High-dimensional multiplex immunofluorescence to reveal immune microenvironment remodeling in matched primary and recurrent serous ovarian cancer.

5606 Background: Recurrent serous ovarian cancer is associated with treatment resistance and poor prognosis, and changes in the tumor immune microenvironment (TIME) during recurrence have been suggested to contribute to these clinical features. However, spatial studies that quantitatively compare immune cell composition between primary and recurrent tumors at the single-cell level within the same patients remain limited. This study aimed to compare the TIME of primary and recurrent serous ovarian cancer using high-dimensional multiplex immunofluorescence. Methods: Tissue microarray (TMA) were constructed from formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained from patients with primary and recurrent serous ovarian cancer. High-dimensional multiplex immunofluorescence staining was performed using the Akoya IO60 panel. Single-cell image analysis was conducted to segment cells and classify them into 19 distinct cell types. Differences in cell proportions were evaluated using unpaired analyses across all cores and paired analyses using matched primary-recurrent samples from the same patients (n=35). For paired analyses, cell proportions were compared using paired t-tests. Results: The 19 cell types included tumor epithelial cells, immune cell populations, and stromal cell populations. In unpaired analyses, recurrent tumors showed differences in immune cell composition compared with primary tumors, with relatively higher proportions of T cell-macrophage-associated populations and B cells. In paired analyses of matched samples (n=35), the proportion of T cell-macrophage-associated populations was significantly increased in recurrent tumors compared with primary tumors (p=0.0042). The proportion of B cells was also higher in recurrent tumors (p=0.0463). No statistically significant differences were observed in other cell types. Conclusions: Using high-dimensional multiplex immunofluorescence–based spatial analysis, we compared the tumor immune microenvironment of primary and recurrent serous ovarian cancer at the patient-matched level. Paired analyses demonstrated increased proportions of T cell-macrophage-associated populations and B cells in recurrent tumors, suggesting selective remodeling of the immune microenvironment during disease recurrence.