Single Circulating Tumor Cells Research Articles

Abstract PO5-18-04: Effect of Digoxin on clusters of circulating tumor cells in patients with metastatic breast cancer (DICCT)

Abstract Background: The presence of circulating tumor cell (CTC) clusters is associated with tumor progression and with a bad prognosis in various cancer types1,2. In breast cancer, a pre-clinical model demonstrated that Na/K-ATPase inhibitors such as digoxin could dissolve CTC clusters and reduce metastasis3. DICCT is a single-arm, therapeutic exploratory phase I study aimed to examine whether digoxin can disrupt CTC clusters in metastatic breast cancer patients (NCT03928210). Material and methods: Metastatic breast cancer patients in whom clusters of CTCs have been detected were eligible. Patients with concomitant heart disease were excluded. After progressive disease in any line of therapy, a window of opportunity before initiation of subsequent systemic therapy was used to administer digoxin treatment for seven (7) days. For CTC detection, blood samples were processed with the FDA-cleared Parsortix system (ANGLE plc, UK). Captured CTCs were stained for EPCAM, HER2, EGFR (positive CTC selection) and CD45 (exclusion marker). To gain insight into the cluster-dissolution ability of digoxin, the primary endpoint was size of CTC clusters (i.e. number of cells within each cluster) under digoxin therapy. The cumulative effect of digoxin treatment on the primary endpoint was assessed by a linear regression model. Results: Nine (9) patients with CTC clusters detected at baseline were enrolled. A target level of digoxin above 0.7 ng/ml could be confirmed at day 7 for all patients. For quantitative and qualitative analysis of CTC clusters, 5.5 to 27 ml of blood were analyzed at baseline, at day 0 pre-treatment, and post 2 hours, 3 days and 7 days of digoxin treatment. The average cluster size ranged between 2 and 11.8 cells (median 2.9) considering all samples from all patients. Digoxin treatment reduced cluster size in 8 patients (88.9%) to different extents, and the proportion of CTC cluster over single CTCs was also reduced in 6 patients (66.7%). The effect of digoxin was evident in both homotypic (cancer cells only) and heterotypic (aggregates of CTCs and white blood cells) clusters. The treatment was well tolerated, and no adverse events related to the study treatment occurred. All patients completed the treatment according to the protocol. Conclusion: DICCT provides the first-in-human proof-of-principle that digoxin induces a partial dissolution of CTC clusters in patients with metastatic breast cancer at drug levels that are safe and well tolerated. Conceptualization of follow-up trials including Na/K-ATPase inhibitors and patient outcome endpoints are underway.

Abstract SY34-01: Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy

Abstract Cellular plasticity and stemness properties enable dynamic changes of circulating tumor cells (CTCs) during cancer dissemination, such as aggregation or cohesion of single CTCs into multicellular CTC clusters with 20-100 times higher metastatic propensity than the singles. Our work demonstrated that stemness glycoproteins on breast cancer stem cells, such as CD44, CD81, and ICAM1 drive CTC aggregation in metastatic breast cancer, such as triple negative breast cancer (TNBC). To determine the glycosylation patterns in CTCs and its association with clinical outcomes, we established multiple CTC analysis approaches including CellSearch, flow cytometry, and immunohistochemistry using patient blood and tissue sections collected at the pre-treatment baseline and after-therapy timepoints. We found that chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of terminal sugar residues α2,6-sialic acids in glycoproteins. CTCs showed dynamic hypo-sialylation in the blood with loss of the sialyl-transferase ST6GAL1, promoting CTC cluster formation with cellular quiescence (proliferative dormancy) and evading chemotherapy in breast cancer. Seeded tumor cells regained ST6GAL1 to enable metastatic colonization. Many adhesion proteins and stemness drivers as glycoprotein substrates of ST6GAL1 drive CTC clustering and metastatic seeding. Neutralizing antibodies against these clustering drivers inhibit CTC cluster formation and improve therapy response, thereby blocking lung metastasis in TNBC. Citation Format: Huiping Liu. Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY34-01.

