Apoptosis-driven microenvironmental conditioning by microvesicles in non-Hodgkin lymphoma
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Date
08/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Patience, Lauren Alexandra
Metadata
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.