# Micro-capsules for versatile multiplexed cytometry

> **NIH NIH R33** · HARVARD MEDICAL SCHOOL · 2024 · $336,075

## Abstract

Project summary
A major goal of cancer research is to define the composition of the tumor micro-environment (TME) across
individuals. Once measured, differences in TME composition can be correlated with prognosis, targeted by
therapy, and used to test or generate novel therapeutic hypotheses. Our appreciation of TME complexity was
significantly advanced with the development of single cell RNA-Sequencing (scRNA-Seq). But scRNA-Seq
remains expensive, noisy at the level of single cells, and has a slow turn-around time (typically weeks). scRNA-
Seq also typically analyzes only 1000s of cells per sample. As a result, scRNA-Seq is not practical for deep
profiling of large patient or animal cohorts, or for routine hypothesis-testing in cancer research. Faster and
more scalable alternatives to scRNA-Seq are flow cytometry (FC) and Cytometry by Time of Flight (CyToF) but
these methods do not resolve the complexity seen in the TME by scRNA-Seq. Thus, there is an unmet need
for rapid, sensitive, highly-multiplexed TME profiling.
The focus of this grant is to address this unmet need by advancing a versatile and novel `micro-capsule'
technology. Capsules represent an evolution of droplet microfluidics, which is a mature technology for carrying
out single cell genomic assays in nanoliter-scale compartments, isolated by oil. Capsules overcome severe
technical limitations of water-in-oil droplets: their fragility to handling, and their complete isolation by immiscible
oil. By contrast, capsules are resilient, semi-permeable compartments that can be dispersed and processed in
any aqueous biological buffer. Prior to this proposal, we optimized capsules to retain cellular mRNA and DNA,
while simultaneously enabling rapid exchange of salts, enzymes, primers and probes with the surrounding
medium. We have now shown that capsules enable multi-step reactions and serial analyses on single cells and
specifically on surface proteins and mRNA molecules. This in turn enables rapid, versatile, highly-multiplexed
cytometry.
In this R33 we will benchmark and optimize two related capsule-derived methods: the first, “CapFlow”,
implements robust multiplexed mRNA flow cytometry with rapid capsule-based signal amplification. The
second, “CapCycle”, extends CapFlow to quantifying the abundance of ≥50 gene transcripts and cell surface
proteins, by replacing flow cytometry with cyclic imaging of immobilized capsules. With these methods,
capsules will enable sensitive, versatile, rapid, low-cost, highly-multiplexed phenotyping of tumor
heterogeneity. Thus, this proposal fills an important analytical gap, and develops a versatile microfluidic
technology with long-term potential to improve biological assays on single biomolecules and cells.

## Key facts

- **NIH application ID:** 10824436
- **Project number:** 5R33CA278392-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Allon Moshe Klein
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $336,075
- **Award type:** 5
- **Project period:** 2023-04-07 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10824436

## Citation

> US National Institutes of Health, RePORTER application 10824436, Micro-capsules for versatile multiplexed cytometry (5R33CA278392-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10824436. Licensed CC0.

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