Mishra Laboratory

Overview

In the Mishra laboratory, we develop bioengineering approaches for cancer diagnostics and cell therapy manufacturing. Specifically, we focus on the application of large-volume microfluidics bioengineering tools for liquid biopsy of cancer cells, immune subset enrichment for cell therapy manufacturing, and enrichment of hematopoietic stem cells for genetic engineering in sickle cell disease. Leveraging the precision and controllability of microfluidics, we recover viable and untouched rare tumor cells from billions of contaminating cells, enabling a comprehensive "cell-based liquid biopsy" with multi-analyte analyses applicable to a diverse array of cancers. The ability to interrogate large numbers of intact cancer cells in individual patients offers unprecedented opportunities for serial multi-omics at the single-cell level, including paired RNA and DNA analyses applied to large numbers of individual tumor cells.

Research Summary

Tumor-cell based liquid biopsies have emerged as a promising tool for cancer diagnostics, treatment selection, and response monitoring. Intact tumor cells provide the full complement of analytes, including DNA, RNA, proteins, and metabolic markers. However, these cells are often extremely rare and exist in large sample volumes. For instance, clinical biofluids, like blood products, require large sampling volumes of tens to hundreds of milliliters, where tumor cells can be as rare as 1 in 50 million nucleated cells. We leverage the precision and controllability of microfluidics, enabled by semiconductor manufacturing techniques, to uncover viable and untouched rare cells at high cellular throughputs (100 million cells/min).

Large-volume microfluidics for tumor-cell based liquid biopsy

Circulating tumor cells (CTCs) are extremely rare (1 cell in 10 mL blood), and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We developed a high-throughput microfluidic platform utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses for processing concentrated large-volume blood products. LPCTC-iChip operates without clogging and activating platelets while providing identical sorting conditions at the single-cell level. In collaboration with Haber, Maheswaran, and Toner labs, this technology has been applied to analyze patient-derived blood products, screening whole blood volume from patients with metastatic cancer, with a median yield of 2,799 CTCs purified per patient. Isolation of 100-fold more CTCs from individual patients enables the characterization of their morphological and molecular heterogeneity, including cell size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

Microfluidic devices for cell and gene therapy manufacturing

Cellular therapies based on the ex vivo editing of hematopoietic stem cells or immune T cells have emerged as a transformative disease-modifying option for treating various diseases such as Sickle Cell Disease and hematologic cancers. Most cell therapy products utilize patients' own blood cells, collected through leukapheresis, as the starting material. This demands precise isolation of rare stem cells or subsets of T cells from high cell-density, large volume (300 to 400 mL) leukopaks. The isolation is followed by genetic modification of the cells and their subsequent culture and infusion back into the patient. Conventional bulk cell sorting methods are highly lossy in recovering rare cells, risk contamination, yield impure products, and prove averse to automation. While these bulk methods suffer from cell loss, microfluidic cell sorting approaches produce high-purity products and enable superior cell yield. To address these challenges, we will bring microfluidic innovations from the field of cancer diagnostics to the field of cell therapy manufacturing. We are focusing on two directions: stem cell purification from blood products for sickle cell gene therapy and the development of a microfluidic CAR-T manufacturing platform.

Select Publications

Mishra A, Huang S-B, Dubash T, Burr R, Edd JF, Wittner BS, et al. Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product. BioRxiv 2024:2024.03.13.583573.

Gopinathan KA, Mishra A, Mutlu BR, Edd JF, Toner M. A microfluidic transistor for automatic control of liquids. Nat 2023 6227984 2023;622:735–41.

Mishra A, Dubash TD, Edd JF, Jewett MK, Garre SG, Karabacak NM, et al. Ultra-high throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells. Proc Natl Acad Sci 2020;117:1–35.

Edd JF, Mishra A, Dubash TD, Herrera S, Mohammad R, Williams EK, et al. Microfluidic concentration and separation of circulating tumor cell clusters from large blood volumes. Lab Chip 2020;20:558–67.

Mishra A, Kwon J-SJ-S, Thakur R, Wereley S. Optoelectrical microfluidics as a promising tool in biology. Trends Biotechnol 2014;32:414–21.