Metastatic Circulating Tumor Cells Research Articles

CXCL12-loaded-hydrogel (CLG): A new device for metastatic circulating tumor cells (CTCs) capturing and characterization

BackgroundCirculating Tumor Cells (CTCs) represent a small, heterogeneous population that comprise the minority of cells able to develop metastasis. To trap and characterize CTCs with metastatic attitude, a CXCL12-loaded hyaluronic-gel (CLG) was developed. CXCR4+cells with invasive capability would infiltrate CLG. MethodsHuman colon, renal, lung and ovarian cancer cells (HT29, A498, H460 and OVCAR8 respectively) were seeded on 150 μl Empty Gels (EG) or 300 ng/ml CXCL12 loaded gel (CLG) and allowed to infiltrate for 16 h. Gels were then digested and fixed with 2 % FA-HAse for human cancer cell enumeration or digested with HAse and cancer cells recovered. CLG-recovered cells migrated toward CXCL12 and were tested for colonies/spheres formation. Moreover, CXCR4, E-Cadherin and Vimentin expression was assessed through flow cytometry and RT-PCR. The clinical trial “TRAP4MET” recruited 48 metastatic/advanced cancer patients (8 OC, 8 LC, 8 GBM, 8 EC, 8 RCC and 8 EC). 10 cc whole blood were devoted to PBMCs extraction (7 cc) and ScreenCell™ filters (3 cc) CTCs evaluation. Ficoll-isolated patient's PBMCs were seeded over CLG and allowed to infiltrate for 16 h; gels were digested and fixed with 2 % FA-HAse, cells stained and DAPI+/CD45-/pan-CK + cells enumerated as CTCs. ResultsHuman cancer cells infiltrate CLG more efficiently than EG (CLG/EG ratio 1.25 for HT29/1.58 for A498/1.71 for H460 and 2.83 for OVCAR8). CLG-recovered HT29 cells display hybrid-mesenchymal features [low E-cadherin (40 %) and high vimentin (235 %) as compared to HT29], CXCR4 two-fold higher than HT29, efficiently migrate toward CXCL12 (two-fold higher than HT29) and developed higher number of colonies (171 ± 21 for HT29-CLG vs 131 ± 8 colonies for HT29)/larger spheres (spheroid area: 26561 ± 6142 μm2 for HT29-CLG vs 20297 ± 7238 for HT29). In TRAP4MET clinical trial, CLG-CTCs were isolated in 8/8 patients with OC, 6/8 with LC, 6/8 with CRC, 8/8 with EC, 8/8 with RCC cancer and 5/8 with GBM. Interestingly, in OC, LC and GBM, CLG isolated higher number of CTCs as compared to the conventional ScreenCell™ (CLG/SC ratio = 1.88 for OC, 2.47 for LC and 11.89 for GBM). Bland and Altman blot analysis and Passing and Bablok regression analysis showed concordance between the methodological approaches but indicate that SC and CLG are not superimposable suggesting that the two systems select cells with different features. ConclusionCLG might represent a new and easy tool to isolate invasive CTCs in multiple cancers such as OC, LC and GBM at today orphan of reliable methods to consistently detect CTCs.

A cell targeting and sorting approach based on the magnetophoretic capturing for early prognostics of metastatic cervical cancer cells

HeLa cervical cancer cells are immortal with telomerase activity and metastatic characteristics similar to circulating tumor cells (CTCs). Here, we report aptamer-modified multilayered magnetic beads (Apt@MBs) that efficiently targeted and captured HeLa cells up to a low concentration of freshly prepared cell suspension (500 cells/mL). Apt@MBs were functionalized with fluorophore-conjugated AS1411-aptamer on an outer layer made up of molybdenum disulfide (MoS2) to target nucleolin on the cell surface of captured HeLa cells. Moreover, this outer MoS2 layer of MBs was nanoporous and could load anticancer drugs inside its porous cavities with the possibility of killing the captured and metastatic CTCs in vivo. An internal core layer of Apt@MBs consisting of Ag–Fe3O4 magnetic particles (MPs) was designed for magnetic manifestations and cell sorting with the possibility of screening CTCs (in the patient's blood samples) for early diagnosis of metastatic cancers. The Apt@MBs after cell capture gave rise to the heavier HeLa-MBs composites to get settled down under gravitational/inertial forces to the bottom of the tube quicker than the free cells (within 10 min). The gravitational settling of HeLa-MBs was further coupled with exposing a magnetic field to effectively capture and enrich the cells at the bottom of the tube (from 91 to 98 % cells). While the fluid containing dead, non-cancerous, or uncaptured cells in the supernatant layers were easily removed by pipetting. The HeLa-MBs after sorting out were resuspended into a fresh culture medium for further incubation or cellular analysis. Moreover, both cisplatin (CP) and epirubicin (EP) loaded Apt@MBs showed the killing of about 50 % of the captured cells. Therefore, we are confident that Apt@MBs can contribute to enumerating patients' blood samples for screening CTCs to timely and efficiently detect metastatic cancers along with the ability to effectively perform prognosis, and treatment of metastatic cancers.

