Circulating Tumor Cells Clusters Research Articles

The Presence of Circulating Tumor Cell Cluster Characterizes an Aggressive Hepatocellular Carcinoma Subtype.

BackgroundGrowing evidence suggests that circulating tumor cell (CTC) clusters may be an important factor in the metastatic process, but their role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to characterize the molecular and clinical features of CTC cluster-positive human HCC and to assess its prognostic value in HCC patients.MethodsThe CTCs and CTC clusters were evaluated in 204 HCC patients using CellSearch™ System. The counts of CTCs and CTC clusters were correlated with different clinical features, while their associations with progression-free survival (PFS) and overall survival (OS) were evaluated integrally and hierarchically by Kaplan–Meier estimates or Cox proportional regression analysis. Five cases each of CTC cluster-negative and cluster-positive patients were selected for RNA-sequencing analysis. The results of gene enrichment analysis were further verified using tissue microarray (TMA) by immunohistochemistry (IHC).ResultsCTCs and CTC clusters were detected in 76 (37.3%) and 19 (9.3%) of 204 preoperative samples, respectively. CTC cluster-positive HCC represented an aggressive HCC phenotype with larger tumor size, more frequent microvascular invasion, and higher tumor stages. The survival of HCC patients utilizing CTCs and CTC clusters individually showed prognostic significance, while joint analysis revealed patients in Group III (CTC ≥ 2 and CTC cluster > 0) had the worst outcome. Stratified analysis of outcomes in Barcelona Clinic Liver Cancer (BCLC) and tumor–node–metastasis (TNM) stages indicated that patients with CTC clusters had significantly poorer prognosis in each stage than those without CTC clusters. Moreover, the RNA sequencing and TMA staining results showed that CTC cluster-positive HCCs were usually associated with Wnt/β-catenin signaling activation.ConclusionThe presence of CTC clusters characterizes an aggressive HCC subtype. CTC clusters may be used as a biomarker in predicting the prognosis on each stage of malignancy in HCC, which provides evidence for formulating therapeutic strategies for more precise treatment.

A Zebrafish Model of Metastatic Colonization Pinpoints Cellular Mechanisms of Circulating Tumor Cell Extravasation.

Metastasis is a multistep process in which cells must detach, migrate/invade local structures, intravasate, circulate, extravasate, and colonize. A full understanding of the complexity of this process has been limited by the lack of ability to study these steps in isolation with detailed molecular analyses. Leveraging a comparative oncology approach, we injected canine osteosarcoma cells into the circulation of transgenic zebrafish with fluorescent blood vessels in a biologically dynamic metastasis extravasation model. Circulating tumor cell clusters that successfully extravasated the vasculature as multicellular units were isolated under intravital imaging (n = 6). These extravasation-positive tumor cell clusters sublines were then molecularly profiled by RNA-Seq. Using a systems-level analysis, we pinpointed the downregulation of KRAS signaling, immune pathways, and extracellular matrix (ECM) organization as enriched in extravasated cells (p < 0.05). Within the extracellular matrix remodeling pathway, we identified versican (VCAN) as consistently upregulated and central to the ECM gene regulatory network (p < 0.05). Versican expression is prognostic for a poorer metastasis-free and overall survival in patients with osteosarcoma. Together, our results provide a novel experimental framework to study discrete steps in the metastatic process. Using this system, we identify the versican/ECM network dysregulation as a potential contributor to osteosarcoma circulating tumor cell metastasis.

Technical Challenges for CTC Implementation in Breast Cancer.

Simple SummaryLiquid biopsy appears as a feasible opportunity to complement tissue biopsy within breast cancer research and real-time monitoring of the disease. In this review, we not only summaries cur-rent trends in the study of CTCs as a potential biomarker of response to systemic therapies but also the technical challenges for their implementation in clinical practice, including the role of free circulating DNA and new approaches based on the isolation of CTCs from body fluids. We also highlight the importance of a consensus on the standardization of liquid biopsy methodologies as a fundamental requirement for their implementation in clinical practice.Breast cancer is the most common neoplasm in women worldwide. Tissue biopsy, currently the gold standard to obtain tumor molecular information, is invasive and might be affected by tumor heterogeneity rendering it incapable to portray the complete dynamic picture by the absence of specific genetic changes during the evolution of the disease. In contrast, liquid biopsy can provide unique opportunities for real-time monitoring of disease progression, treatment response and for studying tumor heterogeneity combining the information of DNA that tumors spread in the blood (circulating tumor DNA) with CTCs analysis. In this review, we analyze the technical and biological challenges for isolation and characterization of circulating tumor cells from breast cancer patients. Circulating tumor cell (CTC) enumeration value is included in numerous clinical studies due to the prognostic’s role of these cells. Despite this, there are so many questions pending to answer. How to manage lymphocytes background, how to distinguish the CTCs subtypes or how to work with frozen samples, are some of the issues that will discuss in this review. Based on our experience, we try to address these issues and other technical limitations that should be solved to optimize the standardization of protocols, sample extraction procedures, circulating-tumor material isolation (CTCs vs. ctDNA) and the very diverse methodologies employed, aiming to consolidate the use of CTCs in the clinic. Furthermore, we think that new approaches focusing on isolation CTCs in other body fluids such as cerebrospinal or ascitic fluid are necessary to increase the opportunities of circulating tumor cells in the practice clinic as well as to study the promising role of CTC clusters and their prognostic value in metastatic breast cancer.

