTC, on the other hand, is a particularly well-suited, but largely unexplored method of measuring TMB and MSI to complement tissue and ctDNA for better overall specificity of detection. In this proof-of-concept study, we show the ability to detect single-cell TMB and MSI. We analyzed 14 CTC and 4 ctDNA samples from 6 metastatic breast cancer patients, as well as 3 single cells and 1 cell pellet sample each from HCT-116 (MSI-High) and WiDr (MSI-Low) cell lines. CTC and cell line cells were enriched with the CellSearch® system and/or isolated with the DEPArray™ system. Whole genome amplified single-cell DNA was sequenced with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space. TMB and MSI scores obtained in CTC and ctDNA were compared to those measured in matched clinical tissue biopsies. Single-cell TMB scores and MSI status were assessable in all CTC tested. CTC TMB scores were highly concordant with the matched tissue samples (r=1.00), as were ctDNA TMB scores (r=0.98) in patients with assessable TMB scores in both biospecimen types compared. Importantly, TMB was detectable in CTC from one patient whose tissue sample was inadequate for clinical sequencing, and from another patient with inadequate, low tumor fraction ctDNA. Intriguingly, one patient harbored 3 TMB-high and 2 TMB-low CTC, potentially indicating intra-patient TMB heterogeneity. The known MSI-low status from clinical tumor tissue sequencing was correctly detected in CTC and ctDNA from all patients. MSI status and scores from single cells of HCT-116 and WiDr cell lines purified with the DEPArray™ system (mimicking CTC isolation), perfectly matched that of the corresponding cell pellet samples (r=1.00). Taken together, these data suggest the potential validity and continued interrogation of potential utility of CTC TMB and MSI detection to complement tissue and ctDNA in guiding checkpoint inhibitor immunotherapy. Citation Format: Andi K. Cani, Emily M. Dolce, Chia-Jen Liu, Brittany Rupp, Elizabeth P. Darga, Costanza Paoletti, Dafydd G. Thomas, Yi-Mi Wu, Dan R. Robinson, Sunitha Nagrath, Arul M. Chinnaiyan, Scott A. Tomlins, Aaron M. Udager, John M. Carethers, Erin F. Cobain, Daniel F. Hayes. Assessment of tumor mutation burden and microsatellite instability by single-cell circulating tumor cell genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 611.

Abstract 2437: Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53

Abstract Human breast cancers often overexpress MDM family proteins MDM2 and MDMX(MDM4). MDM2 is an E3 ubiquitin ligase and MDMX is an E4 that promotes ubiquitination of MDM2 targets (however this is an understudied area). The Cancer Genome Atlas (TCGA) shows high MDMX/MDM2 expression in all breast cancer subtypes. We identified that MDMX depletion reduces circulating tumor cells (CTCs) and metastasis of breast cancers with mtp53. Our ongoing objective is to determine the mechanisms of how MDMX promotes CTCs and metastasis, and the potential MDMX regulated ubiquitinated targets. In TNBC primary tumors, knockdown of mdmx correlates with reduced expression of the G protein coupled receptor, CXCR4. We used mtp53 expressing T47D ER+, and MDA-MB-468, and MDA-MB-231 metastatic TNBC cells and their derived CTC lines, and shRNA-mediated knockdown of MDM2 and MDMX derived lines, to address the potential MDMX targets. We assessed metastatic properties of migration and invasion changed by the knockdown of MDM2/MDMX in parental and CTC cell lines using multi-faceted cell biological and biochemical approaches. Depletion of MDM2 in T47D and MDA-MB-231 cells increased the level of MDMX, demonstrating a critical function of MDM2 for regulating MDMX. MDMX depletion surprisingly did not change MDM2 levels, but did reduce the migration of both MDA-MB-231 cells and their derived CTC lines. Interestingly, the parental MDA-MB-231 cells migrated more rapidly in scratch assays, compared to both their derived CTCs and MDMX-depleted CTCs, suggesting migration to be critical for intravasation, but not for latter cancer cell survival. Experiments are in progress to address if loss of MDMX perturbs CXCL12 ligand communication with the CXCR4 receptor in parental and CTC breast cancer lines. Preliminary data suggests activated MDMX expression correlates with the activation of these metastasis signal transduction pathways. The ubiquitination targets of MDMX/MDM2 in the parental and CTC cell lines are being defined by affinity purification in the presence, and absence, of MDMX/MDM2, and add back of purified MDMX/MDM2 to validate targets. As expected, purified MDMX increases MDM2 E3 ubiquitin ligase activity, and on its own MDMX is inert. Stable isotope labeling in cell culture suggested protein targets involved in DNA replication and repair pathways. This preliminary data informed our interest in studying DNA damage signaling by pharmacologically inhibiting the DNA repair protein PARP. We examined how MDMX/MDM2 pathways respond to DNA damage caused by PARP inhibition. Our preliminary data affirms regulation of the MDMX/MDM2 stress-response circuit by replication stress response pathways, and reveals the requirement of MDMX/MDM2 for their function in the studied cell lines. Disrupting MDMX function has potential to be beneficial for inhibition of breast cancer progression and metastasis. Supported by BCRF-20-011. Citation Format: Rusia Lee, Viola Ellison, Gu Xiao, Dominique Forbes, Pam Leybengrub, Alexandra Kern, Falande Alexandre, George Annor, Jill Bargonetti. Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2437.