Abstract 7507: Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients

Abstract Background: Single circulating tumor cells (sCTC) capture without cell fixation with no blood contaminants like leukocytes are highly challenging. sCTC genome transcriptomics anticipates new avenues in new discovery, targeted therapies, and inclusion of patients in clinical trials where the tissue is inaccessible. We report, an assay to selectively capture live sCTCs at singularity with NGS-based comprehensive mutational profiling in breast cancer (BC) patients. Further, paired ctDNA outcomes using blood with mutational heterogeneity scores of sCTCs adds comprehensive genomics for establishing an accelerated treatment outcomes and resistance signatures. Methods: Retrospectively, sCTC were isolated from 70 breast cancer (BC) patients using OncoRadar platform consisting affinity-based glass beads mediated by anti EpCAM antibody. CTCs were selectively captured and released without fixing and contamination of any leukocytes. Platform possess cleavable disulfide spacer at pH 8.0, between glass beads (diam. 2 mm, area 12.57 mm2) and a ligand. Sensitivity, specificity, positive predictive value (PPV), negative prediction value (NPV), and accuracy were evaluated using MCF-7 cells and CTCs. Assay to isolate CTC has been developed in 96 well plate, for downstream NGS-based comprehensive gene panel analysis using hybridization-based target enrichment. Interestingly, no mutations detected in blood samples (~20%) for CtDNA versus CTC DNA offered non-chip implicated mutations. Results:. Analytical validation using OncoRadar showed higher cell capture efficiency (99%) with spiked MCF-7 cells/ml of blood. Sensitivity and specificity values were 97% and 98% respect, while PPV was 99% and NPV was 7.5% with the accuracy parameter of 99%. Sensitivity, specificity, PPV, and NPV parameters when assessed for CTC isolation from 70 BC patients. NGS-based mutational analysis, sCTC showed the presence of PIK3CA, ESR1, and NF1 mutations, which were absent from paired ctDNA samples. Conclusion: Detection of ctDNA obscure mutations in paired sCTC samples suggests high sensitivity of sCTC-based genomic assays compared to current standard of practice. Heterogeneity scores amongst inter and intra CTCs with MATH scores offer new dimensions in genomics. Combination of ctDNA and sCTC DNA assay offers a comprehensive realistic genomic outcome for more significant therapy decisions, especially when guideline based options may exhausts. Citation Format: Gowhar Shafi, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Bhagwat Jadhav, Sangeeta Prajapati, Moubeen Fauzul, Kanchan Hariramani, Madhura Basavalingegowda, Jayant Khandare. Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients [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 7507.

Abstract 7506: Comprehensive mutational profile of true single circulating tumor cells compared with CtDNA in advanced pancreatic adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is aggressive malignancy characterized by 5-year survival rate of <10%. Comprehending PDAC's mutational landscape through NGS-based assays, acquiring representative tumor tissue biopsies remains arduous. Single circulating tumor cell (sCTC) genomics could provide clinically relevant personalized diagnostics. Here we demonstrate comparative analysis of sCTCs genomics and ctDNA outcomes in paired advanced PDAC patients. Methods: Retrospectively, 25 live sCTCs and sCTC clusters were isolated from 10 advanced PDAC patients' blood using OncoRadar platform consisting glass beads conjugated with antiEPCam antibodies, tuned for releasing CTCs, without cell fixing, an assay in 96 well plate. Whole genomes from sCTCs were amplified using random degenerate primer-based PCR. Amplified genomes and paired ctDNA were subjected to CGP using clinical assay OncoIndx® to identify patient-specific genomic alterations in sCTC and paired ctDNA samples. The raw sequence alignment and variant calling was performed using iCare software®. Results: Live 25 sCTCs and 8 CTC clusters were isolated (≥2 CTCs). Mutational profiling revealed multiple pathogenic (P) and likely pathogenic (LP) variants. sCTCs exhibited at least twice as many P variants compared to paired ctDNA samples, with CHD4, FGFR2, and HNF1α being the most common. The mutational profile of sCTCs showed concordance with at least one variant in paired ctDNA, and was enriched in dysregulated cytoskeleton and cell migration machinery. Specifically, COL7A, KIF5B, and RHOA mutations were prevalent in almost all sCTCs, with exclusive mutations (KIF5B and RHOA) in some cases. KRAS P variants were exclusive to 50% of ctDNA samples, paired sCTCs exhibited NRAS P variants. RAS isoforms were observed in 70% of sCTCs. Gene amplification occurred in CCND1/2, FGFR2, and ERBB2 in 90% of sCTCs, with CCND1 amplification more prevalent in sCTC clusters. Mutations in epigenetic repressors (HDAC1, KDM2B) were ubiquitous in sCTCs, co-occurring with DDR pathway mutations (F test, P 0.003, OR 11). Loss-of-function BRCA mutations were detected in 60% of sCTCs, and were absent in paired ctDNA. Additionally, 70% of sCTCs exhibited therapy-resistant variants (NF1, STK11, ARID1A, and PIK3CA), contrasting with a 10% frequency in paired ctDNA. Conclusions: Genomic profile of sCTCs in advanced PDAC patients revealed a complex mutational landscape, providing additional clinically relevant insights that complemented ctDNA findings. sCTCs exhibited greater tumor mutational heterogeneity compared to paired ctDNA samples, and detected additional actionable pathogenic variants. Results suggest that sCTCs may offer therapeutically relevant molecular information for advanced PDAC patients for whom tissue is unavailable and ctDNA fails to provide actionable therapy options. Citation Format: Jayant Khandare, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Kanchan Hariramani, Madhura Basavalingegowda, Vikas Jadhav, Mohan Uttarwar, Gowhar Shafi. Comprehensive mutational profile of true single circulating tumor cells compared with CtDNA in advanced pancreatic adenocarcinoma [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 7506.

