Patient-derived Circulating Tumor Cells Research Articles

Abstract 3694: Genomic profiling of single cancer cells using the novel single-cell integrated mutational profiling of actionable cancer targets (scIMPACT) assay

Abstract Liquid biopsy provides opportunities to guide therapy decisions using biomarkers such as circulating tumor cells (CTCs) even years after the primary tumor resection. For this, novel methods permitting the genomic profiling of CTCs are needed to characterize systemic cancer spread and identify clinically targetable genomic alterations. Hence, we developed an assay based on the FDA-approved MSK-IMPACT® assay to analyze the genomes of single-cells. We present a series of benchmarking experiments using well-characterized cell lines to demonstrate the performance of our new Single-Cell Integrated Mutation Profiling of Actionable Cancer Targets (scIMPACT) assay.The scIMPACT assay was developed using Ampli1® WGA products of single-cell and pool samples. The workflow consists of an optimized library preparation protocol followed by targeted sequencing with the scIMPACT panel. A bioinformatics mutation calling workflow facilitates the detection of single nucleotide variations (SNVs), indels, and copy number variations (CNVs). To account for WGA associated bias, we developed an in-house proprietary machine learning based classifier algorithm. Two sample collectives were prepared and used for training and validating the workflow. The first collective, including genomic libraries of MDA-MB-453, BT474, BT549 and ZR-75.1 samples, was used for training of the mutation detection workflow. The second collective, consisting of HCC1395/HCC1395BL cell lines representing matched tumor and normal cells, was used to validate the performance of the assay. The scIMPACT assay identified somatic mutations in all WGA products as predicted from bulk gDNA samples and published literature. Notably, evidence of genetic heterogeneity was reported at the single cell level. Pertaining to the SNV detection, the training collective displayed 91% sensitivity and 96% specificity (AUC=0.98) while the validation collective demonstrated similar performance with 84% sensitivity and 96% specificity (AUC=0.90) proving the applicability of our workflow across different datasets. Furthermore, CNV analysis showed high concordance between matching gDNA and WGA samples. Our workflow enables mutational profiling with high specificity and sensitivity and accurate CNV detection in single cells. The scIMPACT assay has been successfully adapted to analyze samples at the single-cell level. Therefore, our method can be applied for the genomic profiling of patient-derived CTCs. Citation Format: Clara Chaiban, Adithi Ravikumar Varadarajan, Jonas Grote, Thomas Ragg, Isabell Blochberger, Vadim Dechand, Jens Warfsmann, Christoph A. Klein, Zbigniew T. Czyż. Genomic profiling of single cancer cells using the novel single-cell integrated mutational profiling of actionable cancer targets (scIMPACT) assay [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 3694.

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.

Ultrahigh-Throughput, Real-Time Flow Cytometry for Rare Cell Quantification from Whole Blood.

We present an ultrahigh-throughput, real-time fluorescence cytometer comprising a viscoelastic microfluidic system and a complementary metal-oxide-semiconductor (CMOS) linear image sensor-based detection system. The flow cytometer allows for real-time quantification of a variety of fluorescence species, including micrometer-sized particles and cells, at analytical throughputs in excess of 400,000 species per second. The platform integrates a custom C++ control program and graphical user interface (GUI) to allow for the processing of raw signals, adjustment of processing parameters, and display of fluorescence intensity histograms in real time. To demonstrate the efficacy of the platform for rare event detection and its utility as a basic clinical tool, we measure and quantify patient-derived circulating tumor cells (CTCs) in peripheral blood, realizing that detection has a sensitivity of 6 CTCs per million blood cells (0.000006%) with a volumetric throughput of over 3 mL/min.

DanioCTC: Analysis of Circulating Tumor Cells from Metastatic Breast Cancer Patients in Zebrafish Xenografts.

Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases.

