Between Dec 19, 2024 and Jan 2, 2025, datasets can be submitted to DRUM but will not be processed until after the break. Staff will not be available to answer email during this period, and will not be able to provide DOIs until after Jan 2. If you are in need of a DOI during this period, consider Dryad or OpenICPSR. Submission responses to the UDC may also be delayed during this time.

Browsing by Subject "Cell Migration"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Capturing Cell Dynamics in Live Pancreatic Adenocarcinoma
    (2020-01) Rodriguez Merced, Nelson
    Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive and lethal cancers and is associated with a robust fibroinflammatory stromal response termed desmoplastic reaction. This stromal response causes the local microenvironment to significantly aid disease progression by providing drug free sanctuaries, immunosuppressive niches, and suppressing cytotoxic T lymphocyte infiltration and distribution, due in part to the increased intra-tumoral pressure and robust extracellular matrix proteins (ECM) density. In order for CD8+ cytotoxic T cells to infiltrate and eliminate cancer cells, they need to migrate efficiently through the dense tumor microenvironment (TME). Thus, altering external (ECM content/architecture) and internal (modulating microtubule (MT) dynamics in immune cell) factors has the potential to enhance efficient infiltration of native or engineered cytotoxic T lymphocytes so they effectively sample the tumor volume to combat disease. Therefore, to analyze the infiltration capabilities in a dense tumor environment, we optimized an approach to culture live tumor slices over 1-4 days in order to perform live cell imaging of carcinoma and immune cell dynamics in complex TMEs with nonlinear optical imaging platforms. From human peripheral blood or tumor-bearing mouse model of PDA, CD4+ or CD8+ cytotoxic T lymphocytes, respectively, were isolated, activated, labeled and later introduced to 3D collagen matrices and live murine PDA tumor slice explants, which has a complex multi-cellular environment and contains elements of the original TME and architecture. Furthermore, we used CRISPR technology to engineer T cells to lack GEF-H1 and alter MT→GEF-H1→RhoA pathway to determine its effect on cell motility. We employed two-photon excitation and second harmonic generation (SHG) imaging to visualize cell dynamics and ECM architecture, and quantify T cell migration behavior through 3D collagen matrices and the native PDA tumor architectures. To test approaches to re-engineer TMEs, we are specifically altering ECM composition and architecture in PDA and quantifying changes in T cell behavior. Thus, combined, these live measures and quantitative analysis will form the basis for our understanding of cell migration in the complex microenvironment and set the mark for our objective to modulate immunity in tumors.
  • Thumbnail Image
    Item
    Distinct developmental functions for cytoplasmic actin isoforms.
    (2010-12) Bunnell, Tina M.
    Actins are among the most highly expressed proteins in eukaryotes and play a critical role in most cellular processes. In mammals there exists six different actin isoforms, of which only the cytoplasmic βcyto- and γcyto-actins are ubiquitously expressed. Remarkably, the cytoplasmic actins differ at only 4 out of 375 amino acids and have been exactly conserved from birds to mammals. It has been postulated that βcyto- and γcytoactin have distinct biological functions; therefore, to test this hypothesis we generated null alleles of the Actb and Actg1 genes. Characterization of the resulting isoformspecific null animals demonstrates that βcyto-actin but not γcyto-actin is essential for embryonic viability. While γcyto-actin is largely dispensable for embryonic development, it does confer growth and survival advantages, as evidenced by the fact that γcyto-actin null embryos exhibited mild developmental delays and decreased postnatal survival Furthermore, γcyto-actin null primary mouse embryonic fibroblasts (MEFs) had a mild growth deficiency and a slight increase in apoptosis, despite total actin levels being maintained. In contrast to γcyto-actin null mice, βcyto-actin null mice were early embryonic lethal, indicating that βcyto-actin is an essential gene required for embryogenesis. The lethality in βcyto-actin null mice is likely due to defects in cell growth and migration as these processes were severely impaired in βcyto-actin knockout primary MEFs. Together, the distinct phenotypes observed in βcyto- and γcyto-actin knockout mice and cells demonstrate that while βcyto- and γcyto-actin can compensate for each other to a limited extent, they also have unique biological functions.