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice. Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo. In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Epithelial-to-Mesenchymal Plasticity in Circulating Tumor Cell Lines Sequentially Derived from a Patient with Colorectal Cancer.

Simple SummaryMetastasis is a complex dynamic multistep process; however, our knowledge is still limited. Very few circulating tumor cells (CTCs) are metastatic precursor cells and represent the intermediate stage of metastasis. Epithelial–mesenchymal plasticity (EMP) has crucial roles in tissue development and homeostasis, and also in metastasis formation. In this study, we explored the EMP phenotype of a unique series of CTC lines, obtained from a patient with colon cancer during the disease course and treatment, by detecting markers involved in the epithelial–mesenchymal and mesenchymal–epithelial (MET) transitions. This study shows that these colon CTC lines have acquired only few mesenchymal features to migrate and intravasate, whereas an increase of MET-related markers was observed, suggesting that metastasis-competent CTCs need to revert quickly to the epithelial phenotype to reinitiate a tumor at a distant site.Metastasis is a complicated and only partially understood multi-step process of cancer progression. A subset of cancer cells that can leave the primary tumor, intravasate, and circulate to reach distant organs are called circulating tumor cells (CTCs). Multiple lines of evidence suggest that in metastatic cancer cells, epithelial and mesenchymal markers are co-expressed to facilitate the cells’ ability to go back and forth between cellular states. This feature is called epithelial-to-mesenchymal plasticity (EMP). CTCs represent a unique source to understand the EMP features in metastatic cascade biology. Our group previously established and characterized nine serial CTC lines from a patient with metastatic colon cancer. Here, we assessed the expression of markers involved in epithelial–mesenchymal (EMT) and mesenchymal–epithelial (MET) transition in these unique CTC lines, to define their EMP profile. We found that the oncogenes MYC and ezrin were expressed by all CTC lines, but not SIX1, one of their common regulators (also an EMT inducer). Moreover, the MET activator GRHL2 and its putative targets were strongly expressed in all CTC lines, revealing their plasticity in favor of an increased MET state that promotes metastasis formation.

N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses.

Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a "liquid biopsy" of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. IMPLICATIONS: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.

High Sensitivity of Circulating Tumor Cells Derived from a Colorectal Cancer Patient for Dual Inhibition with AKT and mTOR Inhibitors.