Abstract 5581: Single-cell genomic analysis of patient-derived circulating tumor cells in pancreatic cancer

Abstract Metastatic pancreatic cancer suffers from an extremely low five-year survival rate of only 3%. While patients with localized disease show marginally better survival rates, 52% of pancreatic cancers are not diagnosed until metastases have already developed. There is therefore a critically unmet need in the early diagnoses of pancreatic cancer patients. Liquid biopsies have emerged as a promising tool for cancer diagnosis through the analysis of various blood-based biomarkers. Among these, circulating tumor cells (CTCs) stand out as a promising target of interest. Over the past decade, CTCs have been identified as a precursor to metastasis. CTCs have also been shown to harbor mutations matching those of the primary tumor, making them a potential surrogate for biopsied tissue. However, CTCs are an extremely heterogenous population of cells, making bulk analysis challenging. In this study, we demonstrate a workflow for the isolation and genomic characterization of single CTCs from pancreatic cancer patients. CTCs are first enriched using a size-based microfluidic system, the Labyrinth, developed in the Nagrath lab. Single CTCs are subsequently isolated using the DEPArray Nxt system, followed by whole genome amplification (WGA) and low-pass sequencing. The pipeline was optimized using three commercially available pancreatic cancer cells lines (Panc-1, Capan-2, and BxPC-3) and two pancreatic cancer CTC cell lines previously developed in-house (CTC-CL-1 and CTC-CL-2). Finally, the workflow was validated using a blood sample from a stage IV pancreatic ductal adenocarcinoma patient. Following WGA, the average nucleic material concentration of the cell lines was 26.24 ± 16.28 ng/μL and 14.43 ng/μL for cells isolated from the patient sample. The average fragment size was 746 ± 232.79 bp for cell line cells and 507 bp for patient sample cells. This was within the expected range of 100-2000 bp. Both CTC cell lines show gains on parts of chromosome 8q (Myc), 10q (FGFR2) and 17q. Myc is a known oncogene and FGFR2 has been associated with cell growth, migration, and invasion. There are also noticeable differences between the two CTC-CL-1 cells, with one cell showing a 3p12.3-3p26.3 gain (CTNNB1), and the other showing a loss of 6q12-6q15 and gain of 6q16.1-6q27 (Myb, ESR1). A patient sample CTC and patient-matched WBC were isolated and sequenced. The CTC has a noticeable gain of chromosome 1q and loss of chromosome 5q. A gain in chromosome 1q has been seen previously in pancreatic cancer and chromosome 5q is the location of multiple oncogenes including APC, CSF1R and FGFR4. The loss of APC is known to affect Wnt/β-catenin signaling, causing an increase in CD34 expression which is linked to increase in cell invasion and migration. These results demonstrate an efficient workflow for the single cell genomic analysis of pancreatic CTCs. Further studies can be conducted to characterize the heterogenous landscape of CTCs in pancreatic cancer. Citation Format: Harrison Ball, Brittany Rupp, Sarah Owen, Kaylee Smith, Valerie Gunchick, Evan T. Keller, Vaibhav Sahai, Sunitha Nagrath. Single-cell genomic analysis of patient-derived circulating tumor cells in pancreatic cancer. [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 5581.

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.

Downregulation of KEAP1 in melanoma promotes resistance to immune checkpoint blockade

Immune checkpoint blockade (ICB) has demonstrated efficacy in patients with melanoma, but many exhibit poor responses. Using single cell RNA sequencing of melanoma patient-derived circulating tumor cells (CTCs) and functional characterization using mouse melanoma models, we show that the KEAP1/NRF2 pathway modulates sensitivity to ICB, independently of tumorigenesis. The NRF2 negative regulator, KEAP1, shows intrinsic variation in expression, leading to tumor heterogeneity and subclonal resistance.

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization.