Abstract 3692: Genomic profiling with the novel single-cell IMPACT assay reveals evidence of genetic heterogeneity among CTCs in late-stage breast cancer patients

Abstract Introduction: Liquid biopsy enables minimally invasive molecular profiling of systemic cancer for diagnosis, therapy selection and longitudinal monitoring of minimal residual disease. Cell-based liquid biopsy methods are particularly well suited to examine the extent of genetic heterogeneity and clonal diversity among metastatic cancer cells, which are believed to be the major determinants of therapeutic failure. However, isolation and molecular profiling of circulating tumor cells (CTCs) from body fluids is challenging due to methodological constrains. Methods: To overcome these challenges, we have developed a workflow for detection, isolation, and mutational profiling of CTCs. The workflow utilizes the FDA-approved CellSearch System® and a novel method for single-cell Integrated Mutation Profiling of Actionable Cancer Targets (scIMPACT). Our workflow was optimized for reliable detection of copy number variations (CNVs) and single nucleotide variations (SNVs) in single CTCs. The method was then applied to 31 CTCs alongside with 15 matched germline samples. Results: After implementation of a customized Bayesian neural network based algorithm, accounting for biases introduced during amplification of single-cell DNA and prioritizing the detected mutations, we achieved a sensitivity of 91% and a specificity of 96%, AUC = 0.98. The CTC analysis revealed presence of CNVs affecting known oncogenes and tumor suppressor genes (including MYC, CCND1, FGF4, TP53 and RB1) as well as SNVs classified as pathogenic (e.g., ARID1A Q775*). Notably, in the examined specimens most of the chromosomal aberrations (51-82%) were clonal across all cells of a given patient. In contrast, most of the detected somatic SNVs (67-96%) were sub-clonal and thus present only in a subset of CTCs. Conclusion: In summary, the novel scIMPACT workflow enabled reliable and accurate genomic profiling of patient-derived CTCs in late-stage breast cancer patients. Our proof-of-concept study revealed evidence of genetic diversity of SNVs among the examined CTCs, which might have been acquired late in progression but relevant for therapy escape in the studied cases. Further analyses are still needed for assessing the generalizability of our finding as well as for deeper understanding of the molecular basis orchestrating the establishment and maintenance of heterogeneous genotypes of CTCs in breast cancer. Citation Format: Zbigniew T. Czyż, Clara Chaiban, Adithi Ravikumar Varadarajan, Cäcilia Köstler, Vadim Dechand, Jonas Grote, Thomas Ragg, Bernhard Polzer, Jens Warfsmann, Christoph A. Klein. Genomic profiling with the novel single-cell IMPACT assay reveals evidence of genetic heterogeneity among CTCs in late-stage breast cancer patients [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 3692.

