Measuring single-cell density with high throughput enables dynamic profiling of immune cell and drug response from patient samples

Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and therefore a fundamental determinant of cell state and function. However, existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples. Here we present an approach for measuring the density of 30,000 single cells per hour with a precision of 0.03% (0.0003 g/mL) by integrating fluorescence exclusion microscopy with a suspended microchannel resonator. Applying this approach to human lymphocytes, we discovered that cell density and its variation decrease as cells transition from quiescence to a proliferative state, suggesting that the level of molecular crowding decreases and becomes more regulated upon entry into the cell cycle. Using a pancreatic cancer patient-derived xenograft model, we found that the ex vivo density response of primary tumor cells to drug treatment can predict in vivo tumor growth response. Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a new biomarker for functional precision medicine. ### Competing Interest Statement S.R.M. and K.L.L. are founders of Travera. S.R.M. is a founder of Affinity Biosensors. K.L.L. receives consulting fees from Bristol Myers Squibb, Blaze Bioscience, and Integragen. MIT has filed a patent application (PCT/US2022/051503) on the method for measuring single-cell density, with S.R.M., W.W., and T.P.M. listed as the inventors. MIT and DFCI have joint filed a patent application (PCT/US2022/051514) on single-cell density as a biomarker for drug response, with S.R.M., K.L.L., W.W., T.P.M., S.H.I, and K.H.C listed as the inventors. The other authors declare no competing interests.