Circulating tumor cells (CTCs) represent an interesting source of biomarkers for diagnosis, prognosis, and the prediction of cancer recurrence, yet while they are extensively studied in oncobiology research, their diagnostic utility has not yet been demonstrated and validated. Their scarcity in human biological fluids impedes the identification of dangerous CTC subpopulations that may promote metastatic dissemination. In this Perspective, we discuss promising techniques that could be used for the identification of these metastatic cells. We first describe methods for isolating patient-derived CTCs and then the use of 3D biomimetic matrixes in their amplification and analysis, followed by methods for further CTC analyses at the single-cell and single-molecule levels. Finally, we discuss how the elucidation of mechanical and morphological properties using techniques such as atomic force microscopy and molecular biomarker identification using nanopore-based detection could be combined in the future to provide patients and their healthcare providers with a more accurate diagnosis.

Integrated Workflow for the Label-Free Isolation and Genomic Analysis of Single Circulating Tumor Cells in Pancreatic Cancer.

As pancreatic cancer is the third deadliest cancer in the U.S., the ability to study genetic alterations is necessary to provide further insight into potentially targetable regions for cancer treatment. Circulating tumor cells (CTCs) represent an especially aggressive subset of cancer cells, capable of causing metastasis and progressing the disease. Here, we present the Labyrinth–DEPArray pipeline for the isolation and analysis of single CTCs. Established cell lines, patient-derived CTC cell lines and freshly isolated CTCs were recovered and sequenced to reveal single-cell copy number variations (CNVs). The resulting CNV profiles of established cell lines showed concordance with previously reported data and highlight several gains and losses of cancer-related genes such as FGFR3 and GNAS. The novel sequencing of patient-derived CTC cell lines showed gains in chromosome 8q, 10q and 17q across both CTC cell lines. The pipeline was used to process and isolate single cells from a metastatic pancreatic cancer patient revealing a gain of chromosome 1q and a loss of chromosome 5q. Overall, the Labyrinth-DEPArray pipeline offers a validated workflow combining the benefits of antigen-free CTC isolation with single cell genomic analysis.

Abstract 5123: Establishing circulating tumor cell-derived xenografts following a period of in vitro culture

Abstract Cancer metastasis—the spread of cancer cells from a primary node to a distant site—is responsible for as much as 90% of all cancer-associated deaths due to treatment resistance and increased tumor burden. Once established, metastatic nodules oftentimes exhibit distinct molecular phenotypes from their original primary tumor, undermining gold-standard methods for determining treatments for patients, which typically rely on molecular characterization of the primary tumor. Unfortunately, a lack of robust, scalable, and appropriate models for studying differences in patient-derived primary and metastatic tumors has hindered progress in identifying metastasis-specific therapies. Previously, we reported the ability to establish in vitro cultures of circulating tumor cells (CTCs), a unique precursor population of cells that exhibits an ability to escape the primary tumor but not yet fully complete metastasis. We have since expanded the application of this GU-CTC platform to culture CTCs from the blood of patients diagnosed with a variety of metastatic cancers, resulting in the successful culture of 31 unique patient-derived CTC cell lines. Furthermore, following an initial short-term culture period, we also performed subcutaneous injection of mixed CTC cultures into immunocompromised mice, resulting in not only primary tumor formation, but also systemic metastatic nodule formation in as short as 3 months. These resulting CTC-derived xenografts (CDXs) have been demonstrated using metastatic pancreatic, colorectal, and lung adenocarcinoma-derived CTCs. Encouragingly, all CDX models also exhibited replicable metastatic patterns upon reimplantation, reinforcing the important role of CTCs as precursors to metastasis. Bulk RNA-sequencing of patient-matched whole blood, cultured CTCs, and CDX-derived primary tumor and metastatic tissues further identified enrichment of an epithelial-mesenchymal transition as well as several cancer-specific gene signatures consistent with an invasive phenotype. This extended GU-CTC platform therefore enables not only the robust and rapid expansion of CTCs through both in vitro and in vivo methods, but also offers an avenue for personalized drug screens in the effort to target cancer metastasis. Citation Format: Jerry Xiao, Richard Schlegel, Seema Agarwal. Establishing circulating tumor cell-derived xenografts following a period of in vitro culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5123.