Platelet-mediated circulating tumor cell evasion from natural killer cell killing via immune checkpoint CD155-TIGIT.

e14552 Background: Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. The interaction between platelets and CTCs is the most extensively studied of all blood cells, and it is well known that CTCs are surrounded by platelets, which form microplots on their surface and play a crucial role in cancer metastasis. The precise mechanisms platelets employ to contribute to CTC immune evasion have not been fully elucidated. Our previous single-cell RNA sequencing data indicated high expression of platelet-related genes in CTCs. Here, our study demonstrated that platelet adhesion to CTCs is prevalent in multiple cancers and is indispensable for CTCs metastasis. Methods: CTC isolation was performed by our self-established ChimeraX-i120 platform. CTC-platelet adhesions were identified by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. The cytotoxicity of natural killer (NK) cells was assessed by the reduction in tumor cell numbers measured using the xCELLigence system. In vitro and ex vivo co-culture systems were established by using platelets and NK cells isolated from the peripheral blood of patients for direct or indirect co-culturing with cell lines and patient-derived organoids (PDOs). The effects of platelets and NK cells on metastasis and immune responses were investigated in vivo by utilizing mouse models. Results: Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in hepatocellular carcinoma patients. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155, therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another two receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. Conclusions: Our results demonstrated CTC evasion from NK cell-mediated innate immunosurveillance mainly via immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

The Tumor Microenvironment Affects Circulating Tumor Cells Metastasis and the Efficacy of Immune Checkpoint Blockade in Non-small Cell Lung Cancer

Abstract: Lung cancer is one of the most lethal malignancies, with non-small cell lung cancer (NSCLC) being the most common histologic subtype. Metastasis leads to poor prognosis for patients with cancer. Tumor cells leave the tumor lesions, invade the surrounding stroma, and enter the bloodstream as circulating tumor cells (CTCs). The development of CTCs is the beginning of metastasis. The internal environment in which tumor cells grow and survive is called the tumor microenvironment (TME). It includes tumor cells, fibroblasts, immune cells, and the extracellular matrix. The TME is complex and dynamic. Moreover, the TME plays an important role in tumor development and metastasis and significantly impacts therapeutic outcomes. Immune checkpoint blockade (ICB) aims to inhibit the interaction of ligands with their corresponding receptors. ICB has the function of restoring the anti-tumor effect of immune cells. This review examines how TME interacts with CTCs, allowing CTCs to evade immunity and facilitating CTC metastasis. TME not only affects the progression of tumor metastasis but also interacts with tumor cells, which may affect the efficacy of immunotherapy.

Fibrin Stiffness Regulates Phenotypic Plasticity of Metastatic Breast Cancer Cells.

The extracellular matrix (ECM)-regulated phenotypic plasticity is crucial for metastatic progression of triple negative breast cancer (TNBC). While ECM faithful cell-based models are available for in situ and invasive tumors, such as cell aggregate cultures in reconstituted basement membrane and in collagenous gels, there are no ECM faithful models for metastatic circulating tumor cells (CTCs). Such models are essential to represent the stage of metastasis where clinical relevance and therapeutic opportunities are significant. Here, CTC-like DU4475 TNBC cells are cultured in mechanically tunable 3D fibrin hydrogels. This is motivated, as in circulation fibrin aids CTC survival by forming a protective coating reducing shear stress and immune cell-mediated cytotoxicity and promotes several stages of late metastatic processes at the interface between circulation and tissue. This work shows that fibrin hydrogels support DU4475 cell growth, resulting in spheroid formation. Furthermore, increasing fibrin stiffness from 57 to 175Pa leads to highly motile, actin and tubulin containing cellular protrusions, which are associated with specific cell morphology and gene expression patterns that markedly differ from basement membrane or suspension cultures. Thus, mechanically tunable fibrin gels reveal specific matrix-based regulation of TNBC cell phenotype and offer scaffolds for CTC-like cells with better mechano-biological properties than liquid.

DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

Abstract 1878: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2520, targeting both PD-L1 and CD47 as cancer immunotherapy

Abstract The programmed cell death protein 1 (PD-1) immune checkpoint is well known in its ability to attenuate T cell-mediated immune responses in tumors expressing its ligand, programmed death-ligand 1 (PD-L1). CD47, a cell surface molecule in the immunoglobulin superfamily, is another established target overexpressed on various malignant cells and acts as an innate inhibitory checkpoint by interrupting the phagocytosis of tumor cells and downstream activation of innate responses. It has been reported that the simultaneous blockade on both CD47 and PD-L1 checkpoints inhibited tumor growth and circulating tumor cells metastasis more potently than a single antibody inhibition or blank control in the 4T1 tumor mouse model in vivo. IMM2520 is an antibody-receptor (mAb-Trap) fusion protein consisting of recombinant human SIRPα and anti- PD-L1 antibody, thereby targeting CD47 and PD-L1 to antagonize both innate and adaptive immune checkpoint pathways. IMM2520 binds to PD-L1 on the tumor cell membrane and restores the immune response of T cells. IMM2520 also reverses the inhibitory effect of tumor cells on macrophages by blocking the binding of CD47 on the tumor cell to SIRPα on macrophages. In vitro studies have demonstrated that IMM2520 not only possesses a potent binding activity to CD47 (EC50 = 0.8 nM) and PD-L1 (EC50 = 0.09 nM) respectively, but also exhibits a strong activity in blocking PD-1/PD-L1 binding (IC50 = 0.06 - 1.0 nM). IMM2520 also showed a potent antibody-dependent cellular cytotoxicity (ADCC, EC50 = 1.54 ng/ml) against Raji-PDL1 and induced a strong antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.04 nM) activity (THP-1 against Raji-PDL1). In vivo efficacy study with CT26-hPD-L1/hCD47 colon cancer model demonstrated that IMM2520 at 3 mg/kg significantly reduced tumor volume (TGITV = 90.12%, P<0.01 vs Vehicle). Interestingly, IMM2520 demonstrated a potent and significantly higher anti-tumor activities than IMM01 (a SIRPα-Fc fusion protein) and IMM2505 (also a CD47XPD-L1 mAb-Trap, but the variable region sequence of this PD-L1 antibody is identical to atezolizumab). IMM2520 at dose of 6 mg/kg completely eradicated the established tumors. In another in vivo efficacy study with MC38-hCD47/hPD-L1 colon cancer model, IMM2520 demonstrated a significant anti-tumor activity in a dose-dependent manner at doses between 2 mg/kg to 20 mg/kg. Given its potent preclinical anti-tumor activity as well as the favorable safety profile, IMM2520 may serve as a potent immunotherapy for multiple cancer types by targeting PD-L1 and CD47 on tumor cells. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Wenqi Zhu, Yanan Yang, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang, Frank Gan. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2520, targeting both PD-L1 and CD47 as cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1878.

A novel whole blood purifier for efficient capture and separation of circulating tumor cells

Circulating tumor cells (CTCs) as important biomarkers for noninvasive clinical diagnosis and prognostic evaluation are significant in predicting the overall survival and progression-free survival of cancer patients. However, the current typical CTCs separation and enrichment techniques were limited to a single collection of small-volume blood samples, which was inadequate to comprehensively profile the distribution of CTCs in the systemic blood. In addition, those techniques cannot reduce metastasis of CTCs unless adjuvant chemotherapy. Herein, inspired by hemodialysis, we designed a whole blood purifier (WBP) composed of a functionalized special spiral-like glass tube modified by anti-epithelial cell adhesion molecule (anti-EpCAM). The WBP allowed real-time capture, enrichment and removal of CTCs from systemic blood circulation, and the purified blood was immediately returned to the body. Furthermore, the WBP did not cause any organic damages in vivo. This approach achieves the high accuracy of liquid biopsy technology and is expected to become an effective clinical adjuvant therapy for tumor metastasis.