Subpopulations of Circulating Cells with Morphological Features of Malignancy Are Preoperatively Detected and Have Differential Prognostic Significance in Non-Small Cell Lung Cancer.

Simple SummaryLung cancer is by far the main cause of cancer-related deaths among both men and women. Early detection of malignant nodules and non-invasive monitoring of disease status is essential to increase the chance of cure. In this study, we analyzed the frequency and the biological features of circulating tumor cells, i.e., cells released from the tumor and in transit in the bloodstream, in patients with a diagnosis of non-small cell lung cancer undergoing surgical resection, with the aim to develop a blood-based diagnostic test and to promptly identify patients at risk of post-operative disease recurrence.Background: Non-small cell lung cancer (NSCLC) frequently presents when surgical intervention is no longer feasible. Despite local treatment with curative intent, patients might experience disease recurrence. In this context, accurate non-invasive biomarkers are urgently needed. We report the results of a pilot study on the diagnostic and prognostic role of circulating tumor cells (CTCs) in operable NSCLC. Methods: Blood samples collected from healthy volunteers (n = 10), nodule-negative high-risk individuals enrolled in a screening program (n = 7), and NSCLC patients (n = 74) before surgery were analyzed (4 mL) for the presence of cells with morphological features of malignancy enriched through the ISET® technology. Results: CTC detection was 60% in patients, while no target cells were found in lung cancer-free donors. We identified single CTCs (sCTC, 46%) and clusters of CTCs and leukocytes (heterotypic clusters, hetCLU, 31%). The prevalence of sCTC (sCTC/4 mL ≥ 2) or the presence of hetCLU predicted the risk of disease recurrence within the cohort of early-stage (I–II, n = 52) or advanced stage cases (III–IVA, n = 22), respectively, while other tumor-related factors did not inform prognosis. Conclusions: Cancer cell hematogenous dissemination occurs frequently in patients with NSCLC without clinical evidence of distant metastases, laying the foundation for the application of cell-based tests in screening programs. CTC subpopulations are fine prognostic classifiers whose clinical validity should be further investigated in larger studies.

Genomic analysis of circulating tumor cells in adenosquamous carcinoma of the prostate: a case report

BackgroundAdenosquamous carcinoma of the prostate (ASCP) is an extremely rare and aggressive prostate cancer variant, whose genomic characteristics have not been elucidated. Although liquid biopsy of circulating tumor cells (CTCs) is an emerging topic in oncology, no study has assessed CTCs in patients with ASCP.Case presentation.A 76-year-old man presented with discomfort in his urethra. His prostate-specific antigen (PSA) level was 13.37 ng/mL. A computed tomography (CT) scan indicated a prostate mass with multiple lymph node and lung metastases. The patient underwent transurethral resection of the prostate and prostatic needle biopsy; both specimens demonstrated Gleason grade group 5 acinar adenocarcinoma of the prostate. Bone scintigraphy indicated bone metastasis in the ischium. Combined androgen blockade was implemented, and his serum PSA level rapidly decreased to 0.01 ng/mL. However, a CT scan 6 months after the initial diagnosis revealed worsening of the disease. The patient therefore underwent repeated prostatic needle biopsy; its specimen demonstrated prostatic adenocarcinoma together with squamous carcinoma components. As immunohistochemical analyses showed the tumor cells to be negative for CD56, chromogranin A, synaptophysin, and PSA, the definitive diagnosis was ASCP. Although the patient underwent chemotherapy (docetaxel and cabazitaxel), he died of the disease 3 months after the diagnosis of ASCP, or 13 months after the initial diagnosis of prostatic adenocarcinoma. His PSA values remained ≤ 0.2 ng/mL. CTCs from the patient’s blood (collected before starting docetaxel) were analyzed and genomically assessed. It showed 5 cytokeratin (CK)+ CTCs, 14 CK− CTCs, and 8 CTC clusters, per 10 mL. Next-generation sequencing identified a total of 14 mutations in 8 oncogenes or tumor suppressor genes: PIK3CB, APC, CDKN2A, PTEN, BRCA2, RB1, TP53, and CDK12. Of 14 mutations, 9 (64%) were detected on CK− CTCs and 5 (36%) were detected on CK+ CTCs.ConclusionsThis is the first report of CTC analysis and genomic assessment in ASCP. Although the prognosis of ASCP is dismal due to lack of effective treatment, genomic analysis of CTCs might lead to effective treatment options and improved survival.

