Rapid and Automated Dissociation of Tumor Tissue for Single Cell Diagnostics

ABSTRACT Solid tumors are diverse ecosystems of different cell types, and this heterogeneity has been implicated as a key factor driving disease progression, metastasis, and drug resistance. Assessing cellular heterogeneity and identifying key driver cells, such as cancer stem cells, has thus become a critical factor for understanding tumor biology. These issues are also important for clinical diagnostics, as targeted therapies must be directed toward the most important cell types if effective cures are to be achieved. Increasingly, single cell analysis methods are being used to define cellular subsets within tumors to address biological and therapeutic questions. However, the need to first convert tissue into single cells is a significant barrier to more widespread use, particularly in clinical settings. Current tumor dissociation methods are long, inefficient, and not standardized. Moreover, there remains a question as to whether different cell subtypes are easier to release than others, leading to results that may not accurately reflect the original tumor. In previous work, we have developed a novel microfluidic device containing an array of branching channels that can dissociate cell aggregates and partially digested tissue in minutes with dramatically higher yield of viable single cells. In this proposal, will significantly improve the power and commercialization potential of this microfluidic tissue dissociation device by combining it with a CD microfluidic technology to create a fully automated CD-lab on a chip (CD-LOC) platform called the Syntrfuge. This will dramatically increase operational versatility, including increasing flow rate by 100x compared to a pump, which will significantly improve dissociation efficiency and decrease processing time. Furthermore, the likelihood of channel clogging will be reduced by direct application of centrifugal forces to tissue fragments and cellular aggregates. Finally, the system will be fully self-contained to maintain sterile conditions and protect the operator. The performance of the Syntrfuge will first be optimized using a murine tumor model, followed by final validation using human tumor tissue specimens. All tests will focus on single cell yield and viability, and the final evaluation will include single cell RNA sequencing. The Specific Aims for this 6 month project include: (1) optimize the Syntrfuge using a murine breast tumor model and (2) Validate performance of the final platform using human tumor tissue specimens. Our microfluidic device platform technology will directly impact single cell analysis of tumor specimens, particularly single cell sequencing, an emerging and potentially transformative molecular analysis tool. This will be achieved by streamlining tumor tissue dissociation through automation, increased efficiency, elimination of pre-processing (i.e. scalpel mincing), and continuous generation of 100% single cells. We are confident that this mechanical processing approach will be viewed favorably by the FDA and provide the data needed to move on to Phase II studies.