High-throughput screening of arrayed single cells for automated analysis of phenotypic heterogeneity - ABSTRACT
The overall objective of this SBIR Phase I proposal is to develop a high-throughput screening platform that can
analyze the phenotypic heterogeneity of single cells exposed to small molecule growth inhibitors. To achieve
this objective, we will conduct the following specific aims: (1) demonstrate a commercially viable approach for
rapidly organizing >100,000 single cells into an array of microfabricated apartments, and (2) demonstrate that
the arrayed cells can be maintained on chip for sufficient time to visualize several cell divisions in each micro-
chamber. These “proof of principle” demonstrations will support a future SBIR Phase II funding application to
develop an integrated system, which can identify the rare cells within a heterogeneous cell population that
evolve resistance to chemotherapy, and then conduct a high throughput screen to understand the biological
basis of their drug resistance. Our market hypothesis is that high-throughput single cell assay platforms can
reduce waste in the drug discovery pipeline by credentialing promising drug candidates more quickly
(days/weeks as opposed to months/years) and thereby advance the best drug candidates into clinical trials.
Considering that the estimated cost of bringing a new drug to market stands at $2.5B, which in large part is
due to the high percentage of drug candidates failing clinical trials (>88%), this single cell proliferation assay
has significant commercial potential. The engineering innovation of this proposal is based on a two-step
process for forming a single cell array, which involves first capturing single cells in fluidic traps of a microfluidic
channel, and then transferring the trapped cells into adjacent apartments with locally applied electromagnetic
forces. This cell organization strategy is gentle, massively parallel, and enables the rapid formation of multi-
component patterns of cells and reagents. The disease model used to benchmark this platform will consist of
acute myeloid leukemia (AML) cells bearing internal tandem duplication (ITD) mutations in FMS-like tyrosine
kinase 3 (FLT3). Initial assay development work will focus on validating that AML cells remain viable inside the
microfluidic device and can proliferate over multiple days. Once demonstrated, we will conduct a preliminary
set of “proof-of-concept” experiments on the proliferation of single AML cells over multiple days when exposed
to quizartinib, a small molecule FLT3 inhibitor to which resistance rapidly emerges. This biological model is
ideal for studying the survival mechanism of pre-existing, but rare, drug resistant clones. Building on these
experiments, we have plans extend this platform in the future to conduct massively parallel RNA-seq of the
arrayed single cells to provide a comprehensive profile of the function and gene expression of each single cell.
The impact of this high throughput drug discovery platform extends far beyond cancer, and can be applied to
develop drug therapies for eradicating latently infected cells responsible for chronic viral infections, such as
HIV, and assist in the development of immunotherapies. This single cell platform can also make a broad
impact in cellular and molecular biology, particularly in areas like cell communication, fate, and decision
making, where cellular heterogeneity influences the outcomes of physiological or disease processes.
