SG33, a vaccine strain of myxoma virus with oncolytic potential, exploits macropinocytosis and clathrin-mediated endocytosis for entry into pancreatic cancer cells.

Oncolytic viruses are being investigated as therapeutic agents in cancer, yet their mechanisms of entry into tumor cells remain incompletely understood. We previously showed that SG33, a veterinary vaccinal strain derived from a pathogenous myxoma virus, displays oncolytic activity in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we investigated the entry pathways of SG33 into primary PDAC-derived cultures. We found that macropinocytosis, an endocytic process frequently upregulated in PDAC, contributes to SG33 uptake. Moreover, SG33 infection itself induced macropinocytosis in a subset of primary PDAC cultures. Mechanistic studies revealed that phosphatidylserine exposed on the viral envelope promotes SG33 internalization through apoptotic mimicry. In PDAC cultures lacking detectable macropinocytosis, SG33 employed clathrin-mediated endocytosis as an alternative entry route. These findings provide the first insights into the entry mechanisms of SG33 into PDAC-derived cells and indicate that this virus can utilize distinct endocytic pathways depending on the cellular context. ImportancePancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with limited therapeutic options. Oncolytic virotherapy is emerging as a promising strategy to overcome treatment resistance, yet the mechanisms by which candidate viruses enter cancer cells remain poorly defined. Here, we characterize the entry route of SG33, a derivative of myxoma virus with potent oncolytic activity in PDAC models. We show that SG33 exploits multiple endocytic pathways, including macropinocytosis (MPC), induces MPC through apoptotic mimicry, and also uses clathrin-mediated endocytosis (CME). These findings provide the first evidence that SG33 has evolved a flexible cell entry strategy, which could enhance its efficacy in the heterogeneous context of PDAC. Understanding the molecular determinants of viral entry is essential for the rational design of improved oncolytic virotherapies. Our study is aligned with this objective and highlights SG33 as a promising candidate to expand the toolbox of virotherapeutic agents against aggressive cancers.