Propagated Circulating Tumor Cells Uncover the Potential Role of NFκB, EMT, and TGFβ Signaling Pathways and COP1 in Metastasis.

Circulating tumor cells (CTCs), a population of cancer cells that represent the seeds of metastatic nodules, are a promising model system for studying metastasis. However, the expansion of patient-derived CTCs ex vivo is challenging and dependent on the collection of high numbers of CTCs, which are ultra-rare. Here we report the development of a combined CTC and cultured CTC-derived xenograft (CDX) platform for expanding and studying patient-derived CTCs from metastatic colon, lung, and pancreatic cancers. The propagated CTCs yielded a highly aggressive population of cells that could be used to routinely and robustly establish primary tumors and metastatic lesions in CDXs. Differential gene analysis of the resultant CTC models emphasized a role for NF-κB, EMT, and TGFβ signaling as pan-cancer signaling pathways involved in metastasis. Furthermore, metastatic CTCs were identified through a prospective five-gene signature (BCAR1, COL1A1, IGSF3, RRAD, and TFPI2). Whole-exome sequencing of CDX models and metastases further identified mutations in constitutive photomorphogenesis protein 1 (COP1) as a potential driver of metastasis. These findings illustrate the utility of the combined patient-derived CTC model and provide a glimpse of the promise of CTCs in identifying drivers of cancer metastasis.

Size-Based Method for Enrichment of Circulating Tumor Cells from Blood of Colorectal Cancer Patients.

Circulating tumor cells (CTCs) are precursors of the metastatic cascade, which is responsible for 90% of all cancer-related deaths. CTCs arise from solid tumors and travel through the bloodstream and lymphatic system. Developments in the isolation and analysis of CTCs promise potential biomarker assays for detection and monitoring of cancer through a minimally invasive procedure. Despite this, the precise role of CTCs in metastasis remains poorly characterized, mainly due to the low density of CTCs (1-10 CTCs per 10 mL of blood) present in patient blood and the lack of robust methods for their isolation in a standard laboratory setting. CellSearch is currently the only FDA-approved CTC enrichment protocol, but limitations of this EpCAM-based method include cost, availability, and the use of a single surface marker for capture. To address these limitations, we have optimized an existing method, MetaCell, which exploits the differences in size of CTCs compared to other blood cells for CTC enrichment from blood. MetaCell contains a membrane with 8 μm pores, and blood is filtered through this kit by capillary action and CTCs, which are typically larger than the pores and remain on top of the membrane, while most leukocytes pass through the pores. The membrane along with these CTCs can be detached and transferred to 6-well plates for culturing or directly used for characterization. Here, we provide a detailed protocol for enrichment of CTCs using the filtration device MetaCell and a procedure for characterization ofCTCs by immunohistochemical staining.

Effect of propofol on lung metastasis of circulating tumor cells after colorectal cancer surgery


 
 
 
 Purpose: To investigate the effect of propofol on lung metastasis of circulating tumor cells (CTCs) after colorectal cancer (CRC) surgery, and the mechanism involved.
 Methods: Specific-pathogen-free (SPF) male BALB/c mice (n = 30; mean age = 7 ± 1 weeks; mean weight = 19 ± 3 g) were used for this study. To establish mouse model of CRC, 100 μL of CRC (CT26) cells was injected into the caudal vein of each mouse. Three groups of 10 mice were used: control, 5 μL/mL propofol, and 10 μL/mL propofol groups. Changes in pulmonary superficial nodules of mice lungs were determined. Colorectal cancer cell (CT26) proliferation and apoptosis were measured using Ki-67 immunohistochemical staining and in situ terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays, respectively.
 Results: The number of pulmonary nodules and proportion of proliferating cells (Ki-67 expression level) were significantly higher in 5 μL/mL propofol-treated mice, relative to control, but markedly lower in 10 μL/mL propofol group than in 5 μL/mL propofol group (p < 0.05). Although apoptotic index increased in 5 μL/mL propofol group, cell apoptosis was comparable among the groups (p > 0.05).
 Conclusion: These results suggest that propofol promotes pulmonary metastasis of CTCs after CRC surgery via stimulation of CTC proliferation in the lungs. Therefore, caution should be exercised in the use of propofol in colorectal cancer surgery.
 
