Drug-resistant extracellular vesicles: a reliable biomarker for predicting therapeutic response in patients with triple-negative breast cancer receiving neoadjuvant chemotherapy

Background: Predicting tumor response for neoadjuvant chemotherapy (NAC) is critical for evaluating prognosis and deciding the treatment strategy in patients with breast cancer; however, there are no reliable circulating biomarkers that can assess therapeutic responses appropriately and effectively. Therefore, we aimed to validate the clinical feasibility of extracellular vesicles (EV) collected through liquid biopsy as biomarkers for predicting tumor response after NAC. Methods: : Drug-resistant sublines were generated from a panel of triple-negative breast cancer (TNBC) cell lines, including HCC1395, MDA-MB-231, and MDA-MB-468. Comparison with parental cells suggested candidate biomarkers to detect resistance to chemotherapeutic drugs. EVs were isolated from cultured cells and plasma samples collected before NAC from breast cancer patients (N=36). The EVs were then coupled to immuno-beads to allow the quantification of drug resistance-related EV markers by flow cytometry. Results: : Drug-resistant TNBC cell lines were stably established showing specific morphology and rapidly growing features. Among the differentially expressed gene profiles between parental and drug-resistant cell lines, drug efflux transporters, such as multidrug resistance protein 1 (MDR1), MDR-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) were highly expressed in resistant cell lines. Drug efflux transporters were identified in not only cell lines but also EVs released from parental cells with immuno-affinity-based EV isolation. Among 20 TNBC patients, the expression of drug-resistant markers in EVs from the patients with the residual disease was relatively high compared to that of patients with pathological complete response (pCR). The optimal combination of drug-resistant EV markers was significantly efficient in predicting patients showing resistance to NAC and represented the best performance with 93% sensitivity, 80% specificity, and 0.93 area under the receiver operating characteristic curve. Conclusions: : The combination of drug-resistant EV markers is effective in predicting the therapeutic response of breast cancer patients who are treated with NAC.