Circulating Tumor Cells Clusters Research Articles

Regulating chemoresistance and cancer stemness: the CDH17-YAP pathway in distinct cellular states of lung cancer CTC clusters

BackgroundDrug resistance in metastatic lung cancer significantly contributes to patient mortality. This study explores the role of circulating tumor cells (CTCs), the precursors to metastasis, in driving this resistance. We aim to delineate the unique biological traits of CTC clusters in lung cancer and elucidate the mechanisms underlying their resistance to chemotherapy.MethodsWe used an ultralow adsorption plate to establish a CTC suspension culture system. Comparisons between adherent and suspension cultures of CTC-TJH-01 cells were made via Cell Counting Kit-8 (CCK-8), western blot, immunofluorescence, and flow cytometry assays to evaluate cell proliferation, drug resistance, and cancer stemness. The tumorigenicity, tumor growth rate, and drug resistance of the CTC clusters were assessed in nude mice. Transcriptomic and proteomic analyses were subsequently conducted to identify differentially expressed genes and proteins in CTC-TJH-01 cells cultured under adherent and suspension conditions. CDH17 gene knockdown in CTC-TJH-01 cells was achieved through RNA interference, and hematoxylin and eosin (HE) staining, immunohistochemistry, and immunofluorescence assays were used to examine the pathological status of these cells.ResultsCTC-TJH-01 cells in suspension formed cell clusters and exhibited decreased proliferation, tumorigenicity, and tumor growth, but increased cancer stemness and drug resistance. CDH17 protein expression was significantly upregulated in these clusters, activating the YAP/TAZ pathway. Knocking down CDH17 not only inactivated this pathway but also significantly increased cell proliferation activity and cisplatin sensitivity in CTC-TJH-01 clusters. Additionally, the tumor growth rate was correlated with cisplatin sensitivity. CDH17 knockdown notably promoted the growth of CTC-TJH-01 xenografts and enhanced their sensitivity to cisplatin, although no significant difference was observed compared with those in the control group.ConclusionsThe results indicate that lung CTC clusters with stem cell-like properties exhibit chemoresistance, which is linked to an activated CDH17-YAP pathway. Additionally, the effectiveness of cisplatin is primarily observed in tumors with relatively high growth rates, highlighting the connection between tumor growth and sensitivity to chemotherapy.Graphical abstract

Gravity-based microfiltration reveals unexpected prevalence of circulating tumor cell clusters in ovarian and colorectal cancer

BackgroundCirculating tumor cells (CTCs) are rare (a few cells per milliliter of blood) and mostly isolated as single-cell CTCs (scCTCs). CTC clusters (cCTCs), even rarer, are of growing interest, notably because of their higher metastatic potential, but very difficult to isolate.MethodWe introduce gravity-based microfiltration (GµF) for facile isolation of cCTCs using in-house fabricated microfilters and 3D printed cartridges. Optimal flow rate and pore size for cCTC isolation are determined by GµF of cultured ovarian single cells and cell clusters spiked in healthy blood. We perform GµF of blood from orthotopic ovarian cancer mouse models and characterize the morphological features of scCTCs and cCTCs, and the expression of molecular markers for aggressiveness. Finally, we analyze blood from 17 epithelial ovarian cancer patients with either localized or metastatic disease, and from 13 colorectal cancer liver metastasis patients.ResultsHere, we show that GµF optimized for cell cluster isolation captures cCTCs from blood while minimizing unwanted cluster disaggregation, with ~85% capture efficiency. We detect cCTCs in every patient, with between 2–100+ cells. We find cCTCs represent between 5–30% of all CTC capture events, and 10-80% of the CTCs are clustered; remarkably, in 10 patients, most CTCs are circulating not as scCTCs, but as cCTCs.ConclusionsGµF uncovers the unexpected prevalence and frequency of cCTCs including sometimes very large ones in epithelial ovarian cancer patients, and motivates additional studies to uncover their properties and role in disease progression.

Digoxin for reduction of circulating tumor cell cluster size in metastatic breast cancer: a proof-of-concept trial.

