Browsing by Subject "multiphoton microscopy"
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Item Pathway to metastasis: carcinoma dissemination via organized collagen tracks(2017-12) Ray, ArjaA vital part of the metastatic cascade that leads to cancer-related deaths is the initial dissemination of cancer cells from a confined lesion into neighboring tissue by migration and invasion. Breast and pancreatic carcinomas are often associated with increased deposition of collagen-I, which can assemble into aligned fiber tracks in mature breast tumors. Such aligned tracks provide contact guidance cues for directed cancer cell migration and dissemination leading to increased invasion, metastasis and decreased disease-free survival in breast cancer patients. Using multiphoton imaging, we demonstrate that organized collagen architectures develop in the pancreatic ductal adenocarcinoma (PDA) stroma even at the pre-invasive stage with single cells and multicellular clusters interacting with aligned collagen tracks in vivo. Mimicking the collagen patterns with microfabricated substrates, we show that for single cells, aligned architectures induce constrained focal adhesion maturation and associated F-actin alignment, consequently orchestrating anisotropic traction stresses that drive cell orientation and directional migration. While such interactions allow single mesenchymal-like cells to spontaneously “sense” and follow topographic alignment, intercellular interactions within epithelial clusters counteract anisotropic cell-substratum forces, resulting in substantially lower directional response. Indeed, anisotropic cell-substratum interactions from organized periductal collagen may contribute to cell extrusion and dissemination from pre-invasive ductal epithelia in PDA. Such contact guided spreading of cancer cells may be inhibited by dismantling the fiber architecture, diminishing its density or by abrogating cell-ECM interactions. To validate our findings in 3D we engineered novel in vitro substrates using a simple method to align 3D collagen gels by guided cellular compaction, to produce highly aligned, acellular collagen constructs as a controlled microenvironment in vitro. Additionally, we integrated the aligned collagen matrices to cell dense tumor-like plugs, allowing tracking of the temporal evolution of the advancing invasion fronts over several days. Live cell imaging and analysis of 3D cell migration revealed profoundly enhanced motility in aligned collagen matrices for the putative cancer stem cell subpopulation. Heterogeneity in cell migration behavior was also observed between cells at the leading edge and those within the tumor boundary, thus demonstrating the versatility of these platforms in capturing the dynamics of contact guided carcinoma dissemination.Item Preclinical Dynamic Contrast Enhanced Imaging for Longitudinal Biophysical Assessments of the Healthy and Malignant Vasculature after Radiotherapy(2021-06) Brooks, JamisonDynamic contrast enhanced (DCE) imaging acquires time-lapsed images of intravenously injected contrast agents to quantify changes in their delivery to and clearance from the tissue. It is widely used in clinical imaging to assess tissue perfusion and vascular permeability. However, minimal work has been done using DCE in a murine model to better understand the effects of radiotherapy on the vasculature and tissue. This is due in part to a lack of DCE imaging systems with the resolution to directly observe the underlying changes in single-vessel structure and function. In this thesis, I develop and use macroscopic dynamic fluorescent imaging (DynFI) and microscopic quantitative multiphoton microscopy (QMPM). These DCE techniques identify changes in tissue perfusion several days after radiotherapy in the healthy bone marrow, leukemic bone marrow, and solid tumor tissue. DCE imaging can be performed using QMPM while simultaneously observing changes in the single-vessel characteristics for direct correlation. Using QMPM, changes in bone marrow vasculature were observed with the onset of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Changes included increased angiogenesis, permeability, decreased mean vessel diameter, decreased single-vessel blood flow, and decreased drug delivery. For mice bearing ALL, this resulted in reduced cellular uptake of a chemotherapy surrogate. The results identified potential biomarkers for leukemia using DCE imaging. Treatments ranging from 2 Gy to 10 Gy of total body irradiation (TBI) increased single-vessel blood flow and drug delivery for mice bearing AML and ALL 2 to 5 days after TBI. For mice bearing ALL, targeted 2 and 4 Gy radiotherapy resulted in improved cellular chemotherapy uptake. Additionally, increased survival was observed for mice bearing ALL when administering 4Gy TBI before chemotherapy compared to chemotherapy before 4Gy TBI. Finally, I develop DynFI with principal component analysis to rapidly identify changes in tumor vasculature before and 2 days after 10 Gy targeted radiotherapy. In conclusion, DCE analysis is an effective tool to measure tissue perfusion and vascular function. Increased availability of DCE imaging for preclinical models will allow for a better understanding of the underlying vascular changes that occur in a clinical setting.