Negative-Selection Enrichment of Circulating Tumor Cells from Peripheral Blood Using the Microfluidic CTC-iChip.

The ability to isolate and analyze rare circulating tumor cells (CTCs) holds the potential to increase our understanding of cancer evolution and allows monitoring of disease and therapeutic responses through a relatively non-invasive blood-based biopsy. While many methods have been described to isolate CTCs from the blood, the vast majority rely on size-based sorting or positive selection of CTCs based on surface markers, which introduces bias into the downstream product by making assumptions about these heterogenous cells. Here we describe a negative-selection protocol for enrichment of CTCs through removal of blood components including red blood cells, platelets, and white blood cells. This procedure results in a product that is amenable to downstream single-cell analytics including RNA-Seq, ATAC-Seq and DNA methylation, droplet digital PCR (ddPCR) for tumor specific transcripts, staining and extensive image analysis, and ex vivo culture of patient-derived CTCs.

Ex Vivo Expanded Circulating Tumor Cells for Clinical Anti-Cancer Drug Prediction in Patients with Head and Neck Cancer.

Simple SummaryThe conventional methods that seek to predict clinical treatment response are based on the number of circulating tumor cells (CTCs) present in liquid biopsies or genetic profiling of extracted CTCs. This paper presents a novel process by which CTCs can be extracted from blood samples taken from head and neck cancer patients and then expanded ex vivo to form organoids that can be tested with a panel of anti-cancer treatments. The resulting drug sensitivity profiles derived from cisplatin treatment of organoids were subsequently found to correlate with clinical treatment response to cisplatin in patients. CTCs extracted from liquid biopsies for ex vivo expansion negates the need for complicated and potentially risky biopsies of tumor material, thereby supporting the application of this procedure for checkups and treatment monitoring.The advanced-stage head and neck cancer (HNC) patients respond poorly to platinum-based treatments. Thus, a reliable pretreatment method for evaluating platinum treatment response would improve therapeutic efficiency and outcomes. This study describes a novel strategy to predict clinical drug responses in HNC patients by using eSelect, a lab-developed biomimetic cell culture system, which enables us to perform ex vivo expansion and drug sensitivity profiling of circulating tumor cells (CTCs). Forty liquid biopsies were collected from HNC patients, and the CTCs were expanded ex vivo using the eSelect system within four weeks. Immunofluorescence staining confirmed that the CTC-derived organoids were positive for EpCAM and negative for CD45. Two illustrative cases present the potential of this strategy for evaluating treatment response. The statistical analysis confirmed that drug sensitivity in CTC-derived organoids was associated with a clinical response. The multivariant logistic regression model predicted that the treatment accuracy of chemotherapy responses achieved 93.75%, and the area under the curves (AUCs) of prediction models was 0.8841 in the whole dataset and 0.9167 in cisplatin specific dataset. In summary, cisplatin sensitivity profiles of patient-derived CTCs expanded ex vivo correlate with a clinical response to cisplatin treatment, and this can potentially underpin predictive assays to guide HNC treatments.

In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment.

