Extracellular Vesicles as Mediators of the Adaptive Survival Response to Hypoxia in Pancreatic Cancer Cells.

다국어 초록 (Multilingual Abstract)

Pancreatic cancer is a highly lethal malignancy and molecular mechanisms underlying its pathobiology are not yet well understood. Pancreatic tumors are characterized by a poor vasculature and fibrous stromal tissue network that creates an extensively hypoxic tumor microenvironment (TME). This unique histopathology requires that tumor cells develop adaptive mechanisms to sustain their growth under extremely hypoxic TME. Extracellular vesicles (EV) are released by all eukaryotic cell types and play important roles in normal physiology and disease pathobiology including that of pancreatic cancer. EV carry active biomaterial within them and transfer it from the donor to recipient cells to exert biological consequences. EV are broadly classified into three subtypes that differ in size distribution and marker profiles suggestive of their distinctive biogenesis. Small EV (SEV; 20 – 150 nm) mostly contain exosomes (Exos) originating through endosomal pathway. Medium-sized EV (MEV; 150 nm – 1 µm) are enriched for microvesicles (MVs) originating by pinching off of the plasma membrane. Large EV (LEV; 1 - 3 µm) are usually apoptotic bodies (ABs) derived from fragmentation of the dying cells. In our studies, we tested the hypotheses that hypoxic stress affects the release kinetics, size distribution and marker profiles of EV in pancreatic cancer cells, and EV play important roles in adaptive hypoxic survival of pancreatic cancer cells via transfer of active biomaterial from the cells that had been subjected to hypoxia. Our data demonstrate that under hypoxic conditions, pancreatic cancer cells (MiaPaCa-2 and AsPC-1) shed greater amount of EV with most noticeable changes recorded for the SEV. Moreover, all EV show a shift towards reduced size depending upon the extent of hypoxia. We also observe all size-based sub-fractions have a mixed profile of expression of subtype-specific markers. Thrombospondin-1 and ARF6 are exclusively detected in large and moderate size fractions, respectively, under both normoxia and hypoxia. However, CD9 is detected in both small and moderate size EV under hypoxia and CD63 is detected in all EV under both normoxia and hypoxia. Furthermore, in release kinetics study, we observe increases in accumulation of EV subtypes under hypoxic condition. In addition, our data demonstrate that EV from the hypoxic cancer cells promote survival of cancer cells under hypoxia and SEV is the most active sub-fraction. We also find that SEV from hypoxic pancreatic cancer cells confer alterations in tumor cell glucose metabolism and ROS accumulation. The data demonstrate hypoxic SEV treatment significantly reduces ROS accumulation and enhances glucose uptake and lactate production in pancreatic cancer cells under severe hypoxia as compared to that provided by normoxic SEV treatment. Mechanistically, we demonstrate that the enhanced release and survival-enhancing potency of SEV is dependent on HIF-1α stabilization. Further, EV analysis identifies differential presence of transcripts of key genes associated with glycolytic metabolism and oxidative stress neutralization suggesting a lateral transfer of biological information. Altogether, our findings establish that hypoxia alters the shedding of EV to support adaptive survival of pancreatic cancer cells, and associated differences could possibly be exploited for effective cancer management.