Circulating tumor cells (CTCs)/circulating tumor endothelial cells (CTECs) and their subtypes in small cell lung cancer: Predictors for response and prognosis.

BackgroundThe aim of the study was to define the clinical significance of circulating tumor cells (CTCs)/circulating tumor endothelial cells (CTECs) and their subtypes in small cell lung cancer (SCLC) patients.MethodsCTCs/CTECs and their subtypes were determined using SE‐iFISH technology in 33 SCLC patients before initial treatment (B1), after two cycles of chemotherapy (B2), at the completion of chemotherapy (B3), and disease progression (B4). The correlations with clinical characteristics, progression‐free survival (PFS), and overall survival (OS) were analyzed.ResultsCTCs and CTECs were detected in 96.6% and 65.5% of patients, respectively. Patients had higher levels of CTCs compared with CTECs in circulation (p < 0.05). Extensive‐stage SCLC patients tended to have higher CTEC counts (p = 0.035), and the detection of CTC‐white blood cell (CTC‐WBC) clusters was associated with a worse response to treatment (p = 0.030). Patients with CTC‐WBC clusters at B1 (17.3 vs. 22.6 months, p = 0.041) and B2 (19.9 vs. 25.2 months, p = 0.018) had significantly shorter OS than those with no detection. Additionally, their presence was revealed as independent predictors for a worse OS in multivariable analyses (B1: HR 9.3, 95% CI: 1.4–48, p = 0.0079; B2: HR 4.4, 95% CI: 1.1–18, p = 0.041). A high CTC level at B4 was an adverse prognostic factor for SCLC patients (PFS: 8.7 vs. 22.5 months, p = 0.0026; OS: 19 months vs. not reached, p = 0.0086). CTC clusters and CTECs also showed prognostic values.ConclusionsThe presence of CTC‐WBC clusters at baseline and after two‐cycle chemotherapy and the total CTC counts at the completion of chemotherapy are strong predictors for the prognostic survival of SCLC patients receiving first‐line treatment.

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

TRAIL-coated leukocytes to kill circulating tumor cells in the flowing blood from prostate cancer patients

BackgroundRadical surgery is the first line treatment for localized prostate cancer (PC), however, several studies have demonstrated that surgical procedures induce tumor cell mobilization from the primary tumor into the bloodstream.MethodsThe number and temporal fluctuations of circulating tumor cells (CTC), cancer associated fibroblasts (CAF) and CTC cluster present in each blood sample was determined.ResultsThe results show that both CTC and CTC cluster levels significantly increased immediately following primary tumor resection, but returned to baseline within 2 weeks post-surgery. In contrast, the CAF level decreased over time. In patients who experienced PC recurrence within months after resection, CTC, CAF, and cluster levels all increased over time. Based on this observation, we tested the efficacy of an experimental TNF-related apoptosis-inducing ligand (TRAIL)-based liposomal therapy ex-vivo to induce apoptosis in CTC in blood. The TRAIL-based therapy killed approximately 75% of single CTCs and CTC in cluster form.ConclusionCollectively, these data indicate that CTC cluster and CAF levels can be used as a predictive biomarker for cancer recurrence. Moreover, for the first time, we demonstrate the efficacy of our TRAIL-based liposomal therapy to target and kill prostate CTC in primary patient blood samples, suggesting a potential new adjuvant therapy to use in combination with surgery.

Metastasis Prevention: Focus on Metastatic Circulating Tumor Cells.

Metastasis is the main cause of cancer death. Metastatic foci are derived from tumor cells that detach from the primary tumor and then enter the circulation. Circulating tumor cells (CTCs) are generally associated with a high probability of distant metastasis and a negative prognosis. Most CTCs die in the bloodstream, and only a few cells form metastases. Such metastatic CTCs have a stem-like and hybrid epithelial-mesenchymal phenotype, can avoid immune surveillance, and show increased therapy resistance. Targeting metastatic CTCs and their progenitors in primary tumors and their descendants, particularly disseminated tumor cells, represents an attractive strategy for metastasis prevention. However, current therapeutic strategies mainly target the primary tumor and only indirectly affect metastasis-initiating cells. Here, we consider potential methods for preventing metastasis based on targeting molecular and cellular features of metastatic CTCs, including CTC clusters. Also, we emphasize current knowledge gaps in CTC biology that should be addressed to develop highly effective therapeutics and strategies for metastasis suppression.

Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis.

Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF-cCAF homotypic clusters, and in heterotypic clusters with CTCs. In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I-IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells. We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.

Abstract 2848: Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer

Abstract Background: Hotspot estrogen receptor-α (ER/ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer and are associated with worse outcome. How these mutations facilitate metastasis remains ambiguous. It is necessary to identify a clear mechanism for therapeutic intervention. Methods: ESR1 mutations were detected by ddPCR. Transcriptome data were derived from cell lines and clinical samples. Y537S and D538G genome-edited MCF7 and T47D cell lines were used for in vitro phenotypic characterization. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and ibidi microfluidic assays. Altered cell-cell adhesion genes were validated using qRT-PCR, immunoblot and immunostaining. Desmosome blocking peptides and carbenoxolone were used for blockade of adhesion. ER cistromes were profiled by ChIP-seq. Tail vein injection was performed on athymic nude mice to evaluate metastasis in vivo. Circulating tumor cell (CTC) clustering propensity in vivo was assessed via intracardiac injection followed by CTC microfilter capture. CTC-cluster gene signatures were generated from ER+ breast cancer CTC RNA-seq data. Results: ESR1 mutations were significantly enriched in distant metastases (12/48) vs local (0/27) recurrences, confirming their critical role in promoting metastasis. Transcriptomic analysis revealed altered cell-cell interaction pathways in ESR1 mutant tumors. ESR1 mutant cells formed more compact spheroids in suspension culture, and exhibited stronger cell-cell adhesion in static condition. Under microfluidic condition with physiological shear stress, mutant ESR1 cells derived more and larger clusters, which were prominently preserved from pre-existing clusters. This effect was correlated with increased expression of desmosome and gap junction genes in mutant cells and pharmacological blockade significantly reduced the enhanced cell-cell adhesion. ER ChIP-seq revealed no de novo mutant ER binding sites at the loci of the target genes, suggesting indirect regulation by mutant ER. This was exemplified by a secondary regulation from cFos/AP1 signaling of GJA1 expression, and an epigenetic regulation at DSC1/DSG1 loci through enhanced H3K4me2 and H3K27ac modification. In vivo studies showed ESR1 mutant cells derived more distant metastases, and MCF7 Y537S cells formed larger CTC clusters with increased compactness compared to WT cells. Finally, ER+ CTC-cluster signatures were enriched in ESR1 mutant tumors, suggesting their unique dependence on this pathway during metastasis. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer enhanced cell-cell adhesion, which facilitates breast cancer metastasis via increased CTCs clustering propensity. These findings provide insights to the development of drugs targeting gap junction in ER mutant tumors. Citation Format: Yang Wu, Zheqi Li, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan M. Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben Ho Park, Jennifer M. Atkinson, Dorraya El-Ashry, Adrian V. Lee, Steffi Oesterreich. Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2848.

Key Characteristics of Circulating Tumor Cell Clusters and Implications for Cancer Metastases

Primary tumors generate metastases by shedding tumor cells into the circulation; these circulating tumor cells (CTCs) implant at distant sites to develop into metastatic lesions. CTCs can travel either as clusters or as single CTCs. Previous studies revealed that the frequency of CTC clusters in a cancer patient positively correlates with the likelihood of developing metastatic lesions. Three key characteristics of CTC clusters - chemoresistance, reduced apoptosis, and epigenetically programmed stemness - enhance their metastatic potential relative to single CTCs: CTC clusters seem to be more resistant to chemotherapy due to their quiescent and necrotic cores, making drug penetration difficult. Their chemoresistance also correlates with specific molecular components of the extracellular matrix. CTC clusters suffer lower rates of apoptosis. This might be attributed to autocrine factors that protect against immune attack and the epithelial-mesenchymal transition. The DNA methylation landscape of CTC clusters closely resembles that of embryonic stem cells. It features hypomethylation of four critical transcription factors associated with stemness and hypermethylation of a set of pro-differentiation genes. Further research might focus on the interdependence of these three characteristics and whether they precede or follow the clustering of CTCs. The answers to these research questions will help drug developers define specific mechanisms that can curb the metastatic potential of CTC clusters.

