Browsing by Subject "Tumor Microenvironment"

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Analyzing CD8+ Cytotoxic T Lymphocyte Migration in The Complex Pancreatic Ductal Adenocarcinoma Tumor Microenvironment
    (2021-05) Zhang, Hongrong
    Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancer types and is estimated to cause more than 432,242 death per year in 2018. Despite increased knowledge in cancer biology and advances in diagnosis tools, most patients were diagnosed at the malignant stage, unsuitable for surgical resection. Moreover, current treatment options like chemotherapy and radiation therapy have limited effect at the malignant stage and only serve as palliative care. Recently, immunotherapy like CAR-T cell therapy has gained great success in some cancer types. To perform the antitumor activities, T cells need to recognize tumor antigens by either establishing contacts with cancerous cells or antigen-presenting cells, which depends on a series of migratory steps from entry to the tumor to the locating of the malignant cells. The dense stroma associated with PDAC creates a great challenge for T cells to effectively migrate in the tumor microenvironment (TME). Also, the mechanism for T cell navigation and migration is not well understood. Here we presented a rapid and cost-effective method to fabricate 3D collagen gel matrix that closely assembles the remodeled extracellular matrix (ECM) in PDAC, allowing high throughput studies. To fully recapitulate the naïve TME, tumor slices from the KPCT genetically engineering PDAC mouse model that shares many of the disease characteristics of human PDAC can be prepared and cultured in an organotypic culture insert for up to six days while maintaining tissue integrity and cell viability. CD8+ can be isolated from tumor-bearing KPC/KPCT mice and activated using Dynabeads T cell activator. Fully activated CD8+ can then be seeded on top of the tumor slice, and attached/infiltrated T cell migration can be monitored using time-lapse imaging utilizing multiphoton laser scanning microscope (MPLSP), and tumor collagen stroma can be virtualized using second harmonic generation (SHG). Additionally, Trackmate was used to track the cytotoxic T lymphocyte migrations in the TME. The 3D speed and motility of the T cells were further analyzed in carcinoma cell-rich and stroma-rich regions of the KPCT TME. Our data suggested that T cell speed and motility both increased significantly in the carcinoma-rich region, where collagen fibers are loosely connected than that of the stroma-rich region. Taken together, the live imaging approach combined with quantitative analysis allows us to gain insight into T cell migration in the complex 3D TME and form new therapeutic approaches.
  • 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
    The Functional Role Of Mir-210 In Hypoxia-Induced Angiogenesis
    (2017-02) Schnettler, Erica
    Hypoxia induced microRNA-210 is implicated in ischemic disorders and in tumor progression. In the present study, we have used a knock out mouse model to investigate how miR-210 regulates angiogenesis. Our studies show that miR-210 sensitizes endothelial cells to bFGF-mediated signaling by targeting FGFRL1 which lacks a receptor kinase domain. FGFRL1 was found to be a negative regulator of bFGF induced pro-angiogenic signaling. In the absence of miR-210, FGFRL1 levels are increased in endothelial cells and as a consequence dampened hypoxia-induced vessel sprouting. miR-210 KO mice showed reduced angiogenesis of FGF-containing matrigel plugs. Furthermore, tumor angiogenesis was attenuated in miR-210 KO animals. These data suggest that miR-210 targets FGFRL1 and sensitizes endothelial cells to bFGF and regulates tumor angiogenesis.
  • Thumbnail Image
    Item
    Targeting the PPARγ and ER pathways via modulation of inflammation in the tumor microenvironment: a novel lung cancer prevention strategy
    (2019-05) Louiselle, Erika
    Lung cancer exceeds all other diagnosed cancers in annual mortality, surpassing the top two annually diagnosed cancers breast and prostate, combined. The vast majority of diagnosed lung cancer cases are in current or former smokers, accounting for 85% of all cases. The number of diagnosed lung cancer cases continues to rise, addressing the need for novel intervention strategies. Despite current advances in chemoprevention for other less-fatal types of cancer, the only currently recognized chemopreventive strategy for lung cancer is smoking cessation. However, former smokers retain a 2.5-fold increased risk of developing lung cancer compared with never smokers, despite cessation efforts. About 40% of all newly diagnosed lung cancers occur in former smokers. As such, chemoprevention strategies for lung cancer are direly needed for the large and ever-growing high-risk population. Preclinical evaluation of existing therapies with established safety and efficacy profiles represents a fruitful opportunity to advance the field. Identification of ERβ expression has been found to be a lucrative method to identify lung cancers that confer poor survival and presents as a potential target for chemopreventive efforts. Preclinical evaluation of anti-estrogens in cell lines and mouse models of lung cancer shows great promise in advancing this class of drugs towards future clinical use in lung cancer prevention. Furthermore, anti-estrogens such as fulvestrant, a complete ER antagonist, have shown anti-tumorigenic activity in lung cancer and others such as tamoxifen have already been successfully implemented in both primary and secondary