Establishment and Molecular Phenotyping of Organoids from the Squamocolumnar Junction Region of the Uterine Cervix

Abstract

The metaplastic epithelium of the transformation zone (TZ) including the squamocolumnar junction (SCJ) of the uterine cervix is a prime target of human papilloma virus (HPV) infection and subsequent cancer development. Due to the lack of adequate in vitro models for SCJ, however, investigations into its physiological roles and vulnerability to carcinogenesis have been limited. By using Matrigel-based three-dimensional culture techniques, we propagated organoids derived from the normal SCJ region, along with metaplastic squamous cells in the TZ. Consisting predominantly of squamous cells, organoids basically exhibited a dense structure. However, at least in some organoids, a small but discrete population of mucin-producing endocervix cells co-existed adjacent to the squamous cell population, virtually recapitulating the configuration of SCJ in a TZ background. In addition, transcriptome analysis confirmed a higher expression level of many SCJ marker genes in organoids, compared to that in the immortalized cervical cell lines of non-SCJ origin. Thus, the obtained organoids appear to mimic cervical SCJ cells and, in particular, metaplastic squamous cells from the TZ, likely providing a novel platform in which HPV-driven cervical cancer development could be investigated.Cancers 2020, 12, 694 2 of 15 endocervix and ectocervix, the newly formed SCJ is shifted, alongside the extension of the TZ toward the endocervix, to the region connecting the TZ and endocervix. The SCJ and the TZ have been regarded as the most important cytological and colposcopic landmarks in the clinic, based on the fact that the large majority of uterine cervical cancers (UCC) and high-grade squamous intraepithelial lesions (HSIL) arise at this region [4,5]. Whereas human papillomavirus (HPV) is a major cause of neoplastic changes in the cervix for both squamous cell carcinoma (SCC) and adenocarcinoma [6], the incidence of UCC is significantly higher than that of cancers arising from other genital tract tissues [7]. However, the precise mechanisms underlying the predisposition of the cervix toward HPV-driven carcinogenesis have remained elusive.Recently, a residual embryonic cell population harboring the capacity to differentiate and the vulnerability to undergo neoplastic transformation was documented in both gastro-esophageal [8] and ecto-endocervical junctions [9]. With regard to the uterine cervix, a small discrete population of cuboidal cells in the SCJ region was histologically identified. By micro-dissection and microarray analysis, over 70 genes were identified as upregulated genes by more than two-fold, compared to adjacent squamous or columnar cell populations. In particular, Cytokeratin7 (KRT7), Anterior gradient protein 2 homolog (AGR2), Cluster differentiation 63 (CD63), Matrix metalloproteinase-7 (MMP7) and Guanine deaminase (GDA) were further demonstrated to specifically mark these cuboidal SCJ cells by immunohistochemistry [9]. Intriguingly, all these five markers remained positive in all HPV-related neoplastic tissues and cervix-d...

Organoids of the Female Reproductive Tract: Innovative Tools to Study Desired to Unwelcome Processes

“…Recently, Maru et al (2020) succeeded in establishing cervical organoids from a limited number of patient-derived biopsies in a medium supplemented with RSPO1, Noggin, EGF, ROCKi and the Notch ligand Jagged-1. The organoid cells expressed validated SCJ markers more robustly than classic cell lines and displayed both differentiated endo-and ectocervical cell types.…”

Organoid systems to study the human female reproductive tract and pregnancy

“…The endocervical organoids express KRT7, whilst the ectocervical ones are positive for basal markers KRT5 and TP63. Organoids have also been derived from the cervical SCJ and grown in a medium containing EGF, RSPO1, Noggin, Y-27632, and Jagged-1 [95]. MMP7 and AGR2 are expressed in these organoids, which are also KRT17 + .…”

“…Organoids can be established from pathological samples and provide powerful tools to study diseases ex vivo although the culture conditions often need optimizing [69,78,95,[102][103][104][105][106][107][108][109][110][111] ( Table 4). A common benign condition is endometriosis where ectopic foci of the endometrium containing both glands and stroma are found in the peritoneal cavity, ovary, and cervix.…”

Changes in Stem Cell Regulation and Epithelial Organisation during Carcinogenesis and Disease Progression in Gynaecological Malignancies

“…Recent comprehensive studies deriving PDOs from cervical cancer cells or normal cervical tissue have been described. Organoids have been developed from normal cervical cells from the SCJ region, and metastatic squamous cells from the transformation zone, providing models in which HPV-driven cervical carcinogenesis could be evaluated [75]. Furthermore, organoids established from human and mouse ecto-and endo-cervical cells revealed that the two epithelial cell types descend from distinct cervical lineage-specific stem cell populations which are regulated by divergent stromal Wnt signals.…”