Cancer is a dynamic disease with heterogenic molecular signatures and constantly evolves during the course of the disease. Single cell proteomic analysis could offer a suitable pathway to monitor cancer cell heterogeneity and deliver critical information for the diagnosis, recurrence, and drug-resistant mechanisms in cancer. Current standard techniques for proteomic analysis such as ELISA, mass spectrometry, and Western blots are time-consuming, expensive, and often require fluorescence labeling that fails to provide accurate information about the multiple protein expression changes at the single cell level. Herein, we report a surface-enhanced Raman spectroscopy-based simple microfluidic device that enables the screening of single circulating tumor cells (CTC) in a dynamic state to precisely understand the heterogeneous expression of multiple protein biomarkers in response to therapy. It further enables identifying intercellular heterogeneous expression of CTC surface proteins which would be highly informative to identify the cancer cells surviving treatment and potentially responsible for drug resistance. Using a bead and cell line-based model system, we successfully detect single bead and single cell spectra when flowed through the device. Using SK-MEL-28 melanoma cells, we demonstrate that our system is capable of monitoring heterogeneous expressions of multiple surface protein markers (MCSP, MCAM, and LNGFR) before and during drug treatment. Integrating a label-free electrochemical system with the device, we also monitor the expression of an intracellular protein (here, BRAFV600E) under drug treatment. Finally, we perform a longitudinal study with 15 samples from five different melanoma patients who underwent therapy. We find that the average expression of receptor proteins in a patient fails to determine the therapy response particularly when the disease progresses. However, single CTC analysis with our device shows a high level of intercellular heterogeneity in the receptor expression profiles of patient-derived CTCs and identifies heterogeneity within CTCs. More importantly, we find that a fraction of CTCs still shows a high expression of these receptor proteins during and after therapy, indicating the presence of resistant CTCs which may evolve after a certain time and progress the disease. We believe this automated assay will have high clinical importance in disease diagnosis and monitoring treatment and will significantly advance the understanding of cancer heterogeneity on the single cell level.

Abstract 585: A robust method for expanding patient-derived circulating tumor cells ex vivo

Abstract In the United States, an estimated 280,000 people will be diagnosed with invasive breast cancer, which has a 5-year survival rate of 27% and 22% in females and males, respectively. Despite the clinical burden of metastasis, the underlying machinery of metastasis remains unclear. This is largely due to a lack of available technologies that can robustly expand patient-derived metastatic cell populations for study ex vivo. One such population is circulating tumor cells (CTCs), which are cells that escape from the primary tumor, intravasate through blood vessel endothelium, and circulate throughout the body. Eventually, CTCs will extravasate at the metastatic site and become the seeds for metastatic colonization. Based on their role in metastasis, CTCs yield great promise for identifying key mechanistic drivers of metastasis. Unfortunately, CTCs exist in low levels within the blood (typically < 100 cells/10mL of blood). Previous methods for culturing CTCs ex vivo have reported low success rates (~ 20%), and often depend on the presence of a high number of CTCs for success. To better interrogate the mechanisms of metastasis, robust methods to propagate CTCs for study are needed. Recently, our lab developed a method using an unbiased selection criterion for isolating and propagating CTCs derived from metastatic breast cancer, yielding 100% success in twelve distinct individuals. This method, based on a density gradient centrifugation, extracts not only CTCs from blood plasma, but also includes potential cancer-associated neutrophils and macrophages. All cultures under our conditions demonstrated growth for > 30 days and were confirmed to have an epithelial and breast phenotype. Notably, 6/12 samples grew longer and were associated with increased CD45 expression using qRT-PCR. We have also recently demonstrated further success in culturing CTCs derived from a variety of other cancers, including colorectal, lung, and pancreatic cancers. This study therefore not only defines a novel technology for expanding CTCs, but also suggests a deficiency in modern isolation methods, which rely on CD45 exclusion, that is limiting culture success in previous methods. Citation Format: Jerry Xiao, Joshua D. Kassner, Kelly Stanton, Richard Schlegel, Paula R. Pohlmann, Seema Agarwal. A robust method for expanding patient-derived circulating tumor cells ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 585.

N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses.

Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a "liquid biopsy" of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. IMPLICATIONS: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.

Ex Vivo Expansion and Drug Sensitivity Profiling of Circulating Tumor Cells from Patients with Small Cell Lung Cancer.

