Date of Award

13-12-2024

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.K.Uma Maheswari

Keywords

Lung Cancer, Chemotherapy, Molecular Targeted Therapy, Mortality, Nanotechnology, Gene silencing, Immunotherapy, Nano-geno-immunotherapy, Research hypothesis, Research Objective

Abstract

Lung cancer ranks among the top causes of cancer-related deaths worldwide. The deteriorating environment and unhealthy lifestyle in the modern era has further contributed to the exponential increase in lung cancer incidence across the globe. Lung cancers fall under two major types based on the location of the tumour in the lung namely, small-cell lung cancer (SCLC) which is more aggressive, and non-small-cell lung cancer (NSCLC) which is more prevalent. Late diagnosis has been a major cause for poor prognosis as the cancer is detected only in an advanced stage.

Conventional therapies like chemotherapy, surgery and radiotherapy against lung cancer are limited by their adverse effects and inability to completely destroy the cancer cells at advanced stages. Additional barriers for therapeutic interventions are presented by the tumour which reprogrammes its microenvironment through recruiting immune cells and fibroblasts that transform into sentinels protecting the tumour cells and suppressing cytotoxic effects of immune cells and therapeutic agents leading to the poor therapeutic outcomes.

In recent years, molecular modulation strategies have gained momentum to overcome the barriers presented by the formidable tumour microenvironment and halting the tumour progression. The present study adopts a multi-modal approach integrating the beneficial properties of gene silencing, immunotherapy, and nanotechnology. One of the key players in this reprogramming is the vascular endothelial growth factor (VEGF) which plays a multi-faceted role in tumour progression and metastasis. We have chosen to partially silence this gene in the tumour cells using a polyplex formed from the biocompatible poly(1-vinylimidazole) with anti-VEGF si-RNA.

Our in vitro studies revealed that the polyplex exhibited superior internalization, endosomal escape and VEGF silencing ability when compared to the free si-RNA in both human derived A549 lung carcinoma cells and mouse derived LL2/LLC Lewis lung carcinoma cells. The polyplex also was capable of rapid internalization into 3D tumour spheroids developed using the overlay method with A549 cells and Matrigel® and silence the target gene. VEGF silencing inhibited the migration of cancer cells and sensitized cancer cells to chemotherapeutic agents. Co-culture studies with Jurkat T-lymphocytes revealed that VEGF silencing enhanced proliferation of Jurkat cells while suppressing cancer cell numbers indicating that VEGF silencing could significantly improve the outcomes of immunotherapy.

We attempted to harness the cytotoxicity of the cell-based immune system to destroy cancer cells. To help the immune cells distinguish cancer cells from normal cells, we employed chitosan nanoparticles conjugated with a MUC1 peptide as a synthetic antigen-presenting entity to trigger the immune response against cancer cells. MUC1 is over-expressed in lung cancer cells and hence we used a 25-mer peptide fragment from MUC1 which could serve as an antigenic epitope to prime the immune cells.

Since, cytotoxicity of the immune cells cannot be realized in the immune-suppressive tumor microenvironment, we used a temporally regulated approach by first silencing the VEGF using the polyplex followed by administration of the MUC1-tagged chitosan nanoparticles. Co-culture studies performed with mouse lung carcinoma cells and mouse macrophages revealed that the multi-modal combination of VEGF silencing and MUC1 presentation activated the M1 phenotype of macrophages and inhibited the epithelial-to-mesenchymal transition of lung cancer cells apart from causing a marked reduction in the tumour cell numbers. In vivo studies performed in an orthotopic lung cancer model in immune-competent C57BL/6 mice confirmed that the combination therapy reduced tumour progression as well as metastasis and restored the normal lung architecture. The VEGF silencing was accompanied by reduction in HIF-1, IL-10, IL-6 and TNF- clearly indicating that this nano-geno-immunotherapy could be a promising therapeutic option for treatment of lung cancers.

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