Kim, C.-J. | bioRxiv Science

bioRxiv2026

A microfluidic platform for multi-marker profiling of extracellular vesicles from single-cell-derived clones

Extracellular vesicles (EVs) carry molecular cargo that can reflect the real-time state of parental cells, yet most in vitro EV analyses rely on bulk approaches and therefore average over pronounced heterogeneity in both cell and EV populations. Here, we present a semi-open microfluidic platform that enables multi-marker profiling of EVs released from single-cell-derived clones, allowing EV signatures to be linked to clonal progeny originating from a single parental cell. The platform integrates aligned cell and EV arrays containing 17,305 wells, assembled with a 3D-printed housing to capture released EVs in one-to-one matched wells. Captured EVs are immunolabeled for canonical tetraspanin markers (CD9, CD63, CD81) and EpCAM, imaged by high-resolution fluorescence microscopy, and quantified using an automated image-analysis pipeline. Applying the platform to single-cell-derived PC3 clones revealed substantial heterogeneity in EV marker co-expression, with hierarchical clustering identifying four distinct tetraspanin co-expression profiles. The fraction of EpCAM-positive EVs increased with PC3 cell proliferation, as assessed by endpoint cell number, whereas free (non-EV-associated) EpCAM showed no correlation. This platform enables near single-EV-level, multi-marker profiling from single-cell lineages and provides a practical approach to simultaneously dissect both cellular and EV heterogeneity.

bioengineering↗

bioRxiv2022

Nuclear morphology and functional characteristics that enable survival of cells in the polyaneuploid cancer cell state following cytotoxic chemotherapy

In this study, we characterized nuclear morphology and function as cancer cells underwent recovery following chemotherapeutic treatment to identify the unique characteristics associated with treatment resistance and successful survival. Cells that survived following treatment and resisted therapy-induced cell death were predominantly mononucleated with increased nuclear/cellular size, enabled by continuous endocycling. We found that cells that survive after therapy release likely employ more efficient DNA damage repair and exhibit a distinct nucleolar phenotype - fewer but larger nucleoli - and increased rRNA levels. These data support a paradigm where soon after therapy release, the treated population mostly contains cells with a high level of widespread and catastrophic DNA damage that leads to apoptosis, while the minority of cells that have successful DDR are more likely to access a pro-survival state. These findings suggest that one way cancer cells can survive systemic therapy is to enter the polyaneuploid cancer cell (PACC) state, a recently-described mechanism of therapy resistance. Cancer cells in this state are physically enlarged, undergo whole-genome doubling resulting in polyaneuploid genomes, and are associated with worse prognosis in cancer patients. The PACC state is accessed when a cancer cell experiences external stress, such as genotoxic chemotherapy; after a period of recovery, cells exit the PACC state and resume proliferation to repopulate the tumor cell pool. Our findings demonstrate the fate of cancer cells following chemotherapy treatment and define key characteristics of the resistant PACC state. This work is essential for understanding and, ultimately, targeting, cancer resistance and recurrence.

cancer biology↗