The presence of circulating tumor cell (CTC) clusters is associated with disease progression and reduced survival in a variety of cancer types. In breast cancer, preclinical studies showed that inhibitors of the Na+/K+ ATPase suppress CTC clusters and block metastasis. Here we conducted a prospective, open-label, proof-of-concept study in women with metastatic breast cancer, where the primary objective was to determine whether treatment with the Na+/K+ ATPase inhibitor digoxin could reduce mean CTC cluster size. An analysis of nine patients treated daily with a maintenance digoxin dose (0.7-1.4 ng ml-1 serum level) revealed a mean cluster size reduction of -2.2 cells per cluster upon treatment (P = 0.003), meeting the primary endpoint of the study. Mechanistically, transcriptome profiling of CTCs highlighted downregulation of cell-cell adhesion and cell-cycle-related genes upon treatment with digoxin, in line with its cluster-dissolution activity. No treatment-related adverse events occurred. Thus, our data provide a first-in-human proof of principle that digoxin treatment leads to a partial CTC cluster dissolution, encouraging larger follow-up studies with refined Na+/K+ ATPase inhibitors and that include clinical outcome endpoints. ClinicalTrials.gov identifier: NCT03928210 .

Challenges in blood fractionation for cancer liquid biopsy: how can microfluidics assist?

Liquid biopsy provides a minimally invasive approach to characterise the molecular and phenotypic characteristics of a patient's individual tumour by detecting evidence of cancerous change in readily available body fluids, usually the blood. When applied at multiple points during the disease journey, it can be used to monitor a patient's response to treatment and to personalise clinical management based on changes in disease burden and molecular findings. Traditional liquid biopsy approaches such as quantitative PCR, have tended to look at only a few biomarkers, and are aimed at early detection of disease or disease relapse using predefined markers. With advances in the next generation sequencing (NGS) and single-cell genomics, simultaneous analysis of both circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) is now a real possibility. To realise this, however, we need to overcome issues with current blood collection and fractionation processes. These include overcoming the need to add a preservative to the collection tube or the need to rapidly send blood tubes to a centralised processing lab with the infrastructure required to fractionate and process the blood samples. This review focuses on outlining the current state of liquid biopsy and how microfluidic blood fractionation tools can be used in cancer liquid biopsy. We describe microfluidic devices that can separate plasma for ctDNA analysis, and devices that are important in isolating the cellular component(s) in liquid biopsy, i.e., individual CTCs and CTC clusters. To facilitate a better understanding of these devices, we propose a new categorisation system based on how these devices operate. The three categories being 1) solid Interaction devices, 2) fluid Interaction devices and 3) external force/active devices. Finally, we conclude that whilst some assays and some cancers are well suited to current microfluidic techniques, new tools are necessary to support broader, clinically relevant multiomic workflows in cancer liquid biopsy.

Modeling the dynamics of circulating tumor cell clusters inside a microfluidic channel.

Circulating tumor cells are central to metastasis, a particularly malign spread of cancer beyond its original location. While rare, there is growing evidence that the clusters of circulating tumor cells are significantly more harmful than individual cells. Microfluidic platforms constitute the core of circulating tumor cell cluster research, allowing cluster detection, analysis, and treatment. In this work, we propose a new mathematical model of circulating tumor cell clusters and apply it to simulate the dynamics of the aggregates inside a microfluidic channel with the external flow of a fluid. We leverage our previous model of the interactions of circulating tumor cells with varying clustering affinities and introduce explicit bonds between the cells that makeup a cluster. We show that the bonds have a visible impact on the cluster dynamics and that they enable the reproduction of known cluster flow and deformation patterns. Furthermore, we demonstrate that the dynamics of these aggregates are sensitive to bond properties, as well as initialization and flow conditions. We believe that our modeling framework represents a valuable mesoscopic formulation with an impact beyond circulating tumor cell clusters, as cell aggregates are common in both nature and applications.

Digoxin Reduces Circulating Tumor Cell Cluster Size in Breast Cancer

Digoxin Reduces Circulating Tumor Cell Cluster Size in Breast Cancer

Formation of motile cell clusters in heterogeneous model tumors: The role of cell-cell alignment.

