Cell State and Cell Type: Deconvoluting Circulating Tumor Cell Populations in Liquid Biopsies by Multi-Omics.

Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient's overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms.

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients' blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/-) and CD45(+/-) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.

Defining A Liquid Biopsy Profile of Circulating Tumor Cells and Oncosomes in Metastatic Colorectal Cancer for Clinical Utility.

Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy's relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.

Multianalyte liquid biopsy to aid the diagnostic workup of breast cancer.

Breast cancer (BC) affects 1 in every 8 women in the United States and is currently the most prevalent cancer worldwide. Precise staging at diagnosis and prognosis are essential components for the clinical management of BC patients. In this study, we set out to evaluate the feasibility of the high-definition single cell (HDSCA) liquid biopsy (LBx) platform to stratify late-stage BC, early-stage BC, and normal donors using peripheral blood samples. Utilizing 5 biomarkers, we identified rare circulating events with epithelial, mesenchymal, endothelial and hematological origin. We detected a higher level of CTCs in late-stage patients, compared to the early-stage and normal donors. Additionally, we observed more tumor-associated large extracellular vesicles (LEVs) in the early-stage, compared to late-stage and the normal donor groups. Overall, we were able to detect reproducible patterns in the enumeration of rare cells and LEVs of cancer vs. normal donors and early-stage vs. late-stage BC with high accuracy, allowing for robust stratification. Our findings illustrate the feasibility of the LBx assay to provide robust detection of rare circulating events in peripheral blood draws and to stratify late-stage BC, early-stage BC, and normal donor samples.

Identification of epithelial and mesenchymal circulating tumor cells in clonal lineage of an aggressive prostate cancer case.

Little is known about the complexity and plasticity of circulating tumor cell (CTC) biology in different compartments of the fluid microenvironment during tumor metastasis. Here we integrated phenomics, genomics, and targeted proteomics to characterize CTC phenotypic and genotypic heterogeneity in paired peripheral blood (PB) and bone marrow aspirate (BMA) from a metastatic prostate cancer patient following the rapid disease progression, using the High-Definition Single Cell Assay 3.0 (HDSCA3.0). Uniquely, we identified a subgroup of genetically clonal CTCs that acquired a mesenchymal-like state and its presence was significantly associated with one subclone that emerged along the clonal lineage. Higher CTC abundance and phenotypic diversity were observed in the BMA than PB and differences in genomic alterations were also identified between the two compartments demonstrating spatial heterogeneity. Single cell copy number profiling further detected clonal heterogeneity within clusters of CTCs (also known as microemboli or aggregates) as well as phenotypic variations by targeted proteomics. Overall, these results identify epithelial and mesenchymal CTCs in the clonal lineage of an aggressive prostate cancer case and also demonstrate a single cell multi-omic approach to deconvolute the heterogeneity and association of CTC phenotype and genotype in multi-medium liquid biopsies of metastatic prostate cancer.

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

Cancer cell enrichment on a centrifugal microfluidic platform using hydrodynamic and magnetophoretic techniques.

Isolation of rare cancer cells is one of the important and valuable stages of cancer research. Regarding the rarity of cancer cells in blood samples, it is important to invent an efficient separation device for cell enrichment. In this study, two centrifugal microfluidic devices were designed and fabricated for the isolation of rare cancer cells. The first design (passive plan) employs a contraction-expansion array (CEA) microchannel which is connected to a bifurcation region. This device is able to isolate the target cells through inertial effects and bifurcation law. The second design (hybrid plan) also utilizes a CEA microchannel, but instead of using the bifurcation region, it is reinforced by a stack of two permanent magnets to capture the magnetically labeled target cells at the end of the microchannel. These designs were optimized by numerical simulations and tested experimentally for isolation of MCF-7 human breast cancer cells from the population of mouse fibroblast L929 cells. In order to use the hybrid design, magnetite nanoparticles were attached to the MCF-7 cells through specific Ep-CAM antibodies, and two permanent magnets of 0.34 T were utilized at the downstream of the CEA microchannel. These devices were tested at different disk rotational speeds and it was found that the passive design can isolate MCF-7 cells with a recovery rate of 76% for the rotational speed of 2100 rpm while its hybrid counterpart is able to separate the target cells with a recovery rate of 85% for the rotational speed of 1200 rpm. Although the hybrid design of separator has a better separation efficiency and higher purity, the passive one has no need for a time-consuming process of cell labeling, occupies less space on the disk, and does not impose additional costs and complexity.

Analytical and Computational Modeling of Sustained-Release Drug Implants in the Vitreous Humor.

Sustained ocular drug delivery systems are necessary for patients needing regular drug therapy since frequent injection is painful, undesirable, and risky. One type of sustained-release systems includes pellets loaded with the drug, encapsulated in a porous shell that can be injected into the vitreous humor. There the released drug diffuses while the physiological flow of water provides the convective transport. The fluid flow within the vitreous is described by Darcy's equations for the analytical model and Brinkman flow for the computational analysis while the drug transport is given by the classical convection-diffusion equation. Since the timescale for the drug depletion is quite large, for the analytical model, we consider the exterior surrounding the capsule to be quasi-steady and the interior is time dependent. In the vitreous, the fluid-flow process is relatively slow, and meaningful results can be obtained for small Peclet number whereby a perturbation analysis is possible. For an isolated capsule, with approximately uniform flow in the far field around it, the mass-transfer problem requires singular perturbation with inner and outer matching. The computational model, besides accommodating the ocular geometry, allows for a fully time-dependent mass-concentration solution and also admits moderate Peclet numbers. As expected, the release rate diminishes with time as the drug depletion lowers the driving potential. The predictive results are sufficient general for a range of capsule permeability values and are useful for the design of the sustained-release microspheres as to the requisite permeability for specific drugs.