Transcriptomic profiling of single circulating tumor cells provides insight into human metastatic gastric cancer

Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.

CNA Profiling of Single CTCs in Locally Advanced Esophageal Cancer Patients during Therapy Highlights Unexplored Molecular Pathways

Background: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy. Methods: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis. Results: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients’ groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients. Conclusions: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients’ outcome.

Sequential Isolation and Characterization of Single CTCs and Large CTC Clusters in Metastatic Colorectal Cancer Patients

Circulating tumor cells (CTCs) detach from a primary tumor or its metastases and circulate in the bloodstream. The vast majority of CTCs are deemed to die into the bloodstream, with only few cells representing viable metastatic precursors. Particularly, single epithelial CTCs do not survive long in the circulation due to the loss of adhesion-dependent survival signals. In metastatic colorectal cancer, the generation of large CTC clusters is a very frequent occurrence, able to increase the aptitude of CTCs to survive in the bloodstream. Although a deepened analysis of large-sized CTC clusters might certainly offer new insights into the complexity of the metastatic cascade, most CTC isolation techniques are unfortunately not compatible with large-sized CTC clusters isolation. The inappropriateness of standard CTC isolation devices for large clusters isolation and the scarce availability of detection methods able to specifically isolate and characterize both single CTCs and CTC clusters finally prevented in-depth studies on the prognostic and predictive value of clusters in clinical practice, unlike that which has been described for single CTCs. In the present study, we validated a new sequential filtration method for the simultaneous isolation of large CTC clusters and single CTCs in patients with metastatic colorectal cancer at failure of first-line treatments. The new method might allow differential downstream analyses for single and clustered CTCs starting from a single blood draw, opening new scenarios for an ever more precise characterization of colorectal cancer metastatic cascade.

Specific alternative splicing and polyadenylation facilitate metastasis mediated by CTC clusters

Circulating tumor cell (CTC) clusters possess a much higher capability to seed metastasis than single CTCs. However, the mechanism underlying this phenomenon is still elusive and no reports have investigated the role of posttranscriptional RNA regulation in CTC clusters. Here, we compared alternative splicing (AS) and alternative polyadenylation (APA) profiles between single CTCs and CTC clusters. 994 and 836 AS events were identified in single CTCs and CTC clusters, separately. About ~20% of AS events exhibited alterations between both cell types. The differential splicing of SRSF6 was a core event that caused AS profiles’ disturbance and made CTC clusters more dangerous. Concerning APA, we identified global 3’ UTRs lengthening in CTC clusters compared with single CTCs. This change was mainly regulated by 14 core APA factors, especially PPP1CA. The altered APA profiles boosted the cell cycle of CTC clusters and reflected that CTC clusters endured less oxidative stress. Our study investigated the posttranscriptional regulation mechanisms in CTC clusters, found that the perturbation of AS and APA contributed to the superiority of CTC clusters compared with single CTCs, and laid the foundation for developing antisense oligonucleotides that inhibit metastasis by reducing CTC clusters.

Circulating Tumor Cell Clusters Are Cloaked with Platelets and Correlate with Poor Prognosis in Unresectable Pancreatic Cancer

Circulating tumor cells (CTCs) are known to be heterogeneous and clustered with tumor-associated cells, such as macrophages, neutrophils, fibroblasts, and platelets. However, their molecular profile and clinical significance remain largely unknown. Thus, we aimed to perform a comprehensive gene expression analysis of single CTCs and CTC clusters in patients with pancreatic cancer and to identify their potential clinical relevance to provide personalized medicine. Epitope-independent, rapid (>3 mL of whole blood/min) isolation of single CTCs and CTC clusters was achieved from a prospective cohort of 16 patients with unresectable pancreatic cancer using a centrifugal microfluidic device. Forty-eight mRNA expressions of individual CTCs and CTC clusters were analyzed to identify pancreatic CTC phenotype. CTC clusters had a larger proportion of mesenchymal expression than single CTCs (p = 0.0004). The presence of CTC clusters positively correlated with poor prognosis (progression-free survival, p = 0.0159; overall survival, p = 0.0186). Furthermore, we found that most CTCs in these patients (90.7%) were cloaked with platelets and found the presence of a positive correlation between the increase in CTC clusters and rapid disease progression during follow-ups. Efficient CTC cluster isolation and analysis techniques will enhance the understanding of complex tumor metastasis processes and can facilitate personalized disease management.