Circulating tumor cell clusters play an important role in the metastatic cascade. These clusters can acquire a migratory and more invasive phenotype, and coordinate their motion to migrate as a collective. Before such clusters can form by collectively detaching from a primary tumor, however, the cluster must first aggregate in the tumor interior. The mechanism of this cluster formation process is still poorly understood. One of the possible ways for cells to cluster is by aligning their direction of motion with their neighboring cells. This work aims to investigate the role of this cell-cell alignment interaction on the formation of motile cell clusters inside the bulk of a tumor using computer simulations. We employ a cellular Potts model in which we model a two-dimensional heterogeneous confluent layer containing both motile and nonmotile cells. Our results indicate that the degree of clustering is governed by two distinct processes: the formation of clusters due to the presence of cell-cell alignment interactions among motile cells, and the suppression of clustering due to the presence of the dynamic cellular environment (comprising the nonmotile cells). We find that the largest motile clusters are formed for intermediate alignment strengths, contrary to what is observed for motile cells in free space (that is, unimpeded by a dense cellular environment), in which case stronger cell-cell alignment always leads to larger clustering. Our findings suggest that the presence of a densely packed cellular environment and strong cell-cell alignment inhibits the formation of large migratory clusters within the primary tumor, providing physical insight into potential factors at play during the early stages of metastasis.

Advanced single-cell and spatial analysis with high-multiplex characterization of circulating tumor cells and tumor tissue in prostate cancer: Unveiling resistance mechanisms with the CoDuCo in situ assay

BackgroundMetastatic prostate cancer is a highly heterogeneous and dynamic disease and practicable tools for patient stratification and resistance monitoring are urgently needed. Liquid biopsy analysis of circulating tumor cells (CTCs) and circulating tumor DNA are promising, however, comprehensive testing is essential due to diverse mechanisms of resistance. Previously, we demonstrated the utility of mRNA-based in situ padlock probe hybridization for characterizing CTCs.MethodsWe have developed a novel combinatorial dual-color (CoDuCo) assay for in situ mRNA detection, with enhanced multiplexing capacity, enabling the simultaneous analysis of up to 15 distinct markers. This approach was applied to CTCs, corresponding tumor tissue, cancer cell lines, and peripheral blood mononuclear cells for single-cell and spatial gene expression analysis. Using supervised machine learning, we trained a random forest classifier to identify CTCs. Image analysis and visualization of results was performed using open-source Python libraries, CellProfiler, and TissUUmaps.ResultsOur study presents data from multiple prostate cancer patients, demonstrating the CoDuCo assay’s ability to visualize diverse resistance mechanisms, such as neuroendocrine differentiation markers (SYP, CHGA, NCAM1) and AR-V7 expression. In addition, druggable targets and predictive markers (PSMA, DLL3, SLFN11) were detected in CTCs and formalin-fixed, paraffin-embedded tissue. The machine learning-based CTC classification achieved high performance, with a recall of 0.76 and a specificity of 0.99.ConclusionsThe combination of high multiplex capacity and microscopy-based single-cell analysis is a unique and powerful feature of the CoDuCo in situ assay. This synergy enables the simultaneous identification and characterization of CTCs with epithelial, epithelial-mesenchymal, and neuroendocrine phenotypes, the detection of CTC clusters, the visualization of CTC heterogeneity, as well as the spatial investigation of tumor tissue. This assay holds significant potential as a tool for monitoring dynamic molecular changes associated with drug response and resistance in prostate cancer.

BIOM-62. CIRCULATING TUMOR CELLS IN MEDULLOBLASTOMA: A NOVEL PERIPHERAL BIOMARKER FOR CNS DISEASE

Abstract BACKGROUND Medulloblastoma is an embryonal tumor of the cerebellum and the most common pediatric malignancy of the central nervous system (CNS). Diagnosis, staging, and disease monitoring require invasive neurosurgical biopsy or MRI. No blood or cerebrospinal fluid (CSF) biomarkers exist. We demonstrate the presence of circulating tumor cell clusters (CTCCs) in patients with medulloblastoma and use a transcriptomic approach to describe their gene expression. Design/Method: We enrolled 34 total patients in a longitudinal study: 24 with medulloblastoma and 10 without malignancy. CTCCs are captured from unprocessed whole blood using a physics-based label-free approach to CTCC isolation. RNA sequencing was performed on 21 samples at time of diagnostic resection from 6 patients with medulloblastoma. Sequencing reads were assessed for quality control, trimmed, and aligned to the human genome. Differential gene expression (DGE) was performed against whole blood from healthy controls. Reactome pathway database of curated, peer-reviewed gene sets are used for gene set enrichment analysis (GSEA). RESULTS The presence of peripherally CTCC patients with medulloblastoma is not previously known and was detected in all patients with medulloblastoma whereas none were observed from patients with non-malignant hematological conditions. RNA-sequencing demonstrate separation on principal component analysis and gene-expression heatmap against whole blood healthy control. DGE shows upregulation of genes indicating stem-like state. GSEA show enrichment of medulloblastoma molecular pathways and gene sets defining integrin, extracellular matrix remodeling, and cell adhesion pathways. Transient elevation of peripheral CTCC followed by return to baseline levels was associated with durable disease response to therapy, whereas persistence or elevation of CTCC in CSF correlated with disease recurrence. CONCLUSION The universal and specific finding of peripherally captured CTCC arising from parent tumor in the CNS within our patient cohort suggests a utility as a tumor biomarker. Future sequencing is planned comparing CTCC transcriptome against parent tumor.