breast cancer prevention modalities. Preclinical and clinical evidence underpinning the importance of managing ER signaling to control lung cancer initiation and progression, although efficacious, alludes to the potential for increased efficacy when used in combination with other agents. Pioglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARγ) agonist belonging to the thiazolidinediones (TZDs) drug class has also been used in preclinical studies to mitigate lung tumorigenesis, progression, and metastasis after a retrospective analysis found that diabetics using TZDs experienced a 33% reduction in lung cancer incidence. PPARγ has also been implicated as a protective pathway in lung cancer initiation and progression in early phase clinical testing. Patients with improved histology scores demonstrated a link between an increased ER gene signature and positive-response to PPARγ activation, which conferred a chemopreventive effect in dysplasias with a persistent and progressive phenotype. This link presents a unique opportunity to utilize two known mechanisms that are efficacious in protecting against carcinogen-induced lung cancer initiation and progression. Cross-talk between PPARγ signaling and estrogen receptor (ER) signaling has also been previously reported in other cancer models. NNK is a principal carcinogen in cigarette smoke, and along with its ability to induce mutations in oncogenes, NNK can act as an inflammatory mediator of the tumor microenvironment (TME) by promoting macrophage infiltration into the lungs. Furthermore, in previously reported NNK-models of lung cancer, immune cells thought to be macrophages that were positive for both aromatase and estradiol were localized to preneoplastic lesions. Taken together, macrophages are hypothesized to play a key role in regulation of the lung TME both through ER-dependent and independent mechanisms, and both ER and PPARγ pathways are also known to be functional. To develop a novel approach to prevent lung cancer, preclinical studies were developed to evaluate the therapeutic potential and chemopreventive capabilities of two FDA-approved agents, pioglitazone and fulvestrant, re-purposed in a lung cancer tumor microenvironment (TME) in vitro model and an NNK-induced adenocarcinoma chemoprevention in vivo model. To test pioglitazone and fulvestrant in a preclinical model simulating the lung TME, we selected a human NSCLC adenocarcinoma cell line with a similar KRAS mutational signature found in smoking-induced lung cancer and a human immortalized macrophage cell line. Additionally, we tested the effects of pioglitazone and fulvestrant in a murine primary cell culture model, utilizing mouse-derived adenocarcinoma cells immortalized from NNK-induced in vivo tumors and primary murine bone marrow-derived macrophages (BMDMs).
  • Thumbnail Image
    Item
    Targeting the Tumor Stroma Using a Monoclonal Antibody Platform Technology
    (2021-04) Hintz, Hallie
    1 in 9 men will be diagnosed with prostate cancer during their lifetime and it remains the second leading cause of cancer death among American men. Men who fail standard-of-care androgen deprivation therapy (ADT) and progress to metastatic castration resistant prostate cancer (mCRPC) are left with few therapeutic options. Current second line therapies only provide a small survival benefit and there is a critical unmet need for new and innovative approaches to treat mCRPC. Imaging is a crucial aspect of mCRPC clinical management used for the detection of recurrent or distant disease. The development of new therapies is also dependent on accurate imaging modalities for patient staging and evaluating treatment response. Our research shows fibroblast activation protein alpha (FAP) is a relevant target for imaging and treating mCRPC. FAP is emerging as the next pan-cancer target given its upregulated expression in cancer associated fibroblasts (CAFs) and localization to the tumor microenvironment. Here we document the discovery and validation of a monoclonal antibody that selectively binds to FAP. The lead antibody, B12, was identified from a naïve murine single-chain variable fragment antibody phage display library screened against recombinant human FAP. The heavy and light chains of B12 were cloned into full-length human immunoglobulin 1 vectors and expressed as a chimeric monoclonal antibody (B12 mAb). B12 mAb was shown to detect FAP expression in cell lines and was rapidly internalized by FAP-expressing cells in vitro. B12 mAb demonstrated cross-reactivity with murine FAP, but not with the highly homologous protease human dipeptidyl peptidase IV. PET/CT imaging with [89Zr]Zr-B12 mAb demonstrated high tumor uptake and long-term retention of the probe in several preclinical animal models. Furthermore, we show its superiority to other clinically investigated imaging probes which suggests clinical translation of B12 mAb as a non-invasive mCRPC imaging probe. Next, we evaluated the therapeutic potential of B12 mAb as an antibody-dependent cell-mediated cytotoxicity (ADCC) inducing agent in combination with an engineered NK-92MI CD64 cell therapy. The immunotherapy demonstrated selective cytotoxicity in vitro and treatment effectively controlled tumor growth in an animal model. Furthermore, we engineered B12 mAb as an antibody-drug conjugate (ADC) and showed cytotoxic effect in several in vitro and in vivo solid tumor models. Overall, this research represents a platform technology for the development of theranostics targeting FAP that could provide urgently needed therapies and imaging probes for mCRPC patients.