Salivary exosomal micrornas as effective biomarkers for predicting chemotherapy response in oral squamous cell carcinoma patients

Abstract

Head and neck cancers (HNC), especially oral squamous cell carcinoma (OSCC), pose a significant global health burden, with persistently high mortality rates. Despite advancements in surgical and therapeutic strategies, clinical outcomes remain poor, largely due to resistance to chemotherapy often leading to high recurrence rates. Conventional treatment assessment approaches majorly tissue biopsy, offer static insights and often fail to capture the evolving nature of tumour biology. Therefore, there is an urgent need for non-invasive biomarkers capable of enabling real-time monitoring, and prediction of treatment response in HNC patients. In this context, saliva has emerged as a promising alternative to blood-based liquid biopsies. It is non-invasive, easy to collect, and allows repeated sampling, making it ideal for real-time disease surveillance. Importantly, saliva contains biologically active components such as exosomes, circulating tumour nucleic acids (CTNAs), and proteins that mirror the tumour microenvironment. Among these, salivary exosomes are recognized for their role in intercellular communication and the transport of oncogenic molecules such as microRNAs (miRNAs). This thesis investigates the clinical utility of salivary exosomal miRNAs, focusing on miR-1307-5p, a previously uncharacterized marker, as a biomarker for prognosis and chemoresistance in OSCC. Through small RNA profiling of paired tumour tissues and salivary exosomes from OSCC patients and healthy controls, miR-1307- 5p emerged as consistently and significantly overexpressed in cancerous samples, particularly in the chemo-resistant patient cohort. This observation was further supported by data from The Cancer Genome Atlas (TCGA), which reinforced its association with chemoresistance. Subsequent validation in a larger, independent cohort of chemo-resistant OSCC patients confirmed these findings and further established a strong correlation with advanced tumour stage, lymph node metastasis, and poor overall survival. Further transcriptome analysis revealed that miR-1307-5p plays a pivotal role in tumour progression. It regulates critical genes involved in cell cycle, apoptosis, and metastasis, including THOP1, EHF, RNF4, GET4, and RNF114. To investigate the functional role of miR-1307-5p in mediating chemoresistance, we first assessed its expression in endogenously chemo-resistant CD44⁺ cancer stemlike cells (CSCs) isolated from OSCC cell line. Notably, miR-1307-5p was significantly upregulated in this subpopulation, suggesting a potential role in driving drug resistance. To delineate its functional impact, we performed transfectionmediated suppression of miR-1307-5p in CD44⁺ CSCs. Knockdown of this miRNA led to pronounced G2/M cell cycle arrest, increased apoptosis along with a marked reduction in invasiveness and angiogenic potential. To evaluate whether miR-1307- 5p suppression could enhance cisplatin sensitivity in these chemo-resistant cells, we treated the transfected CSCs with low dose of cisplatin. Remarkably, this combination induced significant cell cycle arrest, increased apoptosis, and reduced cellular invasion, migration, and angiogenesis, indicating a synergistic effect and reversal of the chemo-resistant phenotype. To delineate the molecular mechanisms underlying this sensitization, transcriptome profiling was performed. KRT4 was the only gene consistently dysregulated in both cell line and patient datasets, showing significant downregulation in chemo-resistant OSCC. Its expression was restored upon miR-1307-5p inhibition, implicating KRT4 as a key mediator of chemoresistance and poor prognosis. Following the discovery of miR-1307-5p enrichment in salivary exosomes from OSCC patients, the functional relevance of exosome-mediated delivery was evaluated in comparison to direct transfection-based assays. OSCC cell lines were first transduced with anti-miR-1307-5p, and exosomes encapsulating the inhibitor were subsequently isolated and co-cultured with CD44⁺ CSCs. Exosome-mediated transfer of anti-miR-1307-5p successfully mirrored the outcomes of transfection, confirming that tumour-derived exosomes can drive chemoresistance through intercellular transfer of miR-1307-5p. These findings provide direct functional evidence for the active role of exosomes in modulating therapeutic response within the tumour microenvironment. To validate the translational relevance of in vitro findings, a novel xenograft model of OSCC was established, an in vivo platform not previously reported. In this model, combined suppression of miR-1307-5p and low-dose cisplatin treatment led to significant tumour regression and enhanced therapeutic response, demonstrating the potential to overcome cisplatin resistance in vivo. Notably, these therapeutic effects were also replicated through exosome-mediated delivery of anti-miR-1307-5p, providing a compelling proof-of-concept for exosome-based therapeutic strategies in OSCC. This study identifies miR-1307-5p as a novel, clinically relevant salivary biomarker for predicting chemoresistance in OSCC, through a non-invasive liquid biopsy approach. It is the first comprehensive investigation to link salivary miR-1307-5p with therapeutic failure and to functionally define its molecular role in regulating cancer stemness and drug resistance. These findings offer new avenues for minimally invasive prognostic strategies in OSCC.