ICIs resistance presents a significant challenge in clinical practice. Circulating extracellular vesicles circRNAs are promising biomarkers for cancer diagnosis and treatment. This study aimed to determine the oncogenic role of circulating extracellular vesicles circ_PPAPDC1A in ICIs resistance diagnosis and prognostic analysis of NSCLC. We used a co-culture system of NSCLC cells and T cells, and a mice xenograft assay, to evaluate the effect of circ_PPAPDC1A on anti-PD-1 antitumor activity. A multicenter cohort study of 40 patients analyzed the associations between extracellular vesicles circ_PPAPDC1A and ICIs resistance. In vitro, circ_PPAPDC1A reduced the sensitivity of NSCLC cells to ICIs by promoting proliferation and inhibiting apoptosis. In vivo, circ_PPAPDC1A promoted NSCLC progression and induced CD8+ T-cell exhaustion. Clinical samples showed circ_PPAPDC1A upregulation in NSCLC patients with ICIs resistance, high diagnostic efficiency (AUC = 0.98), and higher expression associated with shorter PFS. Circulating extracellular vesicles circ_PPAPDC1A may serve as a predictive biomarker for ICIs resistance diagnosis and prognostic analysis.
Non-small cell lung cancer (NSCLC) is currently one of the most prevalent and deadliest malignancies globally. Over 70% of NSCLC patients are already in advanced stages at the time of diagnosis, and first-line chemotherapy regimens based on platinum compounds have an efficacy rate of only 20%-30%, often leading to treatment failure. While the in-depth study of lung cancer gene profiling and the rapid development of targeted therapies have ushered in the era of precise targeted treatment for NSCLC, more than 70% of NSCLC patients with non-driver mutations do not benefit from targeted therapy. In recent years, immune checkpoint inhibitors, with programmed death 1/ligand 1 (PD-1/L1) as a representative, have achieved success in various tumors, including non-small cell lung cancer, significantly improving the five-year survival rate for advanced NSCLC patients from less than 5% to 31.9%. The advent of immune checkpoint inhibitors (ICIs) has brought about a revolutionary change in the diagnosis and treatment and significantly improved the survival rates of advanced NSCLC patients.
However, like other anti-tumor drugs, almost all NSCLC patients who initially respond to ICIs eventually experience disease progression or relapse after a period of immune therapy, leading to the development of ICIs resistance. Currently, there is no unified standard for the definition and classification of ICIs resistance. An expert consensus led by A.J. Schoenfeld divides the diagnostic criteria for ICIs resistance into several components, including treatment type, depth of remission, duration of remission, and treatment continuity, which is the current mainstream definition of ICIs resistance. This definition differs from the SITC definition of immune therapy resistance and is limited to advanced NSCLC. Notably, it differs from the SITC definition in the following ways: 1) Objective remission after ICIs treatment, excluding disease stability; 2) Disease progression within six months after the last ICIs treatment; 3) No need for imaging confirmation of progression after previous remission. Currently, there is a lack of highly sensitive biomarkers for ICIs resistance in clinical practice, primarily relying on imaging examinations. However, imaging examinations often lag significantly behind the actual development of resistance, and the presence of pseudo-progression in tumors renders imaging assessment of ICIs resistance of limited value.
Non-coding RNAs (ncRNAs) are a category of RNA transcribed from the genome but not encoding proteins. They exert biological functions at the post- transcriptional level. Currently, several ncRNAs have been engineered as therapeutics for clinical applications in disease diagnosis and treatment. Circular RNA (circRNA) is a more recently discovered type of ncRNA molecule formed by back-splicing of precursor RNA transcripts, resulting in a covalently closed circular structure lacking the 5'-cap and 3'-poly(A) tail. Importantly, circRNAs are resistant to RNA exonucleases and are not easily degraded. They play a crucial regulatory role in genome stability and the development of tumors. Differing from linear ncRNA molecules like miRNAs, circRNAs exhibit features such as widespread distribution, structural stability, and sequence conservation. Consequently, circRNAs are currently regarded as promising molecular markers and therapeutic targets in the field of cancer diagnosis and treatment.
Extracellular vesicles are a type of vesicular body found in bodily fluids, characterized by a phospholipid bilayer membrane structure, formed through the fusion of cell membranes and multivesicular bodies (MVB). Extracellular vesicles can be released by various cell types, including B cells, T cells, dendritic cells, epithelial cells, stem cells, and tumor cells. Due to their small size, they can effectively evade phagocytosis by monocytes and macrophages. Furthermore, extracellular vesicles can freely traverse vascular barriers and the extracellular matrix, making them widely distributed in bodily fluids such as blood, urine, breast milk, cerebrospinal fluid, and serous cavity fluids. They serve as crucial carriers for intercellular communication, influencing various biological processes and participating in the circulation of biomolecules. Tumor-derived circulating extracellular vesicles are small membrane vesicles released by tumor cells into the extracellular microenvironment, serving as critical mediators for intercellular communication within the tumor microenvironment. circRNA is the primary component carried by tumor-derived extracellular vesicles and serves as the most crucial and efficient vehicle for circRNA intercellular transfer. Furthermore, an increasing body of research has also revealed that tumor-derived extracellular vesicles circRNA exhibits a dual role, acting as both an extracellular vesicles carrier for functional delivery and a regulator of circRNA epigenetic control. As a result, it stands as one of the most promising biomarkers and therapeutic targets in current cancer diagnostics and treatment.
Tumor- derived extracellular vesicles carry a wealth of biomacromolecules, including nucleic acids (DNA, mRNA, ncRNAs, etc.), proteins, lipids, and other biological entities, primarily sourced from parent tumor cells. These molecules are enveloped and protected by the extracellular vehicles (EVs)'s lipid membrane, maintaining their original stability and biological functionality. Among these, circular RNAs (circRNAs) are the principal components carried by tumor-derived extracellular vesicles, representing the most significant and efficient vehicles for circRNA intercellular transfer. Moreover, an increasing body of research has unveiled the dual nature of tumor-derived extracellular vesicles circRNAs, demonstrating their roles in both extracellular vesicles functional transfer and epigenetic regulation. They exhibit high stability, strong specificity, abundant content, non-invasive detectability, and repeatability. As a pivotal constituent of the tumor microenvironment, tumor-derived extracellular vesicles circRNAs play a crucial role in tumor development, progression, and resistance processes. They are currently regarded as the most promising biomarkers and therapeutic targets in the realm of cancer diagnosis and treatment.
Our preliminary study revealed high expression of circ_PPAPDC1A in Osimertinib resistance NSCLC patients, implicating it as one of the significant contributors to Osimertinib resistance. Five NSCLC patients were enrolled in the study, and they underwent second-line standard treatment after developing resistance, which involved ICIs immunotherapy combined with pemetrexed and carboplatin chemotherapy. Clinical practice indicated a negative correlation between circ_PPAPDC1A expression and the response to ICIs. We hypothesized that circ_PPAPDC1A might be associated with ICIs resistance. To validate our hypothesis, we established cell models with circ_PPAPDC1A overexpression and knockdown, along with a co-culture system with T cells to investigate the effect of circ_PPAPDC1A on ICIs sensitivity and its influence on CD8T cells. To further substantiate the potential value of circ_PPAPDC1A as an early diagnostic biomarker for resistance, we initiated a multicenter cohort study to observe the correlation between circulating extracellular vesicles circ_PPAPDC1A and ICIs resistance, as well as its impact on survival prognosis.