A Preliminary Analysis of Circulating Tumor Microemboli from Breast Cancer Patients during Follow-Up Visits.

Most breast cancer-related deaths are caused by distant metastases and drug resistance. It is important to find appropriate biomarkers to monitor the disease and to predict patient responses after treatment early and accurately. Many studies have found that clustered circulating tumor cells, with more correlations with metastatic cancer and poor survival of patients than individual ones, are promising biomarkers. Eighty samples from eleven patients with breast cancer during follow-up visits were examined. By using a microfluidic chip and imaging system, the number of circulating tumor cells and microemboli (CTC/CTM) were counted to assess the distribution in stratified patients and the potential in predicting the disease condition of patients after treatments during follow-up visits. Specific components and subtypes of CTM were also preliminarily investigated. Compared to CTC, CTM displayed a distinguishable distribution in stratified patients, having a better AUC value, in predicting the disease progression of breast cancer patients during follow-up visits in this study. Four subtypes were categorized from the identified CTM by considering different components. In combination with CEA and CA153, enumerated CTC and CTM from individual patients were applied to monitor the disease condition and patient response to the therapy during follow-up visits. The CTM and its subtypes are promising biomarkers and valuable tools for studying cancer metastasis and longitudinally monitoring cancer patients during follow-up visits.

Detection of Circulating Tumor Cells and EGFR Mutation in Pulmonary Vein and Arterial Blood of Lung Cancer Patients Using a Newly Developed Immunocytology-Based Platform.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are powerful molecular targeted therapeutic agents for lung cancer. We recently developed an original immunocytology and glass slide-based circulating tumor cell (CTC) detection platform for both CTC enumeration and EGFR mutation analysis with DNA extracted from CTCs. Using this platform, we conducted a pilot clinical study for CTC enumeration in peripheral blood (PB), pulmonary arterial blood (PA), and pulmonary venous blood (PV) from 33 patients with lung cancer (Stage I-III) who underwent surgery, followed by digital PCR-based EGFR mutation analysis of CTCs in PV from 12 patients. The results showed that CTC levels were significantly higher in PV and PA than in PB (p < 0.05, p < 0.01. respectively), with a notably greater number of small and large CTC clusters (p < 0.01). Genetic analysis of EGFR mutations of CTCs from PV (n = 12) revealed six mutations, including three Exon19del and three L856R, in CTCs and eight EGFR mutations, including five Exon19del and three L856R, in lung tumor tissue. CTC mutation status matched that of tissue samples in nine patients, was unmatched in two patients, and controversial in one patient, indicating a sensitivity of 0.75 (6/8) and specificity of 1.0 (4/4) with some false-negative results for the mutation analysis of CTCs. This immunocytology-based CTC detection platform is a convenient method for detecting both CTC number and EGFR mutation status under microscopy, suggesting its potential as a liquid biopsy tool in the hospital for patients with lung cancer in some clinical settings.

32P Patient derived circulating tumor cell clusters for personalized chemotherapy

32P Patient derived circulating tumor cell clusters for personalized chemotherapy

Label-Free Microfluidic Apheresis of Circulating Tumor Cell Clusters.

Screening liters of blood (i.e., apheresis) represents a generalized approach to promote the reliable access to circulating tumor cell clusters (CTCCs), which are known to be highly metastasis-competent, yet ultrarare. However, no existing CTCC sorting technology has demonstrated high throughput, high yield, low shear stress, and minimal blood dilution simultaneously as required in apheresis. Here, a label-free method is introduced termed Precision Apheresis for Non-invasive Debulking of cell Aggregates (PANDA) to continuously isolate CTCCs from undiluted blood to clean buffer through size sorting, processing 1.4 billion cells per second. The cell focusing is optimized within whole blood leveraging secondary transverse flow and margination. The PANDA chip recovers >90% of spiked ≈24 rare HeLa cell clusters from 100mL undiluted blood samples (equivalent to ≈500 billion blood cells) at 1 Lh-1 throughput, with ≤20s device residence time, ≤15Pa shear stress, and >99.9% return of blood components. The technology lays the groundwork for future routine isolation to increase the recovery of these ultrarare yet clinically significant tumor cell populations from large volumes of blood to advance cancer research, early detection, and treatment.