 
 

Platelet-mediated tumor metastasis mechanism and the role of cell adhesion molecules.

Mounting evidence suggests that platelets play an essential role in cancer metastasis. The interactions between platelets and circulating tumor cells (CTCs) promote cancer metastasis. CTCs induce platelet activation and aggregation, and activated platelets gather and protect CTCs from shear stress and natural killer cells. Finally, platelets stimulate CTC anoikis resistance, epithelial-to-mesenchymal transition, angiogenesis, extravasation, and eventually, metastasis. Cell adhesion molecules (CAMs) have been identified as active players during the interaction of CTCs with platelets, but the specific mechanism underlying the contribution of platelet-associated CAMs to CTC metastasis remains unclear. In this review, we introduce the mechanism of platelet-related tumor metastasis and particularly focus on the role of CAMs in it.

3D Culture of Circulating Tumor Cells for Evaluating Early Recurrence and Metastasis in Patients with Hepatocellular Carcinoma.

PurposeCirculating tumor cells (CTCs) are considered to be a key factor involved in tumor metastasis. However, the isolation and culture of CTCs in vitro remains challenging, and their clinical application for predicting prognosis and survival is still limited. The development of accurate evaluating system for CTCs will benefit for clinical assessment of HCC.MethodsDensity gradient centrifugation and magnetic separation based on CD45 antibody were used to isolate CTCs. 3D culture was used to maintain and amplify CTCs and HCC cells. Cellular immunofluorescence was used to identify CTCs and spheroids. The cutoff value of CTC spheroid was calculated using X-tile software. The relationship between clinicopathological variables and CTC spheroids in HCC patients is analyzed. In vivo models were used to evaluate tumor growth and metastasis of CTC spheroids.ResultsPatient-derived CTCs/HCC cells were isolated and expanded to form spheroids using 3D culture. CTC spheroids could be used to predict short-term recurrence of CTCs compared with conventional CTC enumeration. Different cell lines exhibited different formation rates and grew to different sizes. Identification of CTC spheroids revealed that EpCAM and β-catenin were expressed in spheroids derived from HCC cells and in the HCC/CTCs. EpCAM-positive HCC cells exhibited improved spheroid formation in 3D culture and were more tumorigenic and likely to metastasize to the lung in vivo. Abnormal activation of the Wnt/β-catenin signaling pathway was observed in EpCAM positive cells.ConclusionCTC spheroids could predict prognosis of HCC more precisely compared with conventional CTC enumeration. EpCAM may participate in the formation and survival of CTC spheroids which dependent on Wnt/β-catenin signaling pathway.

Single-Cell Analysis of Highly Metastatic Circulating Tumor Cells by Combining a Self-Folding Induced Release Reaction with a Cell Capture Microchip.

Nondestructive analysis of the single-cell molecular phenotype of circulating tumor cells (CTCs) is of great significance to the precise diagnosis and treatment of cancer but is also a huge challenge. To address this issue, here, we develop a facile analysis system that integrates CTCs' capture and molecular phenotype analysis. An isothermal nucleic acid amplification technique named self-folding induced release reaction (sFiR), which has high-efficiency signal amplification capabilities and can run under physiological conditions, is first developed to meet the high requirements for sensitivity and nondestructivity. By combining the sFiR with immune recognition and a single cell capture microchip, the molecular phenotype analysis of a single CTC is realized. As a model, nondestructive analysis of junction plakoglobin (JUP), an overexpressed membrane protein that is closely related to the metastasis of CTCs, is successfully achieved. Results reveal that this sFiR-based analysis system can clearly distinguish the expression of JUP in different cancer cell lines and can present quantitative information on the expression of JUP. Furthermore, the captured and analyzed CTCs maintain their basic physiological activity and can be used for drug sensitivity testing. Considering the excellent performance and ease of operation of the system, it can provide technical support for CTC-based cancer liquid biopsy and drug development.

USP1 Maintains the Survival of Liver Circulating Tumor Cells by Deubiquitinating and Stabilizing TBLR1.

