Actuatable hydrogel microcages for coupled single-cell sequencing and phenotyping – SNAP-Seq

Single-cell analysis has become an indispensable tool to overcome the cellular heterogeneity of healthy and diseased tissues. Indeed, the bulk analysis of a set of cells cannot give the complete picture of its heterogeneity. Tumors typically comprise multiple types of tumor cells in addition to a wide range of stromal and tumor-infiltrating immune cells. The bulk analysis of the whole tumor would miss much of the underlying diversity.
“Omic” analyses (genomic, epigenomic and transcriptomic) using barcoded next-generation sequencing (NGS) are now possible at high-throughput at the single-cell scale, likewise phenotypic analyses (protein expression, secretion, glycosylation, post-translational modification, metabolism, morphology, response to stimuli…) using microscopy, flow-cytometry and mass-cytometry. Some recent techniques additionally allow analysis of a sub-fraction of the proteome coupled to transcriptomic analysis via barcoded NGS.
There is today a strong need, expressed by the research and clinical community, for a better understanding of complex and heterogeneous biological systems, requiring the acquisition of both phenotypic and omics information for each of the thousand single cells from a sample. The constraints of droplet microfluidics, on which single-cell omics analysis is based today, such as the impossibility of identifying droplets by imaging, explain the absence of such coupling today.
Minos Biosciences is developing a solution to run multimodal single-cell analysis directly coupling phenotypic analysis via optical imaging with omic analysis via barcoded NGS. In its system, single cells can be compartmentalized in over 100,000 thermoactuatable hydrogel micro-cages hosting barcoded primers, adaptors or recombination sites, with a unique barcode specific for each micro-cage.
However, Minos’ solution relies on the use of poly(N-isopropylacrylamide) (PNIPAM), a Lower Critical Solution Temperature (LCST) hydrogel, swollen in water at ambient temperature and collapsed above 32°C. Micro-cages are therefore open at 37°C, whereas it would be highly desirable to analyze the phenotype of living cells under physiologic conditions with closed cages.
The aim of this project is to overcome these limitations by developing novel Upper Critical Solution Temperature (UCST) hydrogels that are collapsed at low temperature and swollen at higher temperature, and to use these polymers to microfabricate micro-cages that are closed at 37°C.
We will develop specific protocols for these UCST-based microfluidic chips, allowing phenotypic analysis of mammalian cells under physiological conditions, including analysis of secreted proteins using a sandwich enzyme-linked immunosorbent assay or an innovative system based on proximity extension combined with Polymerase/Exonuclease/Nickase Dynamic Network Assembly toolbox system.
We will demonstrate the utility of the system to study and refine the newly discovered molecular classification of colorectal cancer (CC). CC is now recognized as a highly heterogeneous disease with respect to its genomic and transcriptional alterations and functional characteristics. Recent results have highlighted that a large proportion of CC is actually presenting intra-tumor heterogeneity with implications in patient prognosis. Several studies have also suggested that multi-omics analysis could allow to elaborate a more comprehensive molecular characterization of both CC cell lines and patient samples.
This project will provide a new tool for multi-omics single cell analysis coupled to image-based phenotypes captured in living cells. We anticipate that this tool will allow to identify new markers to measure intratumor heterogeneity within patients. Moreover, it could also lead to a better understanding of intra-tumor heterogeneity in CC. In particular it could reveal key factors governing the phenotypic plasticity of CC cells, and help to improve therapeutic strategies for CC patients.