High-throughput screening of arrayed single cells for automated analysis of phenotypic heterogeneity

ABSTRACT Celldom is developing a high throughput single cell analysis platform that can screen tens of thousands of single cells per experimentand use high resolution genomic analysis to explore the mechanisms driving heterogeneous responses within a cell populationsuch as resistance to a drugThe company received a Phase I grant and successfully demonstrated a platform that can organize up tosingle cells on a standard cell culture plate sized deviceand then track the growth rates of single cell clones over multiple daysThis Phase II proposal aims to advance a functional platform for phenotyping and genotyping each cell clone in massively parallel format to realize a comprehensive `multi omicsandapossingle cell analysis platformWe hypothesize that Celldom products can seamlessly integrate the isolationstimulationphenotypingand genomic analysis of clonal populations in a combined workflowOnce this is demonstratedCelldomandapos s future products will be positioned as the only platform on the market capable of connecting complex phenotypesi egrowth rate and cell cycle stagingto expression profiles of single cell clones in massively parallel formatThe work plan to achieve this goal is described in three specific aimsIn the first aimwe will quantify heterogeneity of single cell growth rates in drug sensitive and resistant cell linesThis aim will demonstrate a high throughput single cell based drug screening platform validated with an acute myeloid leukemiaAMLcell lineWe will show that the fraction of FLTdrug resistant cells spiked into the parental drug sensitive cell line can be accurately identified in the Celldom platformIn the second aimwe will demonstrate the ability to conduct high throughput mRNA expression analysis using mixtures of each cell cloneThis aim will demonstrate a genomic workflow for transcriptional profiling of each surviving clonal populationWe will print DNA barcodes in each trap site and show that the hydrogels can efficiently entrap cell lysates and limit cross contaminationas demonstrated with a mixture of human and mouse cell populationsIn the third aimwe will demonstrate the ability to combine phenotyping and genotyping in cell lines and primary patient samplesThis aim will demonstrate the ability to quantify heterogeneous proliferation rates in patient derived AML samples exposed to a second generation FLTinhibitor continuously over several daysand then couple the phenotypic data to the transcriptional profiles of the surviving clonal populationsOnce these aims are demonstratedwe will scale our launch in bottom up fashionstarting with academic core labs and key opinion leadersexpanding to biopharma industry as the Celldom system gains acceptance NARRATIVE Cancer patients often relapse because their tumors contain drug resistant cellswhich though initially present at small fractionsbecome enriched during treatment to yield incurable tumorsTraditional approaches to identifyisolateand then examine drug resistant cells can require months of laborintensive workwhich is prohibitive for the early stages of candidate identificationSingle cell assays hold great potential for probing rare cellular eventshowevercompetitor offerings are unable to comprehensively analyze both the phenotype and genotype of each cellIn this Phase II proposalCelldom will solve this problem by developing a validated workflow that can be first translated into the drug developmentand laterclinical marketplaces