Aldehyde dehydrogenase 1A3 detection in extracellular vesicles from breast cancer cell lines by nano-flow cytometry

Exosomes are nanosized extracellular vesicles that carry bioactive molecules reflective of their cells of origin. Developing methods to detect functional enzymatic activity within exosomes can provide a new generation of rapid and informative diagnostic tools. Aldehyde dehydrogenase (ALDH) enzymes, particularly ALDH1A3, are overexpressed in several cancers and contribute to tumor aggressiveness and drug resistance. However, their presence and functionality in cancer-derived exosomes remain poorly characterized. Here, we developed a nano–flow cytometry–based method to detect ALDH activity directly within individual exosomes derived from breast cancer cell lines (SKBR3, MDA-MB-231, and MCF7). Exosomes were isolated by differential ultracentrifugation and validated by nanoparticle tracking analysis, cryogenic transmission electron microscopy, and bead-based immunophenotyping of canonical markers. ALDH enzymatic activity was detected using a resorufin-based fluorescent substrate capable of crossing the exosomal membrane. To ensure specificity, assays were performed in the presence or absence of a selective ALDH inhibitor, confirming that the fluorescent signal originated from ALDH activity within the vesicles. This work provides the first functional evidence of ALDH1A3 enzymatic activity in cancer-derived exosomes and establishes a proof-of-concept platform for rapid, activity-based detection of exosomal enzymes, opening new perspectives for exosome-based diagnostics in breast cancer.