In vitro evidence for the potential of EGFR inhibitors to decrease the TGF-β1-induced dispersal of circulating tumour cell clusters mediated by EGFR overexpression

Most cancer-related deaths are due to the spread of tumour cells throughout the body—a process known as metastasis. While in the vasculature, these cells are referred to as circulating tumour cells (CTCs) and can be found as either single cells or clusters of cells (often including platelets), with the latter having the highest metastatic potential. However, the biology of CTC clusters is poorly understood, and there are no therapies that specifically target them. We previously developed an in vitro model system for CTC clusters and proposed a new extravasation model that involves cluster dissociation, adherence, and single-cell invasion in response to TGF-β1 released by platelets. Here, we investigated TGF-β1-induced gene expression changes in this model, focusing on genes for which targeted drugs are available. In addition to the upregulation of the TGF-β1 signalling pathway, we found that (i) genes in the EGF/EGFR pathway, including those coding for EGFR and several EGFR ligands, were also induced, and (ii) Erlotinib and Osimertinib, two therapeutic EGFR/tyrosine kinase inhibitors, decreased the TGF-β1-induced adherence and invasion of the CTC cluster-like line despite the line expressing wild-type EGFR. Overall, we suggest that EGFR inhibitors have the potential to decrease the dispersal of CTC clusters that respond to TGF-β1 and overexpress EGFR (irrespective of its status) and thus could improve patient survival.

Metal complex lipid-based nanoparticles deliver metabolism-regulating lomitapide to overcome CTC immune evasion via activating STING pathway

Metal complex lipid-based nanoparticles deliver metabolism-regulating lomitapide to overcome CTC immune evasion via activating STING pathway

Identification of circulating tumor cells captured by the FDA-cleared Parsortix® PC1 system from the peripheral blood of metastatic breast cancer patients using immunofluorescence and cytopathological evaluations

Circulating Tumor Cells (CTCs) may serve as a non-invasive source of tumor material to investigate an individual’s disease in real-time. The Parsortix® PC1 System, the first FDA-cleared medical device for the capture and harvest of CTCs from peripheral blood of metastatic breast cancer (MBC) patients for use in subsequent user-validated downstream analyses, enables the epitope-independent capture of CTCs with diverse phenotypes based on cell size and deformability. The aim of this study was to determine the proportion of MBC patients and self-declared female healthy volunteers (HVs) that had CTCs identified using immunofluorescence (IF) or Wright-Giemsa (WG) staining. Peripheral blood from 76 HVs and 76 MBC patients was processed on Parsortix® PC1 Systems. Harvested cells were cytospun onto a charged slide and immunofluorescently stained for identification of CTCs expressing epithelial markers. The IF slides were subsequently WG-stained and analyzed for CTC identification based on morphological features of malignant cells. All testing was performed by operators blinded to the clinical status of each subject. CTCs were identified on the IF slides in 45.3% (≥ 1) / 24.0% (≥ 5) of the MBC patients (range = 0 – 125, mean = 7) and in 6.9% (≥ 1) / 2.8% (≥ 5) of the HVs (range = 0 – 28, mean = 1). Among the MBC patients with ≥ 1 CTC, 70.6% had only CK + /EpCAM- CTCs, with none having EpCAM + /CK- CTCs. CTC clusters were identified in 56.0% of the CTC-positive patients. On the WG-stained slides, CTCs were identified in 42.9% (≥ 1) / 21.4% (≥ 5) of the MBC patients (range = 0 – 41, mean = 4) and 4.3% (≥ 1) / 2.9% (≥ 5) of the HVs (range = 0 – 14, mean = 0). This study demonstrated the ability of the Parsortix® PC1 System to capture and harvest CTCs from a significantly larger proportion of MBC patients compared to HVs when coupled with both IF and WG cytomorphological assessment. The presence of epithelial cells in subjects without diagnosed disease has been previously described, with their significance being unclear. Interestingly, a high proportion of the identified CTCs did not express EpCAM, highlighting the limitations of using EpCAM-based approaches.