Abstract 2303: Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes

Abstract Background: Circulating tumor cell clusters (CTCCs) are aggregated groups of tumor cells that detached from primary tumors and circulate the bloodstream. However, while Circulating Tumor Cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined, with only a few descriptive investigations evaluating their clinical utility. Adding to CTCC complexity is that various subtypes exist, including homotypic clusters consisting of a homogenous population of tumor cells and heterotypic CTCCs made of CTCs with immune/stromal white blood cells (WBCs). Furthermore, CTCs can undergo Epithelial-Mesenchymal Transition (EMT), a process characterized by loss of epithelial traits and upregulation of mesenchymal traits, and can form clustered EMTs (CEMTs). Moreover, CTCs can fuse with macrophages forming Tumor Macrophage Hybrid Cells, aka Cancer-Associated Macrophage Like cells (CAMLs) when in circulation. We enumerated all these types of cells and complex aggregates in the peripheral blood of patients with metastatic breast cancer (mBC). Methods: We enumerated the 6 populations from patient samples enrolled in IRB aproved prospective pilot studies. Whole peripheral blood (7.5mL) was filtered and stained with cytokeratin (CK) & CD45/CD14 to identify CTCs. CTCs were defined as an intact DAPI nuclei and strong filamentous CK. Homotypic CTCCs were defined as ≥2 CTCs attached together. Heterotypic CTCCs were defined as ≥1 CTC attached to ≥1 WBC. EMTs were defined as having DAPI nuclei and weak non-filamentous CK. CEMTS were defined as ≥2 EMTs. CAMLs were defined as having an enlarged polynucleated DAPI, and positive for CD45/CD14 or non-filamentous CK. Median progression free survival (mPFS) and median overall survival (mOS) over 24 months (m) were evaluated. Results: We evaluated the blood of 79 mBC patients. Single CTCs were found in 57% of patients (n=34/79), homotypic CTCCs 15% (n=12/79), heterotypic CTCCs 66% (n=27/79), EMTs 56% (n=44/79), CEMTs 23% (n=18/79) and CAMLs 97% (n=77/79). Over 24 ms, patierns with detectable heterotypic CTCCs & homotypic CTCCs had the shortest response times at mPFS=2.8 & mOS=5.8 ms, or mPFS=2.1 & mOS=4.3 ms, respectively. This was followed by CTCs mPFS=3.7 & mOS=13.7 ms, EMTs mPFS=4.3 & mOS=14.4 ms, CAMLs mPFS=4.3 & mOS=14.4 ms, and CEMT mPFS=4.7 & mOS=13.2 ms. Additionally, CTCCs were rare in HER2+ patients at 4.4% (n=1/23), but no associations to other hormone statuses were found. Conclusion: CTCCs appear to represent an array of subtypes with different biological and clinical meanings. Here we enumerated CTCC subtypes in the blood of mBC patients and stratified the various types to clinical outcomes. These data suggest CTCCs are clinically important and further understanding of their biology in the context of tumor pathogenesis is needed. Citation Format: Daniel L. Adams, Carolina Reduzzi, Aravind Aryasomayajula, R. Katherine Alpaugh, Saranya Chumsri, Cha-Mei Tang, Giuseppe Del Priore, William V. Williams, Massimo Cristofanilli. Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes [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 2303.

Abstract 3693: Molecular and functional characterization of circulating CK+/CD45+ cells in breast cancer

Abstract Background: Circulating tumor cells (CTCs) are considered a key element in metastatic progression and represent a validated prognostic biomarker in breast cancer (BC). CTCs in BC are normally defined as epithelial cells (positive for EPCAM or cytokeratin, CK) lacking the expression of the leukocyte marker CD45. Circulating cells expressing both EPCAM/CK and CD45 (dual positive, DPcells) have however been reported in BC but not investigated. It is believed that DPcells derive from the heterotypic cell fusion of tumor and immune cells, but this hypothesis has not been demonstrated yet. We recently reported, for the first time, the association of DPcells with worse survival in a cohort of 341 BC patients (pts) [Reduzzi et al. ASCO 2022] supporting their tumor origin. Here, we further investigated their malignancy and metastatic potential by molecular profiling and functional experiments. Methods: The study consisted of 2 components: A) Single CTCs and DPcells were enriched from the blood of BC patients with the CellSearch platform, and isolated with the DEPArray system. Single cells underwent whole genome amplification and lowpass whole genome sequencing for single cell copy number alteration (CNA) profiling; B) DPcells and CTCs were investigated in the blood of immunodeficient (NSG) and immunocompetent (FVB) mouse models using various cell lines (LM2, 4T1, MVT1). To test their metastatic potential, DPcells from the primary tumor of FVB donor mice were transplanted into recipient NSG mice, and the metastatic burden in the lungs and liver quantified, in comparison with control CD45- tumor cells. Results: We analyzed 73 DPcells and 18 CTCs collected from 16 BC pts: Aberrant genomes were observed in 28% and 93% of evaluable cells, respectively. When collected from the same pt, CTCs showed clonal CNA profiles, while DPcells were highly heterogeneous. In vivo, DPcells could only be detected in the blood of FVB mice but not in NSG mice. They were very rare, comprising of only about 3% of the total CTC count. When DPcells and control tumor cells were isolated from the primary tumor of 10 donor mice and transplanted into the tail vein of 10 recipient mice, we observed no significant differences in the metastatic colonization of the lungs and liver by the two population, indicating that DPcells do in fact possess metastatic ability, and to a similar extent as control cancer cells. Conclusions: DPcells can have an aberrant genome, supporting their malignancy. Contrary to CTCs, DPcells’ CNA profiles are not clonal and, in vivo, they can only be observed in immunocompetent models; both observations support the tumor/immune fusion hypothesis. Finally, DPcells show metastatic potential in mice, in line with the association with worse survival we previously observed in BC pts. Overall DPcells seem a new CTC subpopulation that should not be neglected anymore. More studies are needed to better understand their origin and phenotypic/molecular features. Citation Format: Carolina Reduzzi, Eleonora Nicolo, Lauren L. Ozimski, Marco Silvestri, Lorenzo Gerratana, Mara S. Serafini, Elisabetta Molteni, Nadia Bayou, Amanda K. Strickland, Huiping Liu, Vera Cappelletti, Nicola Aceto, Massimo Cristofanilli. Molecular and functional characterization of circulating CK+/CD45+ cells in breast cancer [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 3693.

