Supplementary Material for: Tracking Chemotherapy Effects via ALDH1A1, SOX2, CD44v6, and P-gp Expression in Malignant Ascites from High-Grade Serous Carcinoma

Malignant ascites is frequently observed in high-grade serous carcinoma, yet its value as a dynamic source for molecular profiling across treatment stages remains underexplored. The main aim of this study was to evaluate the feasibility of immunocytochemical analysis of malignant ascites samples from patients with high-grade serous carcinoma. A goal was to determine whether marker expression varies before and after chemotherapy and between clinical response groups.
A cohort of 37 malignant ascites samples from 33 high-grade serous carcinoma patients was analysed by immunocytochemistry for ALDH1A1, SOX2, CD44v6, and P-glycoprotein. Full-volume centrifugation of malignant ascites (1–4 L) was used to maximise tumour cell recovery. A biomarker profile was considered positive if ≥1 marker showed >10% tumour cell expression. This profile was significantly more frequent in post-chemotherapy samples (72.2%) than in pre-chemotherapy samples (21.1%; p = 0.003). Stratified analysis revealed increased expression in platinum-sensitive patients following chemotherapy (60% vs. 14.3%, p = 0.032), suggesting chemotherapy-induced reprogramming rather than selection alone. Expression of P-glycoprotein was more frequent in resistant cases, though often limited to <25% of tumour cells. Survival analysis showed no significant difference in overall survival between biomarker-positive patients (p = 0.176).
Our findings suggest that chemotherapy modulates the expression of cancer stem cells and resistance-related markers in high-grade serous carcinoma, potentially contributing to adaptive resistance mechanisms. Malignant ascites, particularly when processed in full, represents a valuable, minimally invasive source of tumour cells for real-time biomarker monitoring. This approach may support early identification of resistance phenotypes and inform personalised therapeutic strategies.