Circulating tumor cells (CTCs) are cells shed from the primary tumor into the bloodstream. While many studies on solid tumor cells exist, data on CTCs are scarce. The mortality of cancer is mostly associated with metastasis and recent research identified CTCs as initiators of metastasis. The PI3K/AKT/mTOR signaling pathway is an intracellular pathway that regulates essential functions including protein biosynthesis, cell growth, cell cycle control, survival and migration. Importantly, activating oncogenic mutations and amplifications in this pathway are frequently observed in a wide variety of cancer entities, underlining the significance of this signaling pathway. In this study, we analyzed the functional role of the PI3K/AKT/mTOR signaling pathway in the CTC-MCC-41 line, derived from a patient with metastatic colorectal cancer. One striking finding in our study was the strong sensitivity of this CTC line against AKT inhibition using MK2206 and mTOR inhibition using RAD001 within the nanomolar range. This suggests that therapies targeting AKT and mTOR could have been beneficial for the patient from which the CTC line was isolated. Additionally, a dual targeting approach of AKT/mTOR inside the PI3K/AKT/mTOR signaling pathway in the colorectal CTCs showed synergistic effects in vitro. Depending on the phenotypical behavior of CTC-MCC-41 in cell culture (adherent vs. suspension), we identified altered phosphorylation levels inside the PI3K/AKT/mTOR pathway. We observed a downregulation of the PI3K/AKT/mTOR signaling pathway, but not of the RAS/RAF/MAPK pathway, in CTCs growing in suspension in comparison to adherent CTCs. Our results highlight distinct functions of AKT isoforms in CTC-MCC-41 cells with respect to cell proliferation. Knockdown of AKT1 and AKT2 leads to significantly impaired proliferation of CTC-MCC-41 cells in vitro. Therefore, our data demonstrate that the PI3K/AKT/mTOR signaling pathway plays a key role in the proliferation of CTC-MCC-41.

Abstract LB-271: Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s)

Abstract Introduction: Circulating tumor cells (CTCs) are liquid biomarkers with high potential for application in precision medicine cancer care. However, no robust methods have been developed to culture CTCs from early-stage cancer patients owing to their rarity and instability. Here, we describe a successful protocol to establish CTC culturing and CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer (NSCLC) patients. Further, comparative multi-omics characterization of these CDX tumors revealed novel diagnostic/therapeutic target(s). Methods: Surgically resected primary tumor fragments from patients with early stage NSCLCs were subcutaneously (s.c.) implanted to immunodeficient NOD SCID gamma (NSG) mice to generate patient-derived xenograft (PDX) models. Once palpable tumor reached a diameter of ~1.5 cm, animals were euthanized and blood was collected, CTCs were enriched with OncoQuick® and cultured in vitro. These patient-derived CTCs were further stained with anti-human antibodies against CK7, Napsin, TTF-1, CK5/6, PDL1 and CD45. Once human CTCs were confirmed, we then injected expanded CTCs s.c. to naïve NSG mice to establish CDX models. Single cell suspension from CDX tumors was cultured and fibroblasts were depleted using specific beads (Miltenyi biotec) to establish stable CTC lines. Further, advanced proteomics, single cell RNA sequencing (transcriptomics) and whole genome sequencing of patient-matched peritumoral and primary tumor tissues along with PDX and CDX tumors were carried out. Results: Human NSCLC primary tumor fragments implanted in NSG mice were tumorigenic at the s.c. implantation site, metastatic to distant organs, and passaged successfully to naïve NSG generations to maintain the PDX colonies. CTCs enriched from the PDX models were successfully cultured in vitro and positive for human NSCLC-specific markers but negative for human leukocyte marker CD45. Cultured PDX-derived CTCs that were xenografted into naïve NSG mice by s.c. demonstrated tumorigenicity and metastatic potential in CDX models. Stable CTC lines established using CDX tumors sustained &amp;gt;30 passages. Global proteomic profiling of tumor tissues revealed 19 new proteins that were upregulated and 9 proteins that are down regulated. Single cell RNA sequence analysis depicts top rank genes that are overexpressed in tumor cluster. Whole genome sequencing of these samples identified somatic variants that needs further validation. Conclusion and future perspectives: This approach establishes CDX models from early-stage NSCLC patients at a high rate for personalized precision medicine. Models provide a unique opportunity to characterize rare patient-derived CTCs that helps in understanding CTC tumor biology and developing effective anti-cancer therapeutic strategies. Multi-omics characterization of these tissues identified a set of novel genes/proteins that needs to be validated in large patient population. Further, studying tumorigenic potential of established CTC lines after knockdown and overexpression of these genes will provide more information about translating these targets into therapeutics. Citation Format: Kanve N. Suvilesh, Yariswamy Manjunath, Vijay Radhakrishnan, Diego M. Avella, Eric T. Kimchi, Kevin F. Staveley-O'Carroll, Guangfu Li, Jussuf T. Kaifi. Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-271.

