PMC8262245_01
Patient's history. The patient underwent resection without adjuvant treatment (Tx). Due to retroperitoneal recurrence, chemotherapy was initiated followed by surgery and adjuvant radiation therapy (RTx). However, the tumor progressed leading to pleural effusion justifying laparotomy and video-assisted thoracoscopy. At that time we established the pleural-effusion derived tumor cell line PF338. *retroperitoneal recurrence; **tumor infiltrate; ***transdiaphragmatic spread and pleural effusion.
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A 59-year-old female patient was diagnosed with UCS and underwent radical resection yielding a pT3aN0M0 FIGO IIIA tumor containing both a dominant stromal sarcomatous and a focal endometroid carcinomatous component (Figure 1). To confirm the origin of the metastatic tumor, we demonstrated that tumor cells were, although only in foci, positive for CD10, a marker for Mullerian system-derived neoplastic mesenchymal cells (Supplementary Figure S1A). S1A). Stainings for estrogen- and progesterone-receptor (ER, PR) indicated only heterogeneous nuclear expression patterns (Supplementary Figure S1A). Congruent to the metastatic tissue, immunohistochemical stainings of the cell block indicated focal positivity for CD10, strong positivity for vimentin and negative staining for E-cadherin (Figure 2A; Supplementary Figure S1B).
Histopathological and in vitro characterization (A) Hematoxylin and eosin (H & E) and immunohistochemical stainings of the primary tumor, the recurrent tumor and of PF338 tumor cells. Whereas the primary tumor expressed both E-cadherin and vimentin, E-cadherin expression was lost in both the metastatic lesion and the cell line (B) PF338 tumor cell growth in vitro.
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To compare the primary tumor lesion at diagnosis with the metastatic tumor we performed immunohistochemical analyses (Figure 2A). Congruent to the metastatic tissue, immunohistochemical stainings of the cell block indicated focal positivity for CD10, strong positivity for vimentin and negative staining for E-cadherin (Figure 2A; Supplementary Figure S1B). In vitro, PF338 tumor cells demonstrated a biphasic growth pattern consisting of an epithelial-like component growing in a monolayer and a mesenchymal-like component growing in multiple layers (Figure 2B; Supplementary Video S1). Despite being sensitive to cisplatin, a significant fraction of cells (15%) remained viable when treated with cisplatin at IC50 concentrations for 10 days (Figure 3D; Supplementary Figure S2).
PF338 tumor cells are sensitive to both cisplatin and paclitaxel (A) Cisplatin reduced the mesenchymal-like cell fraction of PF338 cells (B) Cells were sensitive to cisplatin (IC50: 0.96 microM) and paclitaxel (IC50: 3.81 nM) (C) Cisplatin induced G2/M arrest and paclitaxel apoptosis (D) Tumor cells could still form colonies when treated with IC50 concentrations of cisplatin. Error bars = means +- SE from three repeats. C/D, control, *p < 0.05, **p < 0.01.
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IC50 values of 0.96 microM and 3.81 nM, respectively (Figures 3A,B). Interestingly, whereas cisplatin induced morphology changes to a more uniform, flat phenotype, paclitaxel treatment did not interfere with morphology (Figure 3A). Cell cycle analyses for cisplatin revealed a dose-dependent G2/M arrest, whereas paclitaxel induced apoptosis in a dose-dependent manner (Figure 3C). Despite being sensitive to cisplatin, a significant fraction of cells (15%) remained viable when treated with cisplatin at IC50 concentrations for 10 days (Figure 3D; Supplementary Figure S2). Importantly, phenotypic and expression changes were also observed upon treatment with high-dose SAHA (4 microM) or valproate (Supplementary Figures S3A-D).
PF338 tumor cells are resistant to MEK and TGF-betaRI-kinase inhibition but sensitive to PI3K inhibition (A) selumetinib (Selu), galunisertib (Galu) and BEZ235 (BEZ) did not affect PF338 cell morphology (B) PF338 cells were resistant to selumetinib (A375 cells were used as control) and galunisertib, but sensitive to BEZ235 (IC50: 63.42 nM) (C) BEZ235 induced a modest G0/G1 arrest (D) Galunisertib reduced pSMAD2 expression. Error bars = means +- SE from three repeats. C/D, control, ****p < 0.0001.
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PI3K/mTOR inhibitor BEZ235 due to the mutations in the PI3K pathway and also treated the cells with galunisertib, a TGF-betaRI-kinase inhibitor. PF338 cells were resistant to both selumetinib and galunisertib but strongly sensitive to PI3K/mTOR inhibition (IC50: 63.42 nM) (Figures 4A,B). Cell cycle analyses revealed no changes upon selumetinib or galunisertib treatment and only a modest dose-dependent G0/G1 arrest upon BEZ235 treatment (Figure 4C). However, despite the resistance to galunisertib, pSMAD2 expression was abrogated by the treatment (Figure 4D).
PF338 tumor cells are highly sensitive to HDAC inhibition and change differentiation (A) SAHA treatment changed morphology from biphasic to epithelial (B) Cells were sensitive to SAHA (IC50: 0.38 microM) and olaparib (IC50: 4.60 microM) (C) SAHA induced a G0/1 arrest and with higher concentrations both a G2M arrest and apoptosis; olaparib induced G2/M arrest (D) SAHA dose-dependently upregulated the expression of E-cadherin and beta-catenin. Error bars = means +- SE from three repeats. C/D, control, *p < 0.05, **p < 0.01.
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IC50 of 0.38 and 4.60 microM, respectively (Figures 5A,B). SAHA treatment induced two distinct cell cycle patterns: a dose dependent G0/1 arrest at low drug concentrations and both a G2/M arrest and induction of apoptosis at high concentrations. In contrary, olaparib-treated cells went into G2/M arrest in a dose-dependent manner (Figure 5C). Importantly, PF338 tumor cells changed morphology from the initial biphasic to an epithelial phenotype upon SAHA treatment (Figure 5A; Supplementary Video S2). These phenotypic changes were accompanied by a dose-dependent upregulation of epithelial markers E-cadherin and:to a lesser extent:beta-catenin (Figure 5D).