Adhesion of pancreatic tumor cell clusters by desmosomal molecules enhances early liver metastases formation

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell–cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

Fibrinolytic Platelet Decoys Reduce Cancer Metastasis by Dissociating Circulating Tumor Cell Clusters.

During metastasis, circulating tumor cells (CTCs) can travel in the bloodstream as individual cells or clusters, associated with fibrin and platelets. Clusters have a higher metastatic potential due to their increased ability to withstand shear stress and arrest in small vessels. Moreover, CTC-platelet interaction protects CTCs from shear stress and immune detection. The objective of this project is to develop a fibrinolytic platelet system to leverage platelet-CTC interactions and dissociate CTC clusters. For this approach, tissue plasminogen activator (tPA) is loaded onto two modified platelet systems: platelet Decoys and lyophilized platelets. The activities of the systems are characterized using a Förster Resonance Energy Transfer-based assay and an angiogenic assay. Furthermore, the ability of the system to dissociate cancer cell clusters in vitro is assessed using light transmission aggregometry. The data demonstrates that the fibrinolytic platelets can maintain tPA activity, interact with CTCs, and dissociate cancer cell clusters. Finally, fibrinolytic platelets are assessed in vivo, demonstrating a decreased tumor load and increased survival with tPA-Decoy treatment, which is selected as the optimal treatment based on favorable in vitro results and in vivo trials. Therefore, this fibrinolytic platelet approach is a promising method for leveraging platelet-CTC interactions to disperse CTC clusters and reduce metastasis.

Effect of circulating tumor cells (CTC) and CTC clusters with PD-L1 dynamic biomarker on cellular burden in patients with ovarian cancer.

e17566 Background: In the precision oncology era, monitoring response to the treatments using circulating blood-based biomarkers e.g. circulating tumor DNA (ctDNA) and Circulating Tumor Cells (CTCs) are being rapidly evaluated and established. The leading cause of ovarian cancer patient mortality is owing to the delayed diagnosis, inability of patient selection for targeted therapies, including the use of immune checkpoint blockade (ICB) agents. The treatment of ovarian cancer usually involves a combination of surgery and chemotherapy. However, post operative resection and therapy with curative intent often fails in accounting the Minimal Cellular Disease (MCD). The dissemination of Circulating Tumor Cells (CTC) remains a dogma of Minimal Residual Disease (MRD) that diffuse and cause micro-metastasis through their epithelial to mesenchymal transitions (EMT) and ‘bio-mechanistic activation’ in blood circulation. Simultaneous detection of over-expression of Programmed Cell Death-Ligand 1 (PD-L1) on CTCs as a dynamic biomarker may be expedient for assessing patients for immune checkpoint inhibitor (ICT) therapy. Methods: In retrospective analysis of real-world data, 75 ovarian cancer patient’s peripheral blood was analyzed for the presence of CTCs with and without the expression of PD-L1 and CTC clusters. CTC were positively detected using OncoDiscover platform approved by CDSCO in 1.5 ml peripheral blood. The platform is multifunctional magneto nanosystem mediated by anti-epithelial cellular adhesion molecule (EpCAM) antibody. CTCs were identified as positive if they were EpCAM +ve, CK+ve, PD-L1 +ve, DAPI +ve and CD45-ve. PD-L1 expression on the CTCs was analyzed based on the linear intensity gradients of the fluorescence signals using image acquisition on an automated Zeiss Microscope. Results: At baseline sample analysis, 86 % (n=65) of the patients showed ≥ 1CTCs per 1.5 ml of blood. The CTC distribution in this study ranged from 1-9 CTCs. Whereas, 46.15 % (n=30) patients with CTCs showed the expression of PD-L1. Noteworthy, the highest number of CTCs were observed ~ 26.7 % (n=23) in age group 41-50. Interestingly, 8 % (n=6) of total patients showed the presence of CTC clusters. The presence of CTCs with expression of PD-L1 and CTC clusters did not correlate with factors like staging, follow ups, metastasis, and DFS status. Conclusions: We observed presence of MCD and MRD in CRC patients, in spite of curative intent in ovarian cancers. Detection of CTCs, CTC clusters with over-expression of PD-L1- as a real time dynamic biomarker may be useful for assessing the patients for early metastasis, regression, and selecting patient suitable for immune checkpoint inhibitor (ICT) therapy when the tissue is inadequate or unavailable for better outcome.

Two-color diffuse in vivo flow cytometer.

Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.