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

Abstract

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.

Fig. 1. Patient demographic, pathology, and HDSCA3.0 workflow.

a Patient’s diagnosis, pathology, disease progression, treatment history, and liquid biopsy timepoint. b HDSCA3.0 workflow including immunofluorescence staining and imaging, rare cell detection, CTC classification, molecular profiling, and downstream analyses.

Fig. 2. CTC subtype and enumeration in the paired PB and BMA.

a Immunofluorescence images of CTCs from PB sample. Scale bar: 10 μm. b Immunofluorescence images of CTCs from BMA sample. Color coding: DAPI (blue); CK (red); VIM (white); CD45/CD31 (green). Scale bar: 10 μm. c Enumeration of CTC subtypes in each test of PB and BMA.

Fig. 3. Clonal lineage and its variation between PB and BMA.

a Complex heatmap of single cell copy number profiles from paired PB and BMA grouped by hierarchical clustering. The “c” in clade IDs means compressed profile due to normal cell contamination. Red, copy number gain; blue, copy number loss; white, copy number neutral. VIM + only refers to “VIM + only” rare cells. b Clonal lineage in PB and BMA combination and separate. The number in parenthesis is the number of cells analyzed.

Fig. 4. Association between genotype and phenotype.

a Association of genomic subclones with mesenchymal phenotype or platelet attachment in all CTCs or subpopulations. b Immunofluorescence images and single cell copy number profiles of representative heterogeneous CTC clusters in the BMA sample. Images order: composite, DAPI, CK, VIM, and CD45/CD31. The number is the sequencing cell ID.

Fig. 5. Targeted proteomics of CTC subtypes.

a Heatmap of proteomic expression in CTC subtypes, including AR-N terminal (AR_N), AR-C terminal (AR_C), PSMA, EpCAM, E-Cadherin, N-Cadherin, Vimentin (VIM), PCNA, and β-catenin. b Immunofluorescence and image mass cytometry images of representative CTC subtypes. c Comparisons of proteomic expressions among CTC subtypes. Center line of box: median; upper/lower hinges of box: 75% or 25% quartile; upper/lower whiskers of box: hinge ± 1.5*IQR (inter-quartile range). *p < 0.05, **p < 0.01, ***p < 0.001.