Small cell lung cancer (SCLC) represents one of the most aggressive malignancies among cancer types. Not only tumor sample availability is limited, but also the ability for tumor cells to rapidly acquire drug resistance are the rate-limiting bottlenecks for overall survival in current clinical settings. A liquid biopsy capable of capturing and enriching circulating tumor cells (CTCs), together with the possibility of drug screening, is a promising solution. Here, we illustrate the development of a highly efficient ex vivo CTC expansion system based on binary colloidal crystals substrate. Clinical samples were enrolled from 22 patients with SCLC in the study. The CTCs were enriched and expanded from the collected peripheral blood samples. Expanded cells were analyzed for protein expression and observed for drug sensitivity with the use of immunofluorescence and ATP titer evaluation, respectively. Successful CTC spheroid proliferation was established after 4 weeks within 82% of all the collected peripheral blood samples from enrolled patients. Upon immunofluorescence analysis, the enriched cells showed positive markers for EpCAM, TTF-1, synaptophysin and negative for CD45. Additionally, the expanded CTCs demonstrated marked heterogeneity in the expression of E-cadherin and N-cadherin. In a preliminary case series, the drug sensitivity of patient-derived CTC to cisplatin and etoposide was studied to see the correlation with the corresponding therapeutic outcome. In conclusion, our study demonstrates that it is possible to efficiently expand CTCs from SCLC within a clinically relevant time frame; the biomarker information generated from enriched CTCs can assist the selection of effective drugs and improve disease outcome.

Defining the dimensions of circulating tumor cells in a large series of breast, prostate, colon, and bladder cancer patients.

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient-derived CTCs acquired on cartridges of the FDA-cleared CellSearch® method were retrospectively collected and automatically re-analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch® cartridges containing 71612 CTCs were included. In BC, the median computed diameter (CD) of patient-derived CTCs was 12.4μm vs 18.4μm for cultured cell line cells. For PC, CDs were 10.3μm for CTCs vs 20.7μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5μm and 8.6μm, respectively. Finally, leukocytes were 9.4μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size-based isolation methods.

Abstract LB-271: Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s)

Abstract Introduction: Circulating tumor cells (CTCs) are liquid biomarkers with high potential for application in precision medicine cancer care. However, no robust methods have been developed to culture CTCs from early-stage cancer patients owing to their rarity and instability. Here, we describe a successful protocol to establish CTC culturing and CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer (NSCLC) patients. Further, comparative multi-omics characterization of these CDX tumors revealed novel diagnostic/therapeutic target(s). Methods: Surgically resected primary tumor fragments from patients with early stage NSCLCs were subcutaneously (s.c.) implanted to immunodeficient NOD SCID gamma (NSG) mice to generate patient-derived xenograft (PDX) models. Once palpable tumor reached a diameter of ~1.5 cm, animals were euthanized and blood was collected, CTCs were enriched with OncoQuick® and cultured in vitro. These patient-derived CTCs were further stained with anti-human antibodies against CK7, Napsin, TTF-1, CK5/6, PDL1 and CD45. Once human CTCs were confirmed, we then injected expanded CTCs s.c. to naïve NSG mice to establish CDX models. Single cell suspension from CDX tumors was cultured and fibroblasts were depleted using specific beads (Miltenyi biotec) to establish stable CTC lines. Further, advanced proteomics, single cell RNA sequencing (transcriptomics) and whole genome sequencing of patient-matched peritumoral and primary tumor tissues along with PDX and CDX tumors were carried out. Results: Human NSCLC primary tumor fragments implanted in NSG mice were tumorigenic at the s.c. implantation site, metastatic to distant organs, and passaged successfully to naïve NSG generations to maintain the PDX colonies. CTCs enriched from the PDX models were successfully cultured in vitro and positive for human NSCLC-specific markers but negative for human leukocyte marker CD45. Cultured PDX-derived CTCs that were xenografted into naïve NSG mice by s.c. demonstrated tumorigenicity and metastatic potential in CDX models. Stable CTC lines established using CDX tumors sustained >30 passages. Global proteomic profiling of tumor tissues revealed 19 new proteins that were upregulated and 9 proteins that are down regulated. Single cell RNA sequence analysis depicts top rank genes that are overexpressed in tumor cluster. Whole genome sequencing of these samples identified somatic variants that needs further validation. Conclusion and future perspectives: This approach establishes CDX models from early-stage NSCLC patients at a high rate for personalized precision medicine. Models provide a unique opportunity to characterize rare patient-derived CTCs that helps in understanding CTC tumor biology and developing effective anti-cancer therapeutic strategies. Multi-omics characterization of these tissues identified a set of novel genes/proteins that needs to be validated in large patient population. Further, studying tumorigenic potential of established CTC lines after knockdown and overexpression of these genes will provide more information about translating these targets into therapeutics. Citation Format: Kanve N. Suvilesh, Yariswamy Manjunath, Vijay Radhakrishnan, Diego M. Avella, Eric T. Kimchi, Kevin F. Staveley-O'Carroll, Guangfu Li, Jussuf T. Kaifi. Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s) [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 LB-271.

