Diversity of Epithelial-Mesenchymal Phenotypes in Circulating Tumour Cells from Prostate Cancer Patient-Derived Xenograft Models

Abstract

Metastasis is the leading cause of cancer-related deaths worldwide. The epithelial-mesenchymal plasticity (EMP) status of primary tumours has relevance to metastatic potential and therapy resistance. Circulating tumour cells (CTCs) provide a window into the metastatic process, and molecular characterisation of CTCs in comparison to their primary tumours could lead to a better understanding of the mechanisms involved in the metastatic cascade. In this study, paired blood and tumour samples were collected from four prostate cancer patient-derived xenograft (PDX) models (BM18, LuCaP70, LuCaP96, LuCaP105) and assessed using an EMP-focused, 42 gene human-specific, nested quantitative RT-PCR assay. CTC burden varied amongst the various xenograft models with LuCaP96 having the highest number of CTCs per mouse (mean: 704; median: 31) followed by BM18 (mean: 101; median: 21), LuCaP70 (mean: 73; median: 16) and LuCaP105 (mean: 57; median: 6). A significant relationship was observed between tumour size and CTC number (p = 0.0058). Decreased levels of kallikrein-related peptidase 3 (KLK3) mRNA (which encodes prostate-specific antigen; PSA) were observed in CTC samples from all four models compared to their primary tumours. Both epithelial- and mesenchymal-associated genes were commonly expressed at higher levels in CTCs compared to the bulk primary tumour, although some common EMT-associated genes (CDH1, VIM, EGFR, EPCAM) remained unchanged. Immunofluorescence co-staining for pan-cytokeratin (KRT) and vimentin (VIM) indicated variable proportions of CTCs across the full EMP axis, even in the same model. EMP hybrids predominated in the BM18 and LuCaP96 models, but were not detected in the LuCaP105 model, and variable numbers of KRT+ and human VIM+ cells were observed in each model. SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was enriched at the RNA level in CTCs compared to primary tumours and was the most commonly expressed mesenchymal gene in the CTCs. Co-staining for SERPINE1 and KRT revealed SERPINE1+ cells in 7/11 samples, six of which had SERPINE+KRT+ CTCs. Cell size variation was observed in CTCs. The majority of samples (8/11) contained larger CTCs ranging from 15.3 to 37.8 µm, whilst smaller cells (10.7 ± 4.1 µm, similar in size to peripheral blood mononuclear cells (PBMCs)) were identified in 6 of 11 samples. CTC clusters were also identified in 9/11 samples, containing 2–100 CTCs per cluster. Where CTC heterogeneity was observed in the clusters, epithelial-like cells (KRT+VIM−) were located on the periphery of the cluster, forming a layer around hybrid (KRT+VIM+) or mesenchymal-like (KRT−VIM+) cells. The CTC heterogeneity observed in these models emphasises the complexity in CTC isolation and classification and supports the increasingly recognised importance of the epithelial-mesenchymal hybrid state in cancer progression and metastasis.

A Gene Prognostic Index Associated With Epithelial-Mesenchymal Transition Predicting Biochemical Recurrence and Tumor Chemoresistance for Prostate Cancer

“…Previous studies indicated that the plasticity of EMT tumor cells, which are in a transitory state between epithelial and mesenchymal programs, made it possible to accomplish the invasion-metastasis cascade, rather than the mesenchymal-like tumor cells that have fully completed the EMT program (32,42). Compelling evidence showed that transforming growth factor b (TGF-b) is a potent inducer of EMT and chemoresistance in multiple cancers, including PCa, through small mothers against decapentaplegic homolog-independent or homolog-dependent signaling pathway (9,32,(43)(44)(45)(46)(47).…”

Single-Cell RNA-Seq Analysis Reveals the Acquisition of Cancer Stem Cell Traits and Increase of Cell–Cell Signaling during EMT Progression

“…Rather, non-genetic determinants diversify cell populations into distinct phenotypes or cell states with various degrees of metastatic potential and resistance to various therapies [ 43 , 44 ]. Specifically, phenotypic heterogeneity along the EMT spectrum, or EM plasticity (EMP), is emerging as a distinctive feature of primary tumors [ 45 ], CTCs [ 46 , 47 ], and in the relation between tumors and CTCs [ 48 ]. This heterogeneity can arise due to diverse signaling cues that induce EMT in a spatio-temporal-dependent manner, as seen for instance in time course or wound healing experiments [ 36 , 41 ].…”

The Role of Dielectrophoresis for Cancer Diagnosis and Prognosis

“…In this context, liquid biopsy via isolation of CTCs represents a promising diagnostic tool capable of supplementing state-of-the-art PC diagnostics [ 40 ] and prognosis [ 41 , 42 , 43 , 44 , 45 ].…”