Abstract 584: Circulating tumor cells as prognostic biomarkers in glioblastoma

Abstract Background: Glioblastoma (GBM) is the most common and aggressive type of tumor arising from the central nervous system. GBM remains an incurable disease despite current advancement in therapies, with survival rates of approximately 15 months. Recent literature has highlighted that GBM can release tumoral content which crosses the blood-brain barrier (BBB) and as a consequence can be detected in patients' blood, such as circulating tumor cells (CTCs). CTCs carry tumor information and have shown promise as prognostic and predictive biomarkers in other cancer types. Currently, the prognostic utility of CTCs in GBM is not well understood. Methods: Blood samples from GBM patients (n=18) attending a tertiary referral hospital were collected before and after surgery to investigate the prognostic role of CTCs. CTCs were isolated using spiral microfluidic technology and characterized using immunofluorescence for GFAP, cell surface vimentin and CD45, in addition to fluorescence in situ hybridization (FISH) to detect EGFR amplification. CTC counts of patients with poor outcomes (recurrence or death) versus patients with good outcomes (stable disease and no recurrence) were compared using a negative binomial distribution. Results: CTCs were found in 12 out of 18 patients (66.7%), 8/18 before surgery and 10/17 after surgery. CTC clusters were found in 2 patients. CTCs counts before surgery were significantly higher for patients with poor clinical outcome (p=0.0396). Conclusion: Our preliminary data support the use of this spiral technology to isolate CTCs. CTCs and CTC clusters can be used as prognostic biomarkers for managing GBM patients. Citation Format: Juliana Müller Bark, Arutha Kulasinghe, Paul Leo, Gunter Hartel, Majid E. Warkiani, Rosalind L. Jeffree, Benjamin Chua, Bryan W. Day, Chamindie Punyadeera. Circulating tumor cells as prognostic biomarkers in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 584.

Abstract 599: Investigation of PD-L1 expression in circulating tumor cells isolated using the Parsortix system in metastatic lung and breast cancer patients

Abstract Programmed death-ligand 1 (PD-L1) allows cancer cells to evade the host immune response when upregulated. PD-L1 antagonists have been shown to be effective immunotherapies. The FDA approved the PD-L1 inhibitor nivolumab for treatment of previously treated, non-responding metastatic non-small cell lung cancer (NSCLC) patients, independent of the PD-L1 status identified in tumour tissue, likely due to the controversial value of PD-L1 detection on tissue biopsy that may be out-of-date. Measurement of PD-L1 expression in circulating tumor cells (CTCs) may enable repeat testing to provide up-to-date PD-L1 status and the potential to monitor patients on these therapies. Whilst the epitope-dependent CELLSEARCH® system is the only technology FDA-cleared for the enumeration of CTCs, its application is limited to epithelial CTCs and has limited sensitivity in NSCLC. We developed a research use only assay for the characterisation of PD-L1 expression on CTCs isolated using the Parsortix® system, a label-independent microfluidic device that harvests CTCs of all phenotypes (epithelial and/or mesenchymal) based on size and compressibility. Peripheral blood (8 – 10mL) was drawn into K2EDTA tubes from 17 healthy volunteers, 17 metastatic breast cancer (MBC) patients and 18 metastatic NSCLC patients and processed on Parsortix systems within 24 hours. Harvested CTCs were cytospun and immunofluorescently stained for detection of cytokeratins and PD-L1. Slides were imaged using a BioView AllegroPlus imaging system.No CTCs were identified in the healthy volunteers, highlighting the specificity of the assay. CTCs (≥1) were identified in 70% of the MBC patients and 55% of the NSCLC patients. Importantly, the positivity rate observed in NSCLC patients was 2-fold higher than that in previously described studies using the CELLSEARCH system. A mean of 9 CTCs/tube of blood were identified in the CTC positive MBC patients, with a range of 1 – 128 CTCs. A mean of 11 CTCs/tube of blood were identified in the CTC positive NSCLC patients, with a range of 1 – 23 CTCs. CTC clusters, consisting of 3 – 45 cells per cluster, were observed in both NSCLC and MBC patients. High heterogeneity of PD-L1 expression was observed. The CTC positive patients were classified into three groups: patients with CTCs positive for both cytokeratins (CK+) and PD-L1 (36% in MBC and 40% in NSCLC), patients with a mixed population of CK+, PD-L1+/- CTCs (27% in MBC and 40% in NSCLC) and patients with CK+, PD-L1- CTCs only (36% in MBC and 20% in NSCLC).The optimized assay for detection of PD-L1 expression on CTCs allowed for the identification of PD-L1 positive and PD-L1 negative CTCs in a significant proportion of the NSCLC and MBC patients studied. The ability to isolate significant numbers of PD-L1 positive CTCs in blood lays the groundwork for development of dynamic PD-L1 monitoring to support personalized patient management. Citation Format: Mariacristina Ciccioli, Amy Davis, Ofure Alenkhe, Anne-Sophie Pailhes-Jimenez. Investigation of PD-L1 expression in circulating tumor cells isolated using the Parsortix system in metastatic lung and breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 599.