Abstract 3709: Single cell analysis from CSF of patients with suspected leptomeningeal metastasis

Abstract Introduction: Leptomeningeal metastases (LM) are diagnosed in up to 10% of metastatic cancer patients, the most frequent cancers being carcinomas of breast and lung or melanoma. Due to a dismal prognosis and an overall median survival of only four months new approaches for molecular analysis could help to identify individual treatment options. Material and Methods: In this study, we collected 28 cerebrospinal fluid (CSF) samples from patients with different primary tumors but suspected LM. CSF was divided in two parts for standard neuropathologic cytomorphology and detection of circulating tumor cells (CTCs) by using the CellSearch® CTC Kit, respectively. Subsequently, single Cytokeratin-positive CTCs were isolated for whole genome amplification by Ampli1TM and molecular analysis. Additionally, we were able to isolate cell-free DNA (cfDNA) from CSF in a subset of patients which we amplified by an adapted Ampli1TM library protocol. In a small subset of patients, we collected slices of formalin-fixed paraffin embedded (FFPE) primary tumors and systemic metastases for dissociation and isolation of pure subpopulations by DEPArrayTM technology. Isolated CTCs, cfDNA and subpopulations of tumor and stromal cells were analyzed for copy number variations (CNV) applying the Ampli1TM LowPass protocol on the Illumina MiSeqTM platform. Results: By comparing results from classical cytomorphology and CellSearch® workflow, we could find high concordance (24/28 samples) with a slightly higher tumor cell detection rate by CellSearch® (11 positive cases vs. 9 by cytomorphology). Importantly, we could confirm the malignant origin of isolated cells by CNV analysis in all patients positive in CellSearch® assay. Moreover, by comparing single cell data with cfDNA analysis of a subset of 20 samples, we could also detect a higher sensitivity for tumor detection in CTC-based analysis (8/20 cases positive) in comparison to cfDNA-based analysis (5/20 cases positive). In a comprehensive analysis of multiple samples of an individual patient, including CTCs and cfDNA from CSF at different time points, CTCs from blood and biopsies from multiple visceral metastasis, we could identify divergent genomic evolution between tumor cells from the central nervous syste Conclusion: In this proof-of-concept study we show that a liquid biopsy-based approach for single cell analysis from CSF holds potential for innovative diagnostic assays for patients with suspected LM. Combining CTC quantification with molecular single cell analysis could enable us to define predictive tests to select novel treatment options for patients with LM in the future. Citation Format: Caecilia Koestler, Giancarlo Feliciello, Florian Lueke, Kathrin Weidele, Saida Zoubaa, Peter Hau, Tobias Pukrop, Markus J. Riemenschneider, Christoph A. Klein, Bernhard Polzer. Single cell analysis from CSF of patients with suspected leptomeningeal metastasis [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 3709.

