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  • br To analyze the transcriptome of serum cultured

    2022-05-04


    To analyze the transcriptome of serum-cultured cells, sphere- 
    cultured cells, LL clone AZD8931 and S clone cells, we performed Serial analysis of gene expression sequencing (SAGE-Seq). More than 20,000,000 tags were read per sample, and a total of 18,587 genes were detected in the analysis. Since the primary cancer cells were obtained from uterine endometrioid adenocarcinoma, we analyzed the similarity of gene expression profile to normal gene expression of the en-dometrium. The serum-cultured cells showed gene expression profile close to that of the normal endometrium, and LL clone A cells showed a gene expression profile with less similarity to that of the normal en-dometrium (Fig. 5A).
    Characteristic genes expressed in LL clone cells are shown in Table 1 and those expressed in S clone cells and sphere-cultured cells are shown in Table 2. We performed RT-PCR to confirm the gene expression. Several genes including ADAMTS9, ALOX5AP, BHLHE41, C10orf90, CCR1, CHRNA1, KCNH1 NRP2, UGT2B7 and OTX2 were specifically expressed in sphere-cultured cells and S clone cells. Other genes in-cluding ZMAT4, RFX4, F13A1 and PDGFB were specifically expressed in
    LL clone cells (supplemental Fig. S1). To analyze the characteristics of gene expression, pathway analysis was performed using Metascape (http://metascape.org) (Supplementary Fig. S2). LL clone cells showed a significant gene expression signature of the MAPK cascade, whereas S clone cells showed a significant gene expression signature of stem cell differentiation (supplemental Fig. S2).
    4. Discussion
    Endometroid adenocarcinoma cells with stem cell features have been isolated by ALDH1 (Rahadiani et al., 2011), CD44 and CD133 (Nakamura et al., 2010; Rutella et al., 2009). Recently, novel candidate markers for endometrioid adenocarcinoma CSCs/CICs have been re-ported (Chen et al., 2015; Li et al., 2015; Yusuf et al., 2014). However, previous analysis was performed using long cultured cell lines. In this study, we successfully established distinctive CSC/CIC clones from primary patient tumor sample. The important point of this study is the cell samples had the similar features of the primary patient tumor. The results of histological studies including IHC staining revealed similarity of the primary tumor tissue and xenograft tumor tissues derived from established CSC/CIC clones, suggesting that the CSC/CIC clones es-tablished in this study are reasonable models for analyzing primary endometrioid CSCs/CICs. We previously reported that sphere-cultured cells have the characteristics of CSCs/CICs in cervical cancer (Asano et al., 2016) and ovarian cancer (Mariya et al., 2016), and we therefore we tried to analyze sphere-cultured cells. We established two cell clone types from sphere-cultured cells. Previous studies showed that highly tumorigenic and chemo-resistant cells are enriched in sphere-cultured cells compared with parental serum-cultured cells (Asano et al., 2016; Mariya et al., 2016). However, S clone cells (clones A and I) showed less tumorigenicity than that of LL clone cells (clones A and E). On the other hand, S clone I cells showed greater chemo-resistance than that of LL clone cells. These results indicate that different phenotypes of CSCs/ CICs might be due to the heterogeneity of CSCs/CICs and that different CSC/CIC clone cells might have different phenotypes.
    LL clone cells showed (1) higher tumorigenicity, (2) high expression level of SOX2 (3) high ratio of ALDHhigh cells, and (4) high proliferation rate in vitro. On the other hand, S clone cells showed (1) less tumor-
    igenicity, (2) high expression level of MMP10, (3) lower ratio of ALDHhigh cells and (4) low proliferation rate in vitro. The most re- markable phenotype of LL clone cells is higher tumorigenicity. Previous
    Fig. 4. Functional analysis of serum-cultured cells, sphere-cultured cells, LL clone cells and S clone cells.
    (A) ALDEFLUOR assays of serum-cultured cells, sphere-cultured cells, LL clone cells and S clone cells.
    (B) Cell proliferation assay of serum-cultured cells, sphere-cultured cells, LL clone cells and S clone cells.
    A
    LL clone S clone Serum Sphere
    B
    S clone
    -chemoresistance- Sphere culture cells Primary LL clone patient tumor -tumorigenicity-
    Serum culture
    cells
    Fig. 5. Genetic similarity to normal endometrial tissue and schematic image of this study.
    (A) Spearman's rank correlation coefficients for gene expression profiles of LL clone cells, S clone cells, serum-cultured cells and sphere-cultured cells compared to normal endometrial tissues (Reference ID of the NCBI GEO is GSE 23339).