Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Li, Chi

Committee Co-Chair (if applicable)

Clark, Geoffrey

Committee Member

Clark, Geoffrey

Committee Member

Hood, Joshua

Committee Member

Mitchell, Robert

Committee Member

Yaddanapudi, Kavitha

Author's Keywords

Exosomes; embryonic stem cells; immune-stimulatory granulocyte macrophage-colony stimulating factor; prophylactic vaccine; lung cancer

Abstract

The antigenic similarity between embryos and tumors has raised the idea of using embryonic materials as a preventative vaccine against neoplastic disease. Indeed, a previous study reported that a vaccine comprised of allogeneic murine embryonic stem cells (ESCs) and murine fibroblasts expressing immune-stimulatory granulocyte macrophage-colony stimulating factor (GM-CSF) successfully blocked the outgrowth of an implantable lung cancer (Lewis lung carcinoma; LLC) and lung tumors caused by a combination of a mutagen followed by chronic pulmonary inflammation. However, such a vaccine is obviously impractical for application to humans. The use of fibroblasts to produce GM-CSF is needlessly complicated, and intact live ESCs carry the hazard of generating embryomas/teratomas. Here, we report the successful development of an alternative prophylactic vaccine comprised of exosomes/microvesicles derived from murine ESC line ES-D3 engineered to produce GM-CSF. The prophylactic potential of this exosome-based vaccine against implanted lung cancer (subcutaneous inoculation of LLC) was tested by immunizing mice with ESC-derived exosomes bearing GM-CSF (ES-exo/GM-CSF). Vaccination significantly slowed or blocked the outgrowth of LLC without any detectable side effects in liver function, kidney function and blood cell counts, whereas control exosomes lacking GM-CSF were ineffective against LLC. In an implanted lung tumor model, examination of tumor-infiltrating immune cells showed robust tumor-reactive immune responses in mice vaccinated with the exosomes bearing GM-CSF, including an increase in Th1 cytokine responses, CD8+ T effector responses and CD8+ T effector/T regulatory cell ratio. To further evaluate the efficacy of ES-exo/GM-CSF vaccination in preventing lung tumor development, we investigated an experimental metastasis model where syngeneic LLC cells were administered directly into the bloodstream of wild-type C57BL/6 mice through tail vein injection, primarily resulting in pulmonary metastases. Our studies indicate that vaccination with ES-exo/GM-CSF inhibited metastatic growth of lung tumors. Importantly, control exosomes without GM-CSF failed to provide little protection against metastasized pulmonary malignancies. The efficacy of ES-exo/GM-CSF vaccination was associated with a decrease in tumor-promoting T regulatory cells, myeloid derived suppressor cells (MDSCs) and macrophages in tumor infiltrates as well as an increase in cytokine production from intratumoral CD8+ T cells. Since the pluripotency is one of the most specialized properties shared between ESCs and a subset of lung tumor cells, cancer stem cells (CSCs), we investigated the role of pluripotency of murine ESCs in prophylactic effectiveness of ESC-based vaccine. The pluripotency of murine ESCs was significantly reduced when they were differentiated into neuronal cells. The pluripotency of ESCs was essential for their anchorage-independent growth potential. Importantly, differentiation decreased the efficacy of ESCs against the outgrowth of implanted lung tumors. Furthermore, long-term cancer-preventive potential of ESC vaccine was also inhibited by differentiation. In summary, these data indicate the importance of pluripotency of ESCs in their prophylactic efficacy against lung cancer. While ESC-based vaccine has shown great potential, the antigens responsible for its prophylactic efficacy had not been identified yet. Since it is possible that the antibodies generated by ESC vaccine directly bind tumor cells and initiate antibody-dependent cellular cytotoxicity (ADCC), we employed a novel immunoproteomic strategy to gain a deep understanding of antigenicity of ESC-derived vaccine. We examined the comprehensive profiles of antigens specific for the antibodies induced by vaccines based on intact ES-D3 cells or exosomes of ES-D3 cells. Antigens shared by ES-D3 cells and lung tumor cells were identified, among which lung tumor-associated keratin members 8, 16, 17 are candidate antigens responsible for initiating anti-lung tumor immunity through ADCC. Overall, based on the antigenic similarities between embryos and lung tumors, we developed an effective lung cancer vaccine composed of ESC-derived exosomes bearing GM-CSF. This vaccine was very effective in preventing both primary and metastasized lung tumors by evoking tumor-specific immunity. Therefore, we conclude that a similar vaccine derived from GM-CSF-expressing human ESCs may be applicable to humans with increased risk of developing lung cancer, such as long-term smoking history, lung cancer family history, and carcinogen exposure.

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