Abstract 6561: Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer

Abstract Background: Ovarian Cancer (OC) is one of the leading causes of death among gynecological tumors with an insufficient understanding of OC disease evolution. Since tumor tissue is not always available, blood allows the investigation of single circulating tumor cells (sCTC) over time. We combined immunomagnetic enrichment and image-based sCTC sorting for phenotypic and genotypic analysis of sCTCs in the follow-up of OC to provide insights into sCTC heterogeneity and their malignant character. Patients and Methods: 10 ml blood of high-grade serous OC patients (pts) at the time of primary diagnosis (n=42), after chemotherapy (n=23) and after Avastin (n=11) was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® using CD45 and CD235a antibodies coupled to MACS® MicroBeads. For image-based sCTC sorting (DEPArray®), an immunofluorescence protocol staining for Hoechst, cytokeratin (CK), the folate receptor alpha (FRa), Vimentin (Vim) and CD45 was applied. Whole genome amplification (WGA) was performed in recovered single cells, comprising cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number alteration (CNA) analysis. Ampli1TM low-pass analysis was used to analyze single cell WGA products. 21 primary tumors (PT) were assessed for whole-genome CNA and the Ampli 1TM OncoSeek panel was used for deep sequencing of sCTCs. Results: Phenotypic assessment revealed FRa-positive cells in 28/42 (67%) pts, CK-positive cells in 32/42 (76%) pts and CK-FRa-double-positive cells in 15/42 (36%) pts, respectively, with a significant (p= 0.0205) reduction of cells after chemotherapy only documented for FRa-positive cells. Interestingly, cells with a high nuclear staining but no expression of target antigens were found in 37/42 pts (88%) and significantly (p=0.002) expanded during treatment. Genotyping revealed that only 7% (19/289) of single cells deriving from 13 different pts were aberrant. Whole genome CNA analysis in all pre-treatment sCTCs revealed enriched CNAs in chromosomes 2, 7 and 12. In post-treatment sCTCs, CNA dynamics highlighted the adaptive potential of the homologous recombination pathway and suggested further investigation of MAPK signaling in resistant OC. Low-pass CNA comparison between sCTCs and the PT was feasible in nine pts, 2/9 with persistent PT characteristics in sCTCs at different sampling times. TP53 variants were the most frequently detected ones, persistent CNA in the CDK4 oncogene and emerging CNA in the ALK oncogene were found in sCTCs from two pts. Conclusion: We identified sCTCs in the follow-up of OC and demonstrated interpatient as well as intrapatient heterogeneity of sCTCs. Matched sCTCs and PT data showed similar CNAs to a certain amount. Our low percentage of aberrant cells suggests that higher patient numbers are needed to confirm our findings to understand disease progression. Citation Format: Sabine Kasimir-Bauer, Nikolas H. Stoecklein, Rui P. Neves, Sven T. Liffers, Jan D. Kuhlmann, Pauline Wimberger, Paul Buderath, Rainer Kimmig, Carolin Salmon. Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer [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 6561.

Circulating tumor cell clustering modulates RNA splicing and polyadenylation to facilitate metastasis

Circulating tumor cell (CTC) clusters exhibit significantly higher metastatic potential compared to single CTCs. However, the underlying mechanism behind this phenomenon remains unclear, and the role of posttranscriptional RNA regulation in CTC clusters has not been explored. Here, we conducted a comparative analysis of alternative splicing (AS) and alternative polyadenylation (APA) profiles between single CTCs and CTC clusters. We identified 994 and 836 AS events in single CTCs and CTC clusters, respectively, with ∼20% of AS events showing differential regulation between the two cell types. A key event in this differential splicing was observed in SRSF6, which disrupted AS profiles and contributed to the increased malignancy of CTC clusters. Regarding APA, we found a global lengthening of 3′ UTRs in CTC clusters compared to single CTCs. This alteration was primarily governed by 14 core APA factors, particularly PPP1CA. The modified APA profiles facilitated the cell cycle progression of CTC clusters and indicated their reduced susceptibility to oxidative stress. Further investigation revealed that the proportion of H2AFY mRNA with long 3′ UTR instead of short 3′ UTR was higher in CTC clusters than single CTCs. The AU-rich elements (AREs) within the long 3′ UTR of H2AFY mRNA enhance mRNA stability and translation activity, resulting in promoting cell proliferation and invasion, which potentially facilitate the establishment and rapid formation of metastatic tumors mediated by CTC clusters. These findings provide new insights into the mechanisms driving CTC cluster metastasis.

Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch® and DEPArray™ Systems.

The analysis of circulating tumor cells (CTCs) has shown potential for detection of cancer spread, prognosis, therapeutic target selection, and monitoring of treatment response. CTCs can be obtained repeatedly by simple blood draws as so-called "liquid biopsy." Thus, they can serve as a surrogate material for primary or metastatic tissue biopsies. In addition, isolation of CTCs provides the possibility to investigate those cells which may hold the (molecular) traits responsible for metastatic progression and ultimately patient death. As such, CTCs represent a target of utmost importance in cancer research and therapy. In this chapter, we describe a workflow for the enrichment of CTCs with the FDA-cleared CellSearch® system followed by the isolation of single CTCs using the DEPArray™ technology enabling further molecular single-cell analyses.

A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

Recent Progress in Enhanced Cancer Diagnosis, Prognosis, and Monitoring Using a Combined Analysis of the Number of Circulating Tumor Cells (CTCs) and Other Clinical Parameters.

