Apoptosis-driven microenvironmental conditioning by microvesicles in non-Hodgkin lymphoma

Abstract

Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.