Identification of cell cycle dependent methotrexate resistance in placenta site trophoblastic tumors

Background The purpose is to study the mechanism of chemotherapy resistance in Placental site trophoblastic tumor(PSTT).Methods We established PSTT cell lines by primary culture of a surgically resected PSTT tissues and identified the expression of immune-phenotype markers(HLA-G, β-catenin, CD146, Muc4, hPL, hCG) by immunofluorescence. We measured the IC50 value of methotrexate(MTX), etoposide(VP-16), actinomycin-D(Act-D), cisplatin(DDP), fluorouracil(5-FU) and paclitaxel(TAX) in PSTTs and used a special Mini patient-derived xenograft (Mini PDX) model to evaluate effectiveness of these drugs in vivo. Given that MTX is a cell cycle-dependent chemotherapeutic, we analyzed cell cycle characteristics of PSTT and choriocarcinoma cell lines by flow cytometry and then analyzed RNA profiles and WGS data of the PSTT cell lines to identify the potential mechanism.Results We identified the expression of HLA-G, β-catenin, CD146, hPL and hCG in PSTT cell lines. The IC50 value of MTX was 4.922 mg/ml in PSTT-1, 4.525 mg/ml in PSTT-2, 5.117 mg/ml in PSTT-3, 0.0166 µg/ml in JEG-3 cells (p༜0.001), and 0.01 µg/ml in JAR cells (p༜0.001), with nearly 50,000-fold increase in PSTTs than in choriocarcinoma, indicating that PSTTs are resistant to MTX in vitro. The Mini PDX model revealed that PSTTs are also resistant to MTX in vivo. Cell cycle analysis showed dysregulation of G1/S transition and cell cycle arrest in PSTT cell lines. RNA sequencing profile also identified cell cycle-associated genes which were differentially expressed in PSTT cells than in choriocarcinoma cell.Conclusions We found PSTTs are resistant to MTX in vitro and in vivo compared to choriocarcinoma. Mechanisms could be focused on dysregulation of the G1/S transition and cell cycle arrest.