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.
The cost of bringing a new drug to market is estimated at $2.5B, which in large part is due to the high percentage of drug candidates failing clinical trials (>88%). High-throughput single cell assay platforms have the potential to reduce waste in the drug discovery pipeline by credentialing promising drug candidates at an earlier pre-clinical stage. Building on licensed patent applications of key technology contributors, our goal is to build a minimally viable product (MVP), which can rapidly form an array of 100,000 single cells, maintain cells on chip for up to 7 days, and conduct preliminary studies of the distribution of single cell proliferation rates exposed to small molecule drug compounds. Successful demonstration of these SBIR Phase I aims will pave the way for follow-on SBIR Phase II work focusing on the development of the first-ever single cell based high- throughput drug screening platform, which can identify rare drug-resistant cells and provide insights into the biological basis of their drug resistance.
