Single-cell RNA analysis reveals unexpected hemocyte plasticity and immune cell specialization in a Drosophila overgrowth model

Summary Immune cells play essential roles in maintaining tissue homeostasis and responding to abnormal growth, but how innate immune cells adapt to chronic apoptotic signaling remains poorly understood. In Drosophila melanogaster, hemocytes, particularly plasmatocytes, are recruited to tumor-like overgrowths, yet their transcriptional diversity and lineage dynamics under these conditions remain undefined. Here, we apply single-cell RNA sequencing to nearly 50,000 circulating and sessile hemocytes from larvae bearing undead overgrown eye discs, a model of regenerative overgrowth driven by sustained caspase activity. We resolve 17 transcriptionally distinct hemocyte clusters, including known lineages and 13 previously unrecognized plasmatocyte subtypes. Interestingly, specific plasmatocyte populations are differentially expanded or depleted under overgrowth conditions. Notably, we identify a matrix-remodeling plasmatocyte population marked by high expression of Jonah-family serine proteases. Pseudotime analysis reveals unexpected plasmatocyte plasticity and two novel terminally differentiated effector states. These findings define the immune landscape of tumor-like overgrowth and establish Drosophila as a platform for dissecting innate immune responses to tissue stress and dysregulated growth in vivo. Competing Interest Statement The authors have declared no competing interest.