Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines

Abstract

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via either or both of the CXCR1 and CXCR2 receptors, exerting profound impacts on tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

Dynamic secretome of bone marrow-derived stromal cells reveals a cardioprotective biochemical cocktail

“…The detection can then be accomplished by stopping the experiment at desired times and using secondary antibodies to detect the ligands captured at microspots in the experimental chamber. The finite element simulations executed in COMSOL to model diffusion of proteins (39,40) in the device confirmed that diffusion of proteins with the characteristic sizes matching those of the factors examined here is not distorted by pillars separating the chambers, with the diffusion profiles stable over prolonged time periods (SI Appendix, Fig. S2B).…”

“…Microfluidics can provide 2 important advantages in the analysis of cell secretome: (i) small volume limiting dilution of the secreted compounds and (ii) more predictable liquid handling (34,35). Recently, these advantages have been used to enable quantifiable measurements of cell secretions down to a single-cell level (36)(37)(38)(39)(40). However, important challenges remain.…”

“…Arguably, the information required for this interpolation could be recovered from spatial distributions of diffusing compounds, relying on the intimate connection between space and time in diffusion-mediated transport. Frequently, this information is consciously removed in the design of the measurement devices, aimed at achieving "well-mixed" conditions leading to a rapid equilibration of secreted ligands between distinct detection locations within a device (37,39). However, if this requirement is relaxed and the spatial profiles of secreted ligands are evaluated at each time point, further information can be mined from each experimental measurement, as demonstrated in this study.…”

Automated rare single cell picking with the ALS cellcelector™

“…With the CellCelector it was possible to isolate pure single tumor cells from liquid biopsies of different cancer types including breast , prostate , ovarian , colorectal , and lung cancers.…”

Toward engineered processes for sequencing-based analysis of single circulating tumor cells

“…Should in vitro viability of CTCs be improved, additional live-cell assays could identify (and functionally characterize) candidate single CTCs. For instance, microengraving in an array of subnanoliter wells could be used to evaluate protein secretion from thousands of single cells over ~1–3 hr timescales [43] (i.e. PSA secretion from prostate CTCs) or image cytometry could be used to assess cell motility and single-cell growth kinetics under hypoxic conditions.…”