The prognosis of hepatocellular carcinoma (HCC) is closely associated with the occurrence of distant metastases, which is likely due to circulating tumor cells (CTCs). However, the low number of CTCs is the main obstacle limiting research of the mechanism of CTC metastasis. Here, We evaluated the role of ubiquitin-specific protease 1 (USP1) in promoting CTC survival during blood-borne metastases. We observed that USP1 was frequently upregulated in CTCs and correlated with metastasis and a reduced overall survival rate of patients. Additionally, genetic knockout of USP1 the survival rate of CTCs. Further analyses showed that USP1 mediates oncogenic activity by deubiquitinating and stabilizing transducin β-like 1 X-linked receptor 1 (TBLR1), which plays essential roles in regulating Wnt signaling. These results demonstrated that USP1 may act as an essential factor in promoting the survival of CTCs and suggest that inhibition of USP1 is a potential strategy for HCC treatment.

Abstract 2727: Elucidating genes that mediate brain colonization by metastatic melanoma cells

Abstract Metastatic melanoma shows remarkable tropism for the brain, affecting up to 75% of stage 4 patients, causing devastating symptoms and dramatically reducing survival. Patients with brain metastases have limited treatment options and short-lived unpredictable responses to therapy, probably due to our poor understanding of the biology driving this particular metastatic process. In this work we hypothesize that the brain microenvironment induces particular gene expression changes in melanoma cells as compared to melanoma cells colonizing extra-cranial sites, allowing cells to rewire, survive and proliferate in the brain. To elucidate the molecular basis supporting brain metastasis in melanoma, we enriched for cancer cells from patient blood and established a metastatic circulating tumor cells derived explant (CDX) model of cutaneous melanoma. CDX-derived cell lines were injected intra-dermally in the flank of immunocompromised mice and tumor growth as well as metastatic incidence was quantified. Our data shows that both the CDX and its corresponding derived cell line showed tropism for the brain, liver and kidneys, similar to what was observed in the patient. Notably, the intradermal flank-tumors were predominantly highly pigmented, whereas the liver metastases were either pigmented or non-pigmented. In contrast, the brain metastases located in the parenchyma were exclusively non-pigmented. Cell lines derived from either pigmented or non-pigmented liver lesions as well as non-pigmented lesions from the brain maintained their pigmentation and morphological features in vitro. When injected intradermally into immunocompromised mice, the pigmented cells metastasized to the visceral organs and, even though leptomeningeal metastasis may be observed, these cells did not invade brain parenchyma. In contrast, the non-pigmented cells (derived either from the liver or brain) metastasized to the visceral organs and additionally metastasized to the brain parenchyma; feature that was enhanced following successive in vivo passages. Gene expression analysis revealed an enrichment of genes encoding proteins that regulate extracellular matrix reorganization, angiogenesis and epithelial-to-mesenchymal transition in the non-pigmented cells. Moreover, TGF-beta signalling was found to be one of the most enriched pathways in the cells derived from parenchymal brain metastases, suggesting that increased TGF-beta signalling regulates brain tropism in metastatic melanoma. In conclusion, our experimental model recapitulates the propensity of melanoma to metastasize to the brain parenchyma and our preliminary data suggests that TGF-beta signalling play a key role in supporting this process. Further experiments are currently ongoing for testing our hypothesis that, if corroborated, may open new avenues of treatment options for melanoma patients displaying metastases in the brain. Citation Format: Denys Holovanchuk, Rebecca Lee, Alessio Cannistraci, Nathalie Dhomen, Valeria Pavet, Richard Marais. Elucidating genes that mediate brain colonization by metastatic melanoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2727.

