Chemotherapy-Mediated Purging Before Testicular Organoid Generation Significantly Reduces Cancer Cells While Not Significantly Reducing Germ Cells.

In total, 37-39% of prepubertal patients present with cancer cells in their testes at the time of diagnosing a hematologic malignancy, and elimination of cancer cells from immature testicular tissue (ITT) remains unsuccessful. Treating testicular single-cell suspensions (TCSs) with chemotherapy before generating testicular organoids (TOs) for in vivo maturation after transplantation could be considered to safely restore fertility with cryo-stored ITT. Five different enzymatic digestion protocols (E1 to E5) were compared but did not show significant differences. For subsequent experiments, E3 using 1.4 mg/mL DNase I and 0.5 mg/mL Liberase was chosen based on the highest recovery of viable testicular cells per mg of tissue and lowest enzymatic concentrations. TCSs were contaminated with a human leukemia HL-60 cell line at concentrations of 4 or 10% and exposed for 24 h to cisplatin (1 µM or 3 µM) or carboplatin (10 µM or 30 µM) prior to TO generation and compared to controls. After eighteen days, hematoxylin and eosin staining showed the development of TOs with forming seminiferous tubules (STs) and interstitium in all conditions. Chemotherapy-treated TOs showed altered germ cell (GC) (DDX4+) and somatic (ACTA2+, CYP11A1+) cell-type-specific location. CD43+ HL-60 cells were located outside forming STs in untreated TOs, while in chemotherapy-treated TOs, HL-60 cells were found inside and outside STs. Cancer cell numbers were significantly reduced in 30 µM carboplatin, showing a 3.7-fold reduction, whereas 10 µM carboplatin, 1 µM and 3 µM cisplatin did not demonstrate a statistically significant reduction. DDX4+ GC numbers were not statistically reduced after cisplatin or carboplatin exposure. Chemotherapy did not significantly reduce somatic cell numbers. As substantial residual cancer cell contamination remained, it is unlikely that short-term cisplatin/carboplatin treatment as a stand-alone purging strategy will achieve adequate decontamination for a safe fertility restoration approach. Future research should focus on exploring other chemotherapeutic drugs and/or combinations and elucidating the mechanism of chemotherapy-induced altered TO generation.