More than 18 percent of all cancer deaths are caused by lung cancer, which continues to be the primary cause of cancer-related deaths globally (Lung Cancer, n.d.). With five-year survival rates of only 15 % (and 9 % in the last stage which accounts for 43 % of the cases), the overall survival rate for patients with lung cancer is still appallingly low, despite significant advancements in early identification and treatment (Lung Cancer Statistics | How Common Is Lung Cancer?, n.d.). The primary cause of the high death rate is that lung cancer is detected in late stages when it has spread, and treatment choices are limited. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the two main kinds of lung cancer; NSCLC makes up about 87 % of all cases (Ibodeng et al., 2023). Anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma virus (KRAS) and epidermal growth factor receptor (EGFR) mutations are prevalent among NSCLC subtypes, and therapies that target these mutations, such as tyrosine kinase inhibitors (TKIs), have shown promise (Dalurzo et al., 2021). However, a major obstacle to treating lung cancer is resistance to these treatments, especially in patients with EGFR mutations (L858R or exon 19 deletion) (Laface et al., 2023). Furthermore, the mainstay of lung cancer treatment for many years, chemotherapy, is frequently constrained by serious side effects and the emergence of drug resistance (Anand et al., 2023). Immunotherapy, especially the use of immune checkpoint inhibitors such as PD-1/PD-L1 inhibitors, has transformed the treatment of cancer in recent years; yet, its effectiveness is not universal, and many patients either do not respond or develop resistance (Borgeaud et al., 2023). Therefore, novel therapeutic strategies that combine different therapeutic modalities, such as chemotherapy, immunotherapy, and gene therapy, are critically needed to enhance treatment efficacy and overcome resistance in lung cancer.

The application of microRNAs (miRNAs) in cancer treatment is one such promising strategy. By attaching to complementary sequences in messenger RNAs (mRNAs), miRNAs—small, non-coding RNA molecules—control gene expression post-transcriptionally by causing mRNA degradation or translation inhibition (Ling et al., 2013, Shah et al., 2016). Numerous physiological functions, such as immune response, differentiation, apoptosis, and cell cycle regulation, are influenced by miRNAs. Depending on the genes they target, miRNAs in cancer can either act as tumor suppressors or oncogenes. Since miRNAs have been demonstrated to control important pathways involved in carcinogenesis, metastasis, and resistance to traditional therapies, their therapeutic potential in the treatment of cancer has attracted a lot of attention recently. For example, it has been demonstrated that tumor-suppressive miRNAs, including miR-34a, miR-200c, and miR-145, can stop tumor growth by focusing on important oncogenes and pathways that support the growth, survival, and metastasis of cancer cells (Gao et al., 2024, Mansoori et al., 2021, Sawant and Lilly, 2020). The efficient distribution of miRNAs to target cells is one of the main obstacles in miRNA-based treatments, though. Because miRNAs are unstable in biological fluids, a dependable and effective delivery mechanism is necessary for systemic administration to prevent degradation and guarantee the desired effect.

Extracellular vesicles (EVs), especially exosomes, may encapsulate and shield nucleic acids, including miRNAs from deterioration, and hence may become viable delivery systems for miRNAs (Nathani et al., 2024a, O’Brien et al., 2020). Numerous cell types, including immune cells, cancer cells, and stem cells, produce exosomes, which are tiny (30–150 nm) membrane-bound vesicles. By transporting goods between cells, including proteins, lipids, and nucleic acids, they contribute significantly to intercellular communication (Lee et al., 2024). Further, exosomes may be designed to contain therapeutic cargo, such as miRNAs, and since they naturally can target particular cells and penetrate biological barriers like the blood–brain barrier, they are particularly of interest in the context of cancer therapy (J. Li et al., 2025, Nathani et al., 2024b, Sharma and Mukhopadhyay, 2024, Srivastava et al., 2022). By directly eliminating tumor and virus-infected cells and producing interferon-γ (IFN-γ) to boost immunological responses, NK cells are essential for immune surveillance (Paul & Lal, 2017; R. Wang et al., 2012). They employ processes such as the release of TNF-α, perforin, granzymes, and Fas ligand (FasL) to destroy target cells, and they use a variety of receptors to distinguish between healthy and diseased cells (Prager and Watzl, 2019, Ramírez-Labrada et al., 2022). Although the precise components, such as proteins and miRNAs, responsible for their anticancer activity are still being studied, NK cell-derived extracellular vesicles (NK-EVs) retain many of the cytotoxic qualities of their parent cells, such as the capacity to induce apoptosis in tumor cells through the release of perforin and granzymes (Wu et al., 2021). This makes NK-EVs an appealing platform for cancer immunotherapy. Although cytokine stimulation, especially IL-15, increases NK cell cytotoxicity by triggering signaling pathways such as JAK, PI3K, and MEK, it is yet unknown how cytokines affect NK-EVs and target cell uptake (Nandagopal et al., 2014). It might be possible to boost NK-EVs' anticancer effects, and overcome multidrug resistance, and increase the effectiveness of current treatments like immunotherapy and chemotherapy by loading them with tumor-suppressive miRNAs.

In addition to their potential as miRNA carriers, NK-EVs may improve the efficacy of conventional chemotherapy medications. Advanced non-small cell lung cancer is frequently treated with Carboplatin, a platinum-based chemotherapy drug (Rossi & Di Maio, 2016). However, Carboplatin's harmful side effects and resistance development frequently restrict its usefulness (Zhang et al., 2022a). Carboplatin’s effectiveness may be increased, and side effects may be decreased by combining it with other therapeutic approaches like immunotherapy or miRNA-based therapy. These miRNAs may be loaded into NK-EVs and combined with Carboplatin to produce a synergistic impact that slows tumor development and makes tumor cells more sensitive to chemotherapy, thereby overcoming resistance mechanisms and enhancing patient outcomes.

The goal of this study is to determine whether NK-EVs loaded with miRNAs that target PD-L1 and FOXM1 in conjunction with Carboplatin may be used to treat lung cancer that is resistant to Osimertinib. Patients with EGFR-mutant non-small cell lung cancer, especially those who have grown resistant to first- and second-generation TKIs, are frequently treated with Osimertinib, a third-generation EGFR-TKI (Laface et al., 2023, Santarpia et al., 2017). However, Osimertinib resistance can potentially arise, making therapy choices even more challenging (Gomatou et al., 2023, Leonetti et al., 2019). Using patient-derived xenograft (PDX) and H1975R models of lung cancer, this study intends to assess the therapeutic efficacy of this novel combination strategy in vitro and in vivo by targeting important genes involved in immune evasion and chemoresistance, such as PD-L1 and FOXM1, and combining this approach with Carboplatin. It is hypothesized that miRNA delivery using NK-EVs will improve treatment outcomes, overcome resistance, and increase lung cancer cells' sensitivity to Carboplatin. To gain a better understanding of how this combination therapy works against cancer, the study will also investigate the underlying molecular mechanisms of action, such as the alteration of immunological checkpoints, tumor growth markers, and apoptotic pathways.