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The thesis provides insights in reconstruction and analysis pipelines for processing of
three-dimensional cell and vessel images of megakaryopoiesis in intact murine bone.
The images were captured in a Light Sheet Fluorescence Microscope. The work
presented here is part of Collaborative Research Centre (CRC) 688 (project B07) of
the University of Würzburg, performed at the Rudolf-Virchow Center. Despite ongoing
research within the field of megakaryopoiesis, its spatio-temporal pattern of
megakaryopoiesis is largely unknown. Deeper insight to this field is highly desirable to
promote development of new therapeutic strategies for conditions related to
thrombocytopathy as well as thrombocytopenia. The current concept of
megakaryopoiesis is largely based on data from cryosectioning or in vitro studies
indicating the existence of spatial niches within the bone marrow where specific stages
of megakaryopoiesis take place. Since classic imaging of bone sections is typically
limited to selective two-dimensional views and prone to cutting artefacts, imaging of
intact murine bone is highly desired. However, this has its own challenges to meet,
particularly in image reconstruction. Here, I worked on processing pipelines to account
for irregular specimen staining or attenuation as well as the extreme heterogeneity of
megakaryocyte morphology. Specific challenges for imaging and image reconstruction
are tackled and solution strategies as well as remaining limitations are presented and
discussed. Fortunately, modern image processing and segmentation strongly benefits
from continuous advances in hardware as well as software-development. This thesis
exemplifies how a combined effort in biomedicine, computer vision, data processing
and image technology leads to deeper understanding of megakaryopoiesis. Tailored
imaging pipelines significantly helped elucidating that the large megakaryocytes are
broadly distributed throughout the bone marrow facing a surprisingly dense vessel
network. No evidence was found for spatial niches in the bone marrow, eventually
resulting in a revised model of megakaryopoiesis.
Background
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
Methodology/Principal Findings
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors.
Conclusions/Significance
These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.