Abstract 588: Mesenchymal markers: The new avenue for circulating tumor cells detection

Abstract Introduction: Unlike tissue biopsy, liquid biopsy allows the routine, repeat characterization of cancer at genetic, transcriptional and protein levels. Isolation of circulating tumor cells (CTCs) from a blood draw offers the potential for cancer screening, management and monitoring. Most CTC isolation systems are based on epitope-dependent CTC capture using epithelial markers. However, it is known that tumor cells can undergo epithelial-to-mesenchymal transition (EMT) when extravasating from the primary tumor to enter the bloodstream and eventually establish distant metastases. Epitope-dependent CTC detection platforms have limited sensitivity for detection of mesenchymal CTCs, leading to the inability to harvest clinically relevant CTCs for analysis, particularly in cancers with high mesenchymal phenotype such as non-small cell lung cancer (NSCLC). We developed a research use only assay for enrichment and identification of CTCs based on epithelial and mesenchymal markers using the Parsortix® system, a label-independent microfluidic device that isolates cells based on their size and compressibility. Methods: Peripheral blood (8 – 10mL) was drawn into K2EDTA tubes from 47 metastatic breast cancer (MBC) patients and 48 metastatic NSCLC patients and processed on Parsortix systems within 36 hours after collection. Harvested CTCs were cytospun and immunofluorescently stained using an optimized panel of antibodies for detection of epithelial and mesenchymal markers. Stained slides were imaged using a BioView AllegroPlus automated imaging system. Results: CTCs were captured from both MBC and NSCLC patients. 330 CTCs were identified from 19 MBC patients (mean: 17; range: 1-96). 58 CTCs were identified from 15 NSCLC patients (mean: 4; range: 1-22). Similarly, CTC clusters were observed in 63% (12/19) of the CTC-positive MBC patients and in 33% (5/15) of CTC-positive NSCLC patients. Cluster size ranged from 2-22 CTCs per cluster (2-17 in MBC and 2-22 in NSCLC), and the number of clusters per patient ranged from 1-13 in MBC and 1-2 in NSCLC. Phenotypically, more CTCs expressing only mesenchymal markers were harvested from NSCLC patients compared to MBC patients (38% vs 25%), with a larger proportion of CTCs expressing both epithelial and mesenchymal markers being harvested in both cancer types (59% in NSCLC and 74% in MBC). Only a small percentage of the CTCs harvested expressed just the epithelial markers. Conclusions: This study highlights the importance of the inclusion of mesenchymal markers into CTC characterization, as the majority of the CTCs captured by the Parsortix system from MBC and NSCLC patients expressed mesenchymal markers. The assay developed in this study successfully allowed phenotyping of the harvested cells using epithelial AND mesenchymal markers, with the results suggesting that a large proportion of the CTCs harvested would have been missed with an epithelial-only based approach, particularly in NSCLC. Citation Format: Mariacristina Ciccioli, Natalia Bravo-Santano, Amy Davis, Jolie Lewis, Ross Malcolm, Anne-Sophie Pailhes-Jimenez. Mesenchymal markers: The new avenue for circulating tumor cells detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 588.