Circulating Tumor Cell Migration Requires Fibronectin Acting through Integrin B1 or SLUG

Fibronectin (FN1) is an extracellular matrix protein gaining increasing attention for its multifaceted roles in cancer progression. Using our recently established circulating tumor cell (CTC) lines, we had demonstrated increased FN1 expression and enhanced migration in CTC lines, in comparison to primary tumor cell lines. Whether increased FN1 expression is directly required for CTC migration, and the specific role of FN1’s regulation of integrin B1 (ITGB1) and SLUG (SNAI2) in CTC migration remains unclear. Here, for the first time, we report that the knockdown of FN1, ITGB1, or SLUG expression in CTCs leads to a significant decrease in CTC migration. Knocking down two or all three of these proteins simultaneously did not further inhibit migration. We observed a corresponding increase in CTC migration when recombinant FN1 was added to CTCs. This effect was significantly impeded by prior knockdown of ITGB1 or SLUG. Using knock down experiments and western blotting analysis, we confirmed FN1’s regulation of ITGB1 and SLUG to occur via two separate, independent pathways. Consequently, we can conclude that FN1-dependent enhanced migration of CTCs requires downstream signaling through either ITGB1 or SLUG and that FN1 regulation of ITGB1 and SLUG may have important implications for cancer progression and metastasis.

Expansion of Circulating Tumor Cells from Patients with Locally Advanced Pancreatic Cancer Enable Patient Derived Xenografts and Functional Studies for Personalized Medicine.

Improvement in pancreatic cancer treatment represents an urgent medical goal that has been hampered by the lack of predictive biomarkers. Circulating Tumor Cells (CTCs) may be able to overcome this issue by allowing the monitoring of therapeutic response and tumor aggressiveness through ex vivo expansion. The successful expansion of CTCs is challenging, due to their low numbers in blood and the high abundance of blood cells. Here, we explored the utility of pancreatic CTC cultures as a preclinical model for treatment response. CTCs were isolated from ten patients with locally advanced pancreatic cancer using the Labyrinth, a biomarker independent, size based, inertial microfluidic separation device. Three patient-derived CTC samples were successfully expanded in adherent and spheroid cultures. Molecular and functional characterization was performed on the expanded CTC lines. CTC lines exhibited KRAS mutations, consistent with pancreatic cancers. Additionally, we evaluated take rate and metastatic potential in vivo and examined the utility of CTC lines for cytotoxicity assays. Patient derived expanded CTCs successfully generated patient derived xenograft (PDX) models with a 100% take rate. Our results demonstrate that CTC cultures are possible and provide a valuable resource for translational pancreatic cancer research, while also providing meaningful insight into the development of distant metastasis, as well as treatment resistance.

In-flow measurement of cell-cell adhesion using oscillatory inertial microfluidics.

Multicellular clusters in circulation can exhibit a substantially different function and biomarker significance compared to individual cells. Notably, clusters of circulating tumor cells (CTCs) are much more effective initiators of metastasis than single CTCs, and correlate with worse patient prognoses. Measuring the cell-cell adhesion strength of CTC clusters is a critical step towards understanding their subsistence in the circulation and mechanism of elevated tumorigenicity. However, measuring cell-cell adhesion forces in flow is elusive using existing methods. Here, we report an oscillatory inertial microfluidics system which exerts a repeating fluidic force profile on suspended cell doublets to determine their cell-cell adhesion strength (Fs), without any biophysical modifications to the cell surface and physiological morphology. Using our system, we analyzed a large number (N > 500) of doublets from a patient-derived breast cancer CTC line. We discovered that the cell-cell adhesion strength of CTC doublets varied almost 20-fold between the weakly adhered (Fs < 28 nN) and strongly bound subpopulations (Fs > 542 nN). Our system can be used with other cancer or noncancer cells without restrictions, and may be used for rapid screening of drugs aiming to disrupt the highly-metastatic CTC clusters in circulation.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines

SummaryTumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.