Analysis of circulating tumor cells (CTCs) holds promise to diagnose cancer or monitor its development. Among the methods, counting CTC numbers in blood samples could be the simplest way to implement it. Nevertheless, its clinical utility has not yet been fully accepted. The reasons could be due to the rarity and heterogeneity of CTCs in blood samples that could lead to misleading results from assays only based on single CTC counts. To address this issue, a feasible direction is to combine the CTC counts with other clinical data for analysis. Recent studies have demonstrated the use of this new strategy for early detection and prognosis evaluation of cancers, or even for the distinguishment of cancers with different stages. Overall, this approach could pave a new path to improve the technical problems in the clinical applications of CTC counting techniques. In this review, the information relevant to CTCs, including their characteristics, clinical use of CTC counting, and technologies for CTC enrichment, were first introduced. This was followed by discussing the challenges and new perspectives of CTC counting techniques for clinical applications. Finally, the advantages and the recent progress in combining CTC counts with other clinical parameters for clinical applications have been discussed.

Circulating tumor cells participate in the formation of microvascular invasion and impact on clinical outcomes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. Although the treatment strategies have been improved in recent years, the long-term prognosis of HCC is far from satisfactory mainly due to high postoperative recurrence and metastasis rate. Vascular tumor thrombus, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), affects the outcome of hepatectomy and liver transplantation. If vascular invasion could be found preoperatively, especially the risk of MVI, more reasonable surgical selection will be chosen to reduce the risk of postoperative recurrence and metastasis. However, there is a lack of reliable prediction methods, and the formation mechanism of MVI/PVTT is still unclear. At present, there is no study to explore the possibility of tumor thrombus formation from a single circulating tumor cell (CTC) of HCC, nor any related study to describe the possible leading role and molecular mechanism of HCC CTCs as an important component of MVI/PVTT. In this study, we review the current understanding of MVI and possible mechanisms, discuss the function of CTCs in the formation of MVI and interaction with immune cells in the circulation. In conclusion, we discuss implications for potential therapeutic targets and the prospect of clinical treatment of HCC.

Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

A Novel Filtration Membrane for Clustered Circulating Tumor Cell Extraction: A Prospective Feasibility Study.

Circulating tumor cells (CTCs) have garnered attention as biomarkers for therapeutic response and prognosis in malignant neoplasms. Nonetheless, existing literature predominantly relies on surrogate markers of tumor cells or focuses on single-cell CTC, failing to adequately address the challenge of detecting cluster-forming CTCs, which bear considerable prognostic implications. This prospective study aims to validate the efficacy of a novel filtration membrane, namely Soft Micro Pore Filter (S-MPF®), for rare cell recovery in detecting CTCs through the analysis of clinical samples. Patients with confirmed lung cancer or highly suspected lung cancer based on specific criteria (solid tumor size >2.0 cm, serum carcinoembryonic level >7.5 ng/ml, maximum standard uptake value derived from fluorodeoxyglucose-position emission tomography >2.9) were included in the study. CTCs were extracted from preoperative peripheral arterial blood samples using S-MPF®, and the validity of the filtration system was positively verified. Out of the 25 enrolled patients, 23 had lung cancer. CTC positivity was observed in 17 cases (73.9%), whereas cluster CTC positivity was observed in 16 cases (69.6%), with a median count of two clusters. Single CTC positivity was observed in 11 cases (52.1%), with a median count of one cell. The utilization of the newly developed S-MPF® filtration membrane exhibited a high rate of CTC identification, demonstrating its suitability for clinical applications.

Efficient detection of single circulating tumor cell in blood using Raman mapping based on Aptamer-SERS bio-probe coupled with micropore membrane filtration

Rapid and accurate detection of rare circulating tumor cells (CTCs) in human blood still remains a challenge. We present a surface enhanced Raman spectroscopy (SERS) method based on aptamer-SERS bio-probe recognition coupled with micropore membrane filtration capture for the detection of CTCs at single cell level. The parylene micropore membrane with optimized micropore size installed on a filtration holder could capture bio-probe labeled CTCs by gravity in less than 10 s, and only with very less white blood cells (WBCs) residual. In order to facilitate the synthesis of the aptamer-SERS bio-probe, ethyl acetate dehydration method was established. The bio-probe can be rapidly synthesized within 2 h by binding SH-aptamer to 4- mercaptobenzoic acid (4-MBA) modified AuNPs with the help of ethyl acetate. The SERS bio-probe with selected specific aptamer could distinguish single human non-small cell lung cancer A549 cells from residual WBCs on membrane efficiently and reliably based on their Raman signal intensity difference at 1075 cm−1. Through the filter membrane coupled with aptamer-SERS bio-probe system, even 20 A549 cells in blood solution simulating CTCs sample can be detected, which the recovery rate and recognition rate are more than 90%. This method is rapid, reliable and cost-effective, which indicates a good prospect in clinical application for CTCs detection.

