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 experiment, and use high-resolution genomic analysis to explore the
mechanisms driving heterogeneous responses within a cell population, such as resistance to a drug.
The company received a Phase I grant and successfully demonstrated a platform that can organize
up to 100,000 single cells on a standard cell culture plate sized device, and then track the growth
rates of single cell clones over multiple days. This 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-omics' single cell analysis platform. We hypothesize that Celldom products can
seamlessly integrate the isolation, stimulation, phenotyping, and genomic analysis of clonal
populations in a combined workflow. Once this is demonstrated, Celldom's future products will be
positioned as the only platform on the market capable of connecting complex phenotypes (i.e., growth
rate and cell cycle staging) to expression profiles of single cell clones in massively parallel format.
The work plan to achieve this goal is described in three specific aims. In the first aim, we will quantify
heterogeneity of single cell growth rates in drug sensitive and resistant cell lines. This aim will
demonstrate a high-throughput single cell based drug-screening platform validated with an acute
myeloid leukemia (AML) cell line. We will show that the fraction of FLT3+ drug-resistant cells spiked
into the parental drug-sensitive cell line can be accurately identified in the Celldom platform. In the
second aim, we will demonstrate the ability to conduct high-throughput mRNA expression analysis
using mixtures of each cell clone. This aim will demonstrate a genomic workflow for transcriptional
profiling of each surviving clonal population. We will print DNA barcodes in each trap site and show
that the hydrogels can efficiently entrap cell lysates and limit cross-contamination, as demonstrated
with a mixture of human and mouse cell populations. In the third aim, we will demonstrate the ability
to combine phenotyping and genotyping in cell lines and primary patient samples. This aim will
demonstrate the ability to quantify heterogeneous proliferation rates in patient-derived AML samples
exposed to a second generation FLT3-inhibitor continuously over several days, and then couple the
phenotypic data to the transcriptional profiles of the surviving clonal populations. Once these aims are
demonstrated, we will scale our launch in bottom up fashion, starting with academic core labs and
key opinion leaders, expanding to biopharma industry as the Celldom system gains acceptance.
