EMT-independent detection of circulating tumor cells in human blood samples and pre-clinical mouse models of metastasis

Clinical & Experimental Metastasis https://doi.org/10.1007/s10585-020-10070-y TECHNICAL NOTE EMT‑independent detection of circulating tumor cells in human blood samples and pre‑clinical mouse models of metastasis Jenna Kitz1,2 · David Goodale1 · Carl Postenka1 · Lori E. Lowes3 · Alison L. Allan1,2,4,5 Received: 5 September 2020 / Accepted: 25 December 2020 © The Author(s) 2021 Abstract Circulating tumor cells (CTCs) present an opportunity to detect/monitor metastasis throughout disease progression. The CellSearch® is currently the only FDA-approved technology for CTC detection in patients. The main limitation of this system is its reliance on epithelial markers for CTC isolation/enumeration, which reduces its ability to detect more aggressive mesenchymal CTCs that are generated during metastasis via epithelial-to-mesenchymal transition (EMT). This Technical Note describes and validates two EMT-independent CTC analysis protocols; one for human samples using Parsortix® and one for mouse samples using VyCap. Parsortix® identifies significantly more mesenchymal human CTCs compared to the clinical CellSearch® test, and VyCap identifies significantly more CTCs compared to our mouse CellSearch® protocol regardless of EMT status. Recovery and downstream molecular characterization of CTCs is highly feasible using both Parsortix® and VyCap. The described CTC protocols can be used by investigators to study CTC generation, EMT and metastasis in both pre-clinical models and clinical samples. Keywords Circulating tumor cells · Metastasis · Epithelial-to-mesenchymal transition · CellSearch® · Parsortix® · VyCap Introduction Cancer is the second leading cause of death in the United States; with over 600,000 Americans dying from this disease in 2020 [1]. It is estimated that up to 90% of cancerrelated deaths are due to metastasis, the spread of disease to other sites in the body [2]. This is because current therapies are non-curative against these aggressive cancers. The Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s1058 5-020-10070-y. * Alison L. Allan 1 London Regional Cancer Program, London Health Sciences Centre, London, Canada 2 Department of Anatomy & Cell Biology, Western University, London, Canada 3 Flow Cytometry, London Health Sciences Centre, London, Canada 4 Department of Oncology, Western University, London, Canada 5 Lawson Health Research Institute, London, ON, Canada process of metastasis has been shown to be associated with an epithelial-to-mesenchymal transition (EMT) [3]. During the EMT process, a polarized epithelial cell undergoes morphological and molecular changes that enable it to gain a mesenchymal phenotype [4]; characterized by a greater migratory capacity, increased invasiveness, and elevated resistance to apoptosis [5]. During metastasis and associated EMT, tumor cells can shed from the primary tumor and disseminate throughout the body as circulating tumor cells (CTCs) in the bloodstream [6]. The presence and molecular characteristics of CTCs in patients have been correlated with increased metastatic disease, reduced survival, and therapy response/resistance [7–10]. Although EMT has been shown to be associated with increased metastasis and CTC generation, many technologies used to detect CTCs rely on epithelial characteristics [11]. For example, CellSearch® (Menarini Silicon Biosystems) is currently the only CTC assay approved by the U.S. Food and Drug Administration for clinical CTC analysis [7, 8, 12]. CellSearch® distinguishes CTCs from leukocytes through immunomagnetic selection of cells with an EpCAM+ (epithelial cell adhesion molecule) phenotype followed by differential fluorescent staining for cytokeratins (CK) 8/18/19, CD45 (leukocyte marker), and DNA 13 Vol.:(0123456789) Clinical & Experimental Metastasis (4′,6-diamidino-2-phenylindole [DAPI]). Despite being considered the “gold standard” clinical CTC platform [7, 8, 12], previous studies have shown that in some diseases such as prostate cancer, CTCs are undetectable in ~ 30% of patients despite the presence of widespread metastatic disease [13]. While it is possible that CTCs are truly not present in one third of prostate cancer patients with metastasis, it is more likely that CTCs are present but not detected by the CellSearch® system. This may be because they do not meet the standard CTC definition (EpCAM+/CK+/DAPI+/ CD45−) due to EMT and associated downregulation of epithelial markers [14, 15]. Importantly, several studies have demonstrated that CTCs with a purely mesenchymal phenotype are undetectable by CellSearch®, but that the presence of mesenchymal marker expression on CTCs with a hybrid epithelial-mesenchymal phenotype is indicative of poor prognosis [15–19]. We have previously described the use of this epithelial-based system in capturing both human and mouse CTCs [20, 21] and demonstrated that a CellSearch®-based assay failed to detect a significant number (~ 40–50%) of mesenchymal CTCs. Notably, the CellSearch®-based assay captured the majority of CTCs shed during early-stage disease in vivo, and only after the establishment of metastases were a significant number of undetectable CTCs present [11]. Taken together, this suggests that current clinical assays may be limiting our ability to capitalize on the full potential of CTCs, and that additional technologies that do not rely on epithelial characteristics should be explored. The Parsortix® system (Angle PLC) is a sized-based microfluidics platform that allows for recovery of relatively pure populations of CTCs for downstream molecular analysis based on CTC size and deformability, and is thus independent of EMT status [22]. Whole blood is processed through a filtration cartridge etched with microchannels that are 6.5–10 μM wide [20]. Using microfluidics, CTCs (> 8 μM) are isolated within the cartridge and stained with immunofluorescent antibodies [20]. The VyCap system (VyCap B.V.) is a sized-based CTC isolation and enumeration platform which uses a pump unit to process whole blood through a disposable filter cartridge [23, 24]. CTCs are captured on top of the microsieve which has 160,000 pores; each 5 μM in diameter [23, 24]. The VyCap allows for recovery of CTCs based on CTC size rather than epithelial cell characteristics [24] and is thus similar to the Parsortix® in providing the potential for an EMT-independent approach to CTC capture and analysis. The purpose of this Technical Note is to describe and validate two EMT-independent CTC isolation/enumeration protocols that we have developed for unbiased analysis of CTCs in human blood samples (using Parsortix®) and preclinical mouse models of metastasis (using VyCap). We also provide a summary of advantages/disadvantages and 13 technical considerations that metastasis researchers may find valuable for application of these methods to studies in the areas of CTCs, EMT, and cancer progression. M (...truncated)