Abstract A03: Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers

Abstract Metastasis in head and neck cancer (HNC) patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in HNC. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. n=300 head and neck cancer patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples, which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification). The presence of CTC clusters was associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression-free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence interval [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof-of-principle study, we were able to demonstrate for the first time short-term patient-derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggest that PD-L1 is frequently expressed on CTCs in HNC and an immunoscore may be able to identify patients likely to benefit from immunotherapy—a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression, and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean Paul Thiery, Ken O’Byrne, Chamindie Punyadeera. Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A03.

Abstract B29: Development of CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer patients

Abstract Introduction: Circulating tumor cells (CTCs) are liquid biomarkers with high potential for application in precision medicine cancer care. However, no robust methods have been developed to culture CTCs from early-stage cancer patients owing to their rarity from limited disease burden and instability. Here, we describe a successful protocol to establish CTC culturing and CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer (NSCLC) patients. Methods: Surgically resected primary tumor fragments from patients with early-stage I NSCLCs were subcutaneously (s.c.) implanted to immunodeficient NOD SCID gamma (NSG) mice to generate patient-derived xenograft (PDX) models. Once palpable tumor reached a diameter of 1.5 cm, animals were euthanized and blood was collected via retro-orbital vein puncture. CTCs were enriched with OncoQuick® and cultured in vitro. These patient-derived CTCs were further characterized by immunostaining with antihuman antibodies against CK 8/18/19, Napsin, TTF-1, and CD45. Once human CTCs were confirmed, we then injected expanded CTCs s.c. and intravenous (i.v.) into naïve NSG mice to establish CDX models. Tumor growth and metastatic potential in CDX models were measured by MRI, autopsy, and histopathology. Results: Human NSCLC primary tumor fragments implanted in NSG mice were tumorigenic at the s.c. implantation site, metastatic to distant organs, and passaged successfully to naïve NSG generations to maintain the PDX colonies. CTCs enriched from the PDX models were successfully cultured in vitro and positive for human NSCLC-specific markers (CK 8/18/19, Napsin, and TTF-1), but negative for human leukocyte marker CD45. Cultured PDX-derived CTCs that were xenografted into naïve NSG mice by s.c. and i.v. implantation demonstrated tumorigenicity and metastatic potential in CDX models. Conclusion and Future Perspectives: This approach establishes CDX models from early-stage NSCLC patients at a high rate for personalized precision medicine. Models provide a unique opportunity to characterize rare patient-derived CTCs from curable, nonmetastatic NSCLC patients towards understanding CTC tumor biology and developing effective anticancer therapeutic strategies. Further, combined genetic and proteomic characterization of primary, PDX, and CDX tumors will be highly useful in discovering novel diagnostic/therapeutic target(s) specific to NSCLC. Citation Format: Suvilesh Kanve Nagaraj, Yariswamy Manjunath, Vijay Radhakrishnan, Diego Avella, Eric Kimchi, Kevin Staveley-O'Carroll, Guangfu Li, Jussuf Kaifi. Development of CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B29.