Abstract 592: Circulating tumor cells of esophageal and gastric cancers

Abstract Background: The availability of circulating tumor cells (CTCs) creates a powerful non-invasive approach to monitor tumor progression, therapeutic response, relapse, metastasis, and, in some cases, provide early diagnosis. Esophageal cancer (EC) is the fifth most common cause of cancer-related death in men 40-59 years of age, with a ~90% death rate. Gastric cancers (GC) have a 30% five-year survival rate. Smoking, alcohol, age, H. pylori infection, and specific diets are established risk factors for esophageal and gastric cancers. We aim to isolate, propagate, and identify RNA expression profiles in CTCs from EC and GC patients. This study permits the potential clinical application of identifying markers that may define prognosis, metastatic potential, and therapeutic efficacy. Materials and methods: Venous blood samples from the esophageal and gastric cancer patients (n=6) were collected before and a week after chemotherapy. A negative enrichment method was performed to isolate CTCs using EasySep™ Direct Human CTC Enrichment Kit (StemCell Technologies). Label-free and viable CTCs were cultured in Epicult-C (StemCell Technologies) containing proliferation supplement and hydrocortisone. After rRNA depletion, strand-specific paired-end sequencing libraries for approximately 100M read-depth will be prepared by SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) for RNA sequencing. Libraries will be constructed using the Illumina Nextera XT kit. The Washington DC VAMC Institutional Review Board approved this study and written informed consent was obtained from patients. Results: We observed a dynamic range of CTCs in the same patient during chemotherapy treatments. The fluctuation of the number of cells obtained before and a week after infusion may represent the effect of therapy on both primary and circulating tumor cells. Samples with clusters of CTCs preserved their structures during cell culture. Our most extended CTC sample culture approached 55 days, which obtained a patient with intestinal-type gastric cancer. Conclusion and future directions: We expect to identify differentially expressed genes in CTCs and the primary tumor, along with alternatively spliced isoforms in both primary tumor and CTCs. The fluctuation of the number of cells obtained before and a week after infusion may represent the effect of therapy on circulating tumor cells. RNA sequencing of CTCs will be performed and serve as baseline data for functional studies. This study permits monitoring therapeutic efficacy, identifying therapy-resistant or therapy-sensitive clones/markers that may allow the selection of appropriate therapeutic regimens. We will continue to follow patients and obtain tissues from lymph node or distant metastasis to analyze the evolution of their tumors. Citation Format: Hayriye Verda Erkizan, Robert G. Wadleigh. Circulating tumor cells of esophageal and gastric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 592.

CD81 Facilitates Tumor Cell Clustering and Metastasis in Triple Negative Breast Cancer

Tumor-initiating cells with reprogramming plasticity and/or de-differentiation attributes have been thought to initiate primary tumor development as well as to regenerate secondary tumors in metastatic organs; however, the molecular mechanisms are not fully understood. We previously found that breast tumor-initiating cell marker, CD44, directs multicellular aggregation and cluster formation of circulating tumor cells (CTCs), which further enhance stemness and survival of such cells, enabling metastatic colonization to the lungs. To further elucidate the molecular network underlying CTC cluster formation, we performed global proteomic profiling and discovered that the tetraspanin protein CD81, which is normally enriched in exosomes (small extracellular vesicles), is a new driver of cancer initiation and metastasis as a facilitator and target of CD44. Loss of CD81 compromises tumorigenicity and mammosphere formation of triple negative breast cancer (TNBC) cells. Assisted by machine learning-based algorithms and mutagenesis approach, we found that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. Notably, genetic knockout of CD44 or CD81 results in loss of both CD81 and CD44 in secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells, such as mammosphere formation. In addition, RNA sequencing analysis showed that CD81 knockdown up-regulates expression of a cell differentiation marker, SEMA7a, whose down-regulation partially rescues mammosphere formation inhibition by CD81 depletion. Clinically, CD81 expression was observed in >80% of CTCs and specifically enriched and co-expressed along with CD44 in clustered CTCs of breast cancer patients. Mimicing the phenotypes of CD44 deficiency, loss of CD81 also inhibited tumor cell aggregation and lung metastasis of TNBC in both human and mouse tumor models, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights a new driving role of CD81 in cancer exosome-induced stemness, clustered CTCs, and metastasis initiation of TNBC, reported for the first time to our knowledge.

Clusters, Assemblies and Aggregates of Tumor Cells in the Blood of Breast Cancer Patients; Composition, Mode of Action, Detection and Impact on Metastasis and Survival

Circulating tumor cells (CTCs) are gaining momentum as a diagnostic tool and therapeutic target. CTC clusters are more metastatic, but harder to study and characterize, because they are rare and the methods of isolation are mostly focused on single CTCs. This review highlights the recent advances to our understanding of tumor cell clusters with the emphasis on their composition, origin, biology, methods of detection, and impact on metastasis and survival. New approaches to therapy, based on cluster characteristics are also described.

Diversity of Epithelial-Mesenchymal Phenotypes in Circulating Tumour Cells from Prostate Cancer Patient-Derived Xenograft Models.

