Mucinous adenocarcinoma developed from human fallopian tube epithelial cells through defined genetic modifications

Abstract

Recent studies have suggested that some ovarian and pelvic serous carcinomas could originate from the fimbriated end of the distal fallopian tube. To test this hypothesis, we immortalized a normal human fallopian tube epithelial (FTE) cell line by using retrovirus-mediated overexpression of the early region of the SV40 T/t antigens and the human telomerase reverse transcriptase subunit (hTERT). These immortalized FTEs were then transformed by ectopic expression of oncogenic human HRAS (V12) . Tumorigenicity of the immortalized and/or transformed cells was subsequently tested by anchorage-independence growth assay and inoculation into nude mice via subcutaneous and intraperitoneal injection. As expected, the HRAS (V12) -transformed FTEs produced tumors through both subcutaneous and intraperitoneal injections, whereas no tumor growth was observed in immortalized FTEs. Unexpectedly, histopathological examination of tumors resulting from subcutaneous as well as intraperitoneal injections revealed largely poorly differentiated mucinous adenocarcinoma mixed with undifferentiated carcinoma. The tumor implants invaded extensively to the liver, colon, spleen, omentum, adrenal gland and renal capsule. Immunohistochemical staining of tumor cells showed positive staining for the epithelial cell markers cytokeratin AE1/AE3 and Müllerian lineage marker PAX8. Our study demonstrates that FTEs can generate poorly differentiated mucinous adenocarcinoma mixed with undifferentiated carcinoma through genetic modifications. Thus, we provide the first experimental evidence that fimbrial epithelial cells of the fallopian tube could be a potential source of ovarian mucinous adenocarcinoma.

Figure 1. Transformation of human primary fallopian tube epithelial cells by oncogenic HRAS^V12. (A) Expression of SV40 T/t antigens in immortalized FTEs (FTE187-hT-SV) and expression of HRAS in immortalized as well as transformed (FTE187-hT-SV-H) FTEs was detected by western blot analysis. (B) FTE187-hT-SV-H cells were strongly colonized on soft agar plates, whereas their immortalized counterparts lacked the ability to grow in semi-solid culture. (C) When 10,000 cells were plated on soft agar plates, FTE187-hT-SV-H cells generated > 1,000 colonies exceeding 50 µm within 30 d, compared with ~30 colonies formed by FTE187-hT-SV. Results represent the average of three independent experiments. ****p < 0.0001, as determined by one-way ANOVA (GraphPad Prism 5.0, GraphPad Software Inc.). (D) Bilateral subcutaneous (1) and intraperitoneal (2, arrow) tumors generated by HRAS^V12-transformed FTEs in immune-deficient mice. (E) Tumor growth curves of subcutaneous tumors formed by FTE187-hT-SV and FTE187-hT-SV-H cells. *p < 0.05, ****p < 0.0001, as determined by two-way ANOVA (GraphPad Prism 5.0). n = 5.

Figure 2. Immunohistological characteristics of HRAS^V12-transformed FTEs and tumor xenografts. Immunohistochemical staining to cytokeratin, p53, SV40, WT-1, PAX8 and PAX2 in FTE187-hT-SV-H cells (A–C, G–I) as well as in tumor xenographs generated by FTE187-hT-SV-H cells (D–F, J–L).

Figure 3. Immunohistochemical examination of tumors generated by HRAS^V12-transformed primary fallopian tube epithelial cells. Hematoxylin and eosin (H&E), immunochemical staining showing histological and immunological features of FTE187-hT-SV-H-generated tumor xenographs. (A–D) H&E staining shows tumor enriched in mucinous substances and mucinous substance within the cytoplasm of a tumor cell (thick arrow) that is characteristic of mucinous carcinoma. (E and F) H&E staining shows an area of undifferentiated carcinoma; (G) tumor cells (red arrows) invade the liver (black arrows, hepatocytes). (H and I) Immunochemical staining for cytokeratin showing positive spindle cells (thick arrow) in an area of undifferentiated carcinoma (H) and positive epithelial cells in an area of undifferentiated diffierentiated carcinoma (thick arrow). (J–L) Periodic acid schiff and Alcian blue pH 2.5 staining of tumor sections from FTE187-hT-SV-H-generated intra-peritoneal tumors in mice showing acidic mucinous substances as stained blue, neutral polysaccharides as stained magenta and nuclei as stained purple. Black arrows show the accumulation of mucinous substance within the cytoplasm of tumor cells.