HOW MICROVESICLES ARE "BUDDING" INTO THE CANCER CONVERSATION: THE ROLE OF GLYCOCALYX-INDUCED MEMBRANE BENDING

Abstract

Cancer intercellular communication has been recognized to occur by direct cell-to-cell contact as well as local paracrine signaling between cancer and normal cells. However, tumor-derived extracellular vesicles (EVs), which are lipid bilayer vesicles released from cancer cells, can travel long distances in the body and cytosolically deliver oncogenic cargos, such as DNA, RNA, and proteins, to normal cells and contribute to tumor progression and metastases. Microvesicles, a specific EV subtype, directly shed from the plasma membrane, but how this occurs is not fully elucidated. To help address this, there is a need for consistent convention and methodology to clearly differentiate and fully characterize subtypes of EVs to understand their contributions in cancer and what direct cancer-related paradigms impact their expression. Here, we assess a range of techniques commonly used for detection of EVs to confirm reliable methods for characterization of vesicles. We combined scanning electron microscopy and cryo-transmission electron microscopy for visualization of EVs along with nanoparticle tracking analysis for diameter distributions and quantification. We found these methods to be reliable in characterizing and differentiating EV subtypes. We then used these techniques along with our genetically encoded toolbox of synthetic to native glycocalyx biopolymers to understand the role of the glycocalyx in membrane bending and microvesicle biogenesis. We demonstrate that molecular crowding on the plasma membrane by flexible glycocalyx biopolymers allows membrane bending to occur. Moreover, in coordination with the actin cytoskeleton, glycocalyx biopolymers induce membrane bending required for generation of shapes, including tubules and pearling instabilities that are consistent with microvesicles. Next, we explored properties that were found to be important for inducing membrane bending and MV shedding. Particularly, we found that the MV shedding was dependent on the flexible polymer domain. Further, we investigated whether extracellular cues such as physical confinement modulates MV shedding. Ultimately, these studies highlight the glycocalyx from a biophysical perspective in understanding microvesicle biogenesis in cancer. Thus, these key insights can potentially lead to new targeted therapeutic approaches in the mitigation of cancer metastasis.