Simple SummarySpread of prostate cancer to other parts of the body is responsible for the majority of deaths. Tumour cell epithelial mesenchymal plasticity (EMP) increases their metastatic potential and facilitates their survival in the blood as circulating tumour cells (CTCs). The aim of this study was to molecularly characterise CTCs in a panel of prostate cancer patient-derived xenografts using genes associated with epithelial and mesenchymal phenotypes, and to compare the EMP status of CTCs with their matched primary tumours. The study highlights high heterogeneity in CTC enumeration and EMP gene expression between tumour-bearing mice and within individual blood samples, and therefore caution should be taken when interpreting pooled CTC analyses. Critically, tumour cells were present in the epithelial-mesenchymal hybrid state in the circulation. The study also demonstrates that there is high variation in CTC size, which would introduce sample bias to size-based CTC isolation techniques.Metastasis is the leading cause of cancer-related deaths worldwide. The epithelial-mesenchymal plasticity (EMP) status of primary tumours has relevance to metastatic potential and therapy resistance. Circulating tumour cells (CTCs) provide a window into the metastatic process, and molecular characterisation of CTCs in comparison to their primary tumours could lead to a better understanding of the mechanisms involved in the metastatic cascade. In this study, paired blood and tumour samples were collected from four prostate cancer patient-derived xenograft (PDX) models (BM18, LuCaP70, LuCaP96, LuCaP105) and assessed using an EMP-focused, 42 gene human-specific, nested quantitative RT-PCR assay. CTC burden varied amongst the various xenograft models with LuCaP96 having the highest number of CTCs per mouse (mean: 704; median: 31) followed by BM18 (mean: 101; median: 21), LuCaP70 (mean: 73; median: 16) and LuCaP105 (mean: 57; median: 6). A significant relationship was observed between tumour size and CTC number (p = 0.0058). Decreased levels of kallikrein-related peptidase 3 (KLK3) mRNA (which encodes prostate-specific antigen; PSA) were observed in CTC samples from all four models compared to their primary tumours. Both epithelial- and mesenchymal-associated genes were commonly expressed at higher levels in CTCs compared to the bulk primary tumour, although some common EMT-associated genes (CDH1, VIM, EGFR, EPCAM) remained unchanged. Immunofluorescence co-staining for pan-cytokeratin (KRT) and vimentin (VIM) indicated variable proportions of CTCs across the full EMP axis, even in the same model. EMP hybrids predominated in the BM18 and LuCaP96 models, but were not detected in the LuCaP105 model, and variable numbers of KRT+ and human VIM+ cells were observed in each model. SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was enriched at the RNA level in CTCs compared to primary tumours and was the most commonly expressed mesenchymal gene in the CTCs. Co-staining for SERPINE1 and KRT revealed SERPINE1+ cells in 7/11 samples, six of which had SERPINE+KRT+ CTCs. Cell size variation was observed in CTCs. The majority of samples (8/11) contained larger CTCs ranging from 15.3 to 37.8 µm, whilst smaller cells (10.7 ± 4.1 µm, similar in size to peripheral blood mononuclear cells (PBMCs)) were identified in 6 of 11 samples. CTC clusters were also identified in 9/11 samples, containing 2–100 CTCs per cluster. Where CTC heterogeneity was observed in the clusters, epithelial-like cells (KRT+VIM−) were located on the periphery of the cluster, forming a layer around hybrid (KRT+VIM+) or mesenchymal-like (KRT−VIM+) cells. The CTC heterogeneity observed in these models emphasises the complexity in CTC isolation and classification and supports the increasingly recognised importance of the epithelial-mesenchymal hybrid state in cancer progression and metastasis.

Circulating tumor cells, circulating tumor DNA, and disease characteristics in young women with metastatic breast cancer.

Clinical and genomic data from patients with early-stage breast cancer suggest more aggressive disease in premenopausal women. However, the association between age, disease course, and molecular profile from liquid biopsy in metastatic breast cancer (MBC) is not well characterized. Patients were classified as premenopausal (<45years), perimenopausal (45-55years), or postmenopausal (> 55years). Cohort 1 consisted of patients with MBC who consented for prospective serial evaluation of circulating tumor cells (CTCs) using CellSearch™. Cohort 2 included patients who, as part of routine care, had circulating tumor DNA (ctDNA) sequenced by the Guardant360™ assay. Clinicopathologic data were collected from retrospective review to compare disease features between premenopausal and postmenopausal women. Premenopausal women represented 26% of 138 patients in Cohort 1 and 21% of 253 patients in Cohort 2. In Cohort 1, younger patients had a shorter time to metastases and a higher prevalence of lung and brain metastases. Overall, there were similar rates of ≥ 5 CTCs/7.5mL, HER2 + CTC expression, and CTC clusters between pre- and postmenopausal women. However, for those with triple negative breast cancer, premenopausal women had a higher proportion of ≥ 5 CTCs/7.5mL. In Cohort 2, premenopausal women had a higher incidence of FGFR1 (OR 2.75, p = 0.022) and CCND2 (OR 6.91, p = 0.024) alterations. There was no difference in the ctDNA mutant allele frequency or the number of detected alterations between these age groups. Our data reveal that premenopausal women diagnosed with MBC have unique clinical, pathologic, and molecular features when compared to their postmenopausal counterparts. Our results highlight FGFR1 inhibitors as potential therapeutics of particular interest among premenopausal women.