Clinical Significance of Circulating Tumor Cell Induced Epithelial-Mesenchymal Transition in Patients with Metastatic Colorectal Cancer by Single-Cell RNA-Sequencing

Background: Circulating tumor cells (CTCs) are a prognostic marker in patients with metastatic colorectal cancer (mCRC). However, little is known about the characterization of CTCs in mCRC at the single-cell level using RNA sequencing. The purpose of this study was to validate the capability to detect and isolate single CTCs for single-cell RNA sequencing (scRNA-seq) and to identify clinical significance at a single CTC level. Methods: Single CTCs from 27 mCRC patients were collected by CTC-FIND, which is comprised of filter separation and immunomagnetic depletion to collect ultra-pure CTC samples. To address tumor heterogeneity, CTCs were collected without relying on any traditional CTC markers, such as epithelial and mesenchymal cell antigens, and were undertaken by scRNA-seq using SMART-Seq v4. Results: We identified 59 single CTCs which were classified into four groups by epithelial, epithelial-mesenchymal transition (EMT) and stem cell-related gene expression. Patients receiving second or later-line treatment who had EMT gene expressing CTCs had a significantly shorter PFS and OS. Conclusions: Exploiting CTC-FIND with SMART-Seq v4 showed that scRNA-seq of CTCs may shed new insight into tumor heterogeneity of mCRC and that the presence of CTCs expressing EMT-related genes at the single-cell level could have prognostic value in mCRC patients.

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.

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

Profiling heterogenous sizes of circulating tumor microemboli to track therapeutic resistance and prognosis in advanced gastric cancer

Circulating tumor microemboli (CTM) aggregated by ≥ 2 circulating tumor cells (CTCs) are more migratory than single CTCs. Aside from the plasticity in their molecular characteristics, which have been considered tumor migration, CTM also possesses high size heterogeneity. This study, therefore, systematically investigated the heterogeneous sizes of CTM and their involvement in therapeutic resistance in 114 patients with advanced gastric cancer (GC) using a pre-established surface molecule-independent subtraction enrichment (SE)-iFISH strategy. CTM, which was pre-therapeutically detected in 33.3% of GC patients, can further form in another 34.78% of patients following chemo-/targeted therapies. The presence of CTM is relevant to liver metastasis as well as higher CTC levels (≥ 5/6 mL). Further size-based profiling of GC-CTM revealed that CTM with 2 CTCs (CTM2) was the dominant subtype, accounting for 50.0% of all detected GC-CTMs. However, CTM with 3–4 CTCs (CTM3–4) specifically associates with chemo-/targeted therapeutic resistance and inferior prognosis. Patients with ≥ 1 CTM3–4/6 mL have shorter median progression-free survival and median overall survival. Unlike CTM2 and CTM3–4, which are detectable in pre-therapy and post-therapy, larger aggregated CTM≥5 (CTM with ≥ 5 CTCs) was only intra-therapeutically detected in four HER2+ GC patients, of which three experienced liver metastases. Obtained results suggested that the cluster size of GC-CTM should be dynamically profiled beyond pre-therapeutic whole CTM enumeration in terms of chemo-/targeted resistance or metastasis monitoring. GC-CTM3–4 could be a potential indicator of therapeutic resistance, while the dynamic presence of GC-CTM≥5 implies liver metastasis in HER2+ GC patients.