Improved Characterization of Circulating Tumor Cells and Cancer-Associated Fibroblasts in One-Tube Assay in Breast Cancer Patients Using Imaging Flow Cytometry.

Circulating tumor cells (CTCs) and circulating cancer-associated fibroblasts (cCAFs) have been individually considered strong indicators of cancer progression. However, technical limitations have prevented their simultaneous analysis in the context of CTC phenotypes different from epithelial. This study aimed to analyze CTCs and cCAFs simultaneously in the peripheral blood of 210 breast cancer patients using DAPI/pan-keratin (K)/vimentin (V)/alpha-SMA/CD29/CD45/CD31 immunofluorescent staining and novel technology-imaging flow cytometry (imFC). Single and clustered CTCs of different sizes and phenotypes (i.e., epithelial phenotype K+/V- and epithelial-mesenchymal transition (EMT)-related CTCs, such as K+/V+, K-/V+, and K-/V-) were detected in 27.6% of the samples and correlated with metastases. EMT-related CTCs interacted more frequently with normal cells and tended to occur in patients with tumors progressing during therapy, while cCAFs coincided with CTCs (mainly K+/V- and K-/V-) in seven (3.3%) patients and seemed to correlate with the presence of metastases, particularly visceral ones. This study emphasizes the advantages of imFC in the field of liquid biopsy and highlights the importance of multimarker-based analysis of different subpopulations and phenotypes of cancer progression-related cells, i.e., CTCs and cCAFs. The co-detection of CTCs and cCAFs might improve the identification of patients at higher risk of progression and their monitoring during therapy.

Laser transfer for circulating tumor cell isolation in liquid biopsy.

75Cancer research has found in the recent years a formidable ally in liquid biopsy, a noninvasive technique that allows the study of circulating tumor cells (CTCs) and biomolecules involved in the dynamics of cancer spread like cell-free nucleid acids or tumor-derived extracellular vesicles. However, single-cell isolation of CTCs with high viability for further genetic, phenotypic, and morphological characterization remains a challenge. We present a new approach for single CTC isolation in enriched blood samples using a liquid laser transfer (LLT) process, adapted from standard laser direct write techniques. In order to completely preserve the cells from direct laser irradiation, we used an ultraviolet laser to produce a blister-actuated laser-induced forward transfer process (BA-LIFT). Using a plasma-treated polyimide layer for blister generation, we completely shield the sample from the incident laser beam. The optical transparency of the polyimide allows direct cell targeting using a simplified optical setup, in which the laser irradiation module, standard imaging, and fluorescence imaging share a common optical path. Peripheral blood mononuclear cells (PBMCs) were identified by fluorescent markers, while target cancer cells remained unstained. As a proof of concept, we were able to isolate single MDA-MB-231 cancer cells using this negative selection process. Unstained target cells were isolated and culture while their DNA was sent for single-cell sequencing (SCS). Our approach appears to be an effective approach to isolate single CTCs, preserving cell characteristics in terms of cell viability and potential for further SCS.

Deciphering the Biology of Circulating Tumor Cells through Single-Cell RNA Sequencing: Implications for Precision Medicine in Cancer.

Circulating tumor cells (CTCs) hold unique biological characteristics that directly involve them in hematogenous dissemination. Studying CTCs systematically is technically challenging due to their extreme rarity and heterogeneity and the lack of specific markers to specify metastasis-initiating CTCs. With cutting-edge technology, single-cell RNA sequencing (scRNA-seq) provides insights into the biology of metastatic processes driven by CTCs. Transcriptomics analysis of single CTCs can decipher tumor heterogeneity and phenotypic plasticity for exploring promising novel therapeutic targets. The integrated approach provides a perspective on the mechanisms underlying tumor development and interrogates CTCs interactions with other blood cell types, particularly those of the immune system. This review aims to comprehensively describe the current study on CTC transcriptomic analysis through scRNA-seq technology. We emphasize the workflow for scRNA-seq analysis of CTCs, including enrichment, single cell isolation, and bioinformatic tools applied for this purpose. Furthermore, we elucidated the translational knowledge from the transcriptomic profile of individual CTCs and the biology of cancer metastasis for developing effective therapeutics through targeting key pathways in CTCs.