Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits

Abstract

Ample evidence supports genetic and functional heterogeneity in primary tumors, but it remains unclear whether circulating tumor cells (CTCs) also exhibit the same hierarchical organization. We examined the functional diversity of viable, single CTCs using an array of subnanoliter wells (nanowells). The compartmentalization of single cells by nanowells allowed clonal comparison and mapping of heterogeneity of single cells or preformed clusters of cells. By measuring the short-term viability, invasiveness and secretory profiles of individual CTCs, it was evident that only a rare subset of CTCs possessed malignant traits indicative of metastatic potential in late-stage, progressing metastatic castration-resistant prostate cancer (mCRPC) patients. These CTCs were resistant to anoikis after being in the circulation, were invasive in their epithelial state, or secreted proteases capable of cleaving peptide substrates. Every CTC observed, however, did not exhibit such metastatic potential, suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients.

Cancer-associated fibroblasts as target and tool in cancer therapeutics and diagnostics

“…The hydrogels formed from peptide crosslinkers and polyethylene glycol monomers permit cell-controlled invasion and proteinase specific matrix peptides may further allow for selective control of invasion. Arrays of subnanoliter wells (nanowells) embedded with a fibroblast-derived matrices or a hydrogel containing fibroblasts or other organ-specific host cells allow the characterization of the functional diversity of CTCs [117]. In addition, microfluidic culture devices may further increase the realistic recapitulation of potential metastatic target organs such as the liver, brain and lung [10].…”

Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

“…Although several methods, such as fluorescence microscopy and flow cytometry 1,34 , are available for discerning the cellular properties (such as the level of a specific protein) of intact cells, even in this setting, microdevices provided early value by confining cellular products so that they could be detected 20 and aiding in the processing of low-input samples 5 . Illustrative applications range from nanowell- and droplet-based methods for measuring the amount of antibody produced by individual B cells 33 to characterizing surface marker expression in rare populations of cells using nanowells 35 .…”

“…Similar methodologies, focused on simultaneously profiling mutually informative classes of variables, such as DNA and RNA or RNA and the epigenome 78 , seem to be just around the corner (BOX 1), and may soon guide our understanding of cellular function. Importantly, many other informative properties of cells have been measured with microfluidic devices — for example, morphology 127 , proliferation dynamics 128 , motility 129 , invasiveness 35 , interactions 123 , calcium dynamics 130 and familial relationships 131 — all of which may help to determine cellular identity in the future.…”

Profiling protein expression in circulating tumour cells using microfluidic western blotting

“…Strikingly, most single-cell protein assays (for unmodified endogenous targets) are single-stage immunoassays, including enzyme-linked immunosorbent assays (with direct or sandwich readout) and immunocytochemistry, as well as newer immunoassay formats designed to improve multiplexing using spatial barcoding1516 or mass cytometry17. CTC protein analyses primarily focus on surface and secreted proteins1819. Although important, the capability to multiplex and assay a wide range of protein targets (including intracellular signalling pathways) has been limited20.…”