Non-small cell lung cancer (NSCLC) comprises over 80 % of all newly diagnosed lung cancer cases globally, and its frequency has remained high over the last two decades [1,2]. NSCLC has a poor prognosis, with more than 70 % of patients diagnosed at late stages and a 5-year overall survival rate of below 3 % [3].

Current NSCLC treatment options include a surgical excision, chemo, radio-, and targeted therapies for driver mutations (e.g., EGFR, ALK), and immunotherapy, especially immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 or CTLA-4 [4]. Although these techniques contributed to better outcomes for patients, substantial limits remain. Chemotherapy and radiation frequently cause systemic damage and are unable to prevent recurrence [5]. Targeted therapies function exclusively in those who carry certain genetic mutations and are commonly impeded by acquired resistance [6]. Despite their revolutionary nature, ICIs serve only a subset of patients, with many developing primary or acquired resistance. A crucial underlying process of such resistance is the severely immunosuppressive tumor microenvironment (TME), which has been defined by the increase of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), M2-polarized macrophages, and the expression of various ICIs [7,8]. This immunosuppressive microenvironment reduces cytotoxic T lymphocyte (CTL) function and promotes immunological escape, emphasizing the necessity for innovative therapies focused on remodeling the TME to restore efficient anti-tumor immunity [9]. One approach is to investigate particular genetic factors and signaling pathways involved in tumor progression and immunomodulation [10]. These approaches may provide new therapeutic targets and methods for improving NSCLC outcomes.

MicroRNAs (miRNAs) have been identified as important regulators in tumor biology [11]. These highly conserved, small non-coding RNAs, which are around 20–25 nucleotides long, mainly regulate gene expression by binding to the 3′ untranslated region (3’ UTR) of target mRNAs, resulting in either mRNA destruction or translational inhibition [12]. several studies have shown that miRNAs regulate tumor formation via post-transcriptionally altering oncogenes or tumor suppressor genes [13]. As a result of their dual involvement in driving or preventing tumor growth, miRNAs have emerged as promising cancer therapeutic targets.

Among them, the miR-146 family, which includes miR-146a and miR-146b in humans, has received much interest for its role in cancer development and invasion [14]. Increased or reduced miR-146a expression has been linked to carcinogenesis and metastatic potential in a variety of malignancies. In pancreatic, colorectal, and prostate cancers, miR-146a decreases epithelial-mesenchymal transition (EMT), which is a crucial driver of cancer cell migration and metastasis [[15], [16], [17]]. In NSCLC cell lines and patient tissue specimens, miR-146a downregulation or polymorphisms are linked to major lung cancer characteristics such as uncontrolled proliferation, resistance to apoptosis, increased migration, metastasis, and altered energy metabolism [[18], [19], [20], [21]]. In addition to its tumor-specific functions, miR-146a plays an important role in maintaining the balance of inflammatory pathways and innate immunity by targeting IRAK1 and TRAF6, crucial downstream factors in the IL-1 and TNF signaling cascades [22]. MiR-146a suppresses the NF-κB pathway, limiting pro-inflammatory cytokine release and cell proliferation while increasing apoptosis [23]. MiR-146a's inflammatory regulatory activities are mediated by targets directly of the oncogenic pathway, including STAT3 and MYD88, resulting in decreased cell growth, increased apoptosis, and lowered metastasis [24,25]. MiR-146a also has a major function in immune modulation by targeting PD-L1 on cancer cells [26] and crucial adaptor proteins like IRAK1 and TRAF6 in immune cell types, thereby reducing elevated inflammation and reducing immunosuppressive mechanisms [27].

Furthermore, miR-146a controls T cell-mediated immune responses by functioning as an autonomous regulator within T cells, influencing proliferation and function [28]. MiR-146a levels are elevated in CD4+ and CD8+ T lymphocytes following inflammatory and viral stimuli [29]; however, the exact mechanism of this induction is unknown.

Recent research has identified extracellular vesicles, particularly Extracellular vesicles (EVs), as important mediators of intercellular communication in cancer [30]. EVs, especially exosomes (30–150 nm), have been identified as important intercellular interaction mediators in the TME. Tumor-derived exosomes (TEX) perform a paradoxical dual function. On the one hand, they enhance tumor development and immunosuppression by delivering tumor-associated proteins, lipids, and nucleic acids to the target cells, allowing for metastasis, invasion, and the training of stromal and immune cells to develop a tumor-promoting phenotype. On the other hand, this endogenous intercellular transport pathway provides a novel treatment option [31]. Exosomes are being developed as complex drug delivery systems by leveraging their innate biocompatibility, low immunogenicity, circulatory stability, and natural tropism toward parent or comparable tumor cells [32]. Exosomes have better biological membrane properties than synthetic nanoparticles, which improves tissue penetration and reduces removal [33]. However, the translation of nanomaterials from basic research to clinical applications in the biomedical arena presents a number of complex hurdles and future perspectives [34]. For exosomes, obstacles remain in standardizing isolation procedures, scaling up manufacturing, and accurately controlling cargo loading [35]. Recent improvements in exosome engineering aim to maximize their delivery capacity while minimizing their innate pro-tumorigenic cargo, establishing them as prospective nanocarriers for cancer treatment. Their intrinsic affinity for tumor tissues increases the efficacy of targeted miRNA delivery.

Given the importance of miR-146a in NSCLC and the potential of exosome-mediated delivery, we thus propose that carrying miR-146a through TEX will function as a native tumor-resident delivery system to enable dual-targeting of the MYD88/STAT3 oncogenic axis and the PD-L1 immunosuppressive axis, thus reverting malignant phenotype and restoring anti-tumor immunity in NSCLC. We assessed the efficacy of miR-146a-loaded tumor exosomes (TEXmiR-146a) as a potential cell-free treatment strategy for inhibiting NSCLC cell proliferation, migration, and invasion while also enhancing antitumor immune responses.