AFM-compatible microfluidic platform for affinity-based capture and nanomechanical characterization of circulating tumor cells

Circulating tumor cells (CTCs) carried by the patient’s bloodstream are known to lead to the metastatic spread of cancer. It is becoming increasingly clear that an understanding of the nanomechanical characteristics of CTCs, such as elasticity and adhesiveness, represents advancements in tracking and monitoring cancer progression and metastasis. In the present work, we describe a combined microfluidic–atomic force microscopy (AFM) platform that uses antibody–antigen capture to routinely isolate and nanomechanically characterize CTCs present in blood samples from prostate cancer patients. We introduce the reversible assembly of a microfluidic device and apply refined and robust chemistry to covalently bond antibodies onto its glass substrate with high density and the desired orientation. As a result, we show that the device can efficiently capture CTCs from patients with localized and metastatic prostate cancer through anti-EpCAM, anti-PSA, and anti-PSMA antibodies, and it is suitable for AFM measurements of captured intact CTCs. When nanomechanically characterized, CTCs originating from metastatic cancer demonstrate decreased elasticity and increased deformability compared to those originating from localized cancer. While the average adhesion of CTCs to the AFM tip surface remained the same in both the groups, there were fewer multiple adhesion events in metastatic CTCs than there were in their counterparts. The developed platform is simple, robust, and reliable and can be useful in the diagnosis and prognosis of prostate cancer as well as other forms of cancer.

Circulating tumor cells in hepatocellular carcinoma: single-cell based analysis, preclinical models, and clinical applications.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors and metastatic lesions and provide significant information about tumor progression and metastasis. CTCs contribute to tumor metastasis through the epithelial-to-mesenchymal transition (EMT). CTC clusters and stem-like phenotypes lead to a more aggressive and metastatic potential. CTCs retain the heterogeneity and imitate the nature of corresponding primary tumors. Therefore, it is important to use single-cell based analysis to obtain information on tumor heterogeneity and biology. CTCs are also good candidates for building preclinical models (especially 3D organoid cultures) for drug screening, disease modeling, genome editing, tumor immunity research, and organ-like biobank establishment. In this article, we summarize the current CTC capture technology, dissect the phenotypes associated with CTC metastasis, and review the progress in single-cell based analysis and preclinical modeling of the pattern and kinetics of CTCs. In particular, we discuss the use of CTCs to assess the progression of hepatocellular carcinoma (HCC).

Dual blockage of both PD-L1 and CD47 enhances immunotherapy against circulating tumor cells

Carcinoma metastasis is triggered by a subpopulation of circulating tumor cells (CTCs). And single immune checkpoint therapy is not good enough to inhibit CTC-induced metastasis. Here, we demonstrate that simultaneously blocking CD274 (programmed death ligand 1, PD-L1 or B7-H1) and CD47 checkpoints which were respectively signal of “don’t find me” and “don’t eat me” on CTCs by corresponding antibodies could enhance the inhibition tumor growth than single CD274 or CD47 antibody alone. In vitro flow cytometry data proved that CD47 and CD274 were overexpressed on the tested mouse tumor cell lines. The antibodies could effectively block the expressions of CD47 and CD274 on the cell surface and stably attached to tumor cell surface for several hours. The simultaneous blockade on both CD47 and CD274 checkpoints inhibited tumor growth and CTCs metastasis more potently than a single antibody inhibition or blank control on 4T1 tumor mouse model in vivo. Our results demonstrated that simultaneous dual targeting immune checkpoints, i.e., CD47 and CD274, by using specific antibodies may be more effective as an immunotherapeutics on CTCs than a CD47 or CD274 alone.

Inertial focusing of cancer cell lines in curvilinear microchannels

Circulating tumor cells (CTCs) are rare cancer cells, which originate from the primary tumors and migrate to the bloodstream. Separation of CTCs from blood is critical because metastatic CTCs might hold different genomic and phenotypic properties compared to primary tumor cells. In this regard, accurate prognosis and effective treatment methods are necessary. For this purpose, focusing biological particles and cells using microfluidic systems have been implemented as an efficient CTCs enumeration and enrichment method. Passive, continuous, label-free and parallelizable size-dependent focusing based on hydrodynamic forces is preferred in this study to sort cancer cells while avoiding cell death and achieving high throughput. The focusing behavior of MDA-MB-231 (11–22 μm), Jurkat (8–17 μm), K562 (8–22 μm), and HeLa (16–29 μm) was examined with respect to different Reynolds numbers and Dean numbers. The effect of curvature on cell focusing was carefully assessed. The focusing positions of the cells clearly indicated that isolations of MDA cells from MDA-Jurkat cell mixtures as well as of HeLa cells from HeLa-Jurkat cell mixtures were possible by using the curvilinear channels with a curvature angle of 280° at the Reynolds number of 121. © 2019 The Author(s)