Establishing a papillary craniopharyngioma cell line by SV40LT-mediated immortalization.

BACKGROUND: Craniopharyngioma represents a troublesome tumor of the intracranial sellar region. There are currently no available well-characterized craniopharyngioma cell lines. This lack of reliable, immortal cell lines is a major reason for the slow progress in fundamental research related to craniopharyngioma. METHODS: We describe the development of an immortal papillary craniopharyngioma (PCP) cell line by transfecting primary PCP cells with the pLenti-simian virus 40 large T antigen(SV40LT). RESULTS: Three clones have been cultured for more than 14 months so far, while non-transfected cells ceased proliferation within three months of isolation. The established immortal PCP cell lines were identified to have BRAFV600E mutations, while no mutations in tumor suppressor genes were found in primary cells or immortal cells. Immortal cells had higher proliferation rates and formed tumors when implanted in the bran of nude mice. BRAF inhibition in immortal PCP cells altered cell morphology, inhibited cell proliferation and promoted apoptosis. CONCLUSION: We successfully developed PCP cell lines by SV40LT-mediated immortalization. These cell lines represent a powerful tool for fundamental and therapeutical studies on craniopharyngioma.

Feasibility of primary human cell cultures as a model for adamantinomatous craniopharyngioma research: Evidence from RNA-Seq analysis.

Adamantinomatous craniopharyngioma (ACP) is a benign epithelial tumor of the sellar region. Whether primary human cell cultures can be used as a stable research model has yet to be determined. The characteristics of three cultured craniopharyngioma primary cell (CPC) lines were identified using immunofluorescence. The culture duration for each CPC line was 10, 20 and 30 days. Cell lines and paired parental tumor tissues were subsequently analyzed using transcriptome sequencing (RNA-Seq). Transcriptomic differences between ACP tissues and CPC lines were compared. CPCs maintained the original epithelial lineage markers, including pan-cytokeratin and epithelial cell adhesion molecule. However, the Pearson's correlation coefficient of transcriptomes between each pair of CPC lines and ACP tissues decreased from 0.657 (cultured for 10 days) to 0.61 (cultured for 20 days) and further to 0.547 (cultured for 30 days). The number of differentially expressed genes between ACP tissues and CPCs was increased from 1,247 (cultured for 10 days) to 1,643 (cultured for 20 days) and then to 1,949 (cultured for 30 days). The results of Gene Set Enrichment Analysis demonstrated that the diversity of gene sets increased with longer culture time. Significant differences in the majority of signature gene sets were not observed between ACP tissues and CPCs, with the exception of keratinization phenotype [normalized enrichment score (NES)=-2.02, false discovery rate (FDR)=0.0038] and epithelial cell phenotype (NES=-1.82, FDR=0.032). Cell proliferation (NES=1.78, FDR=0.028) and mitosis (NES=1.93, FDR=0.012) were enhanced in CPCs. Therefore, primary human cell cultures can be used as a suitable research platform for ACP, however further experiments are required.

Identification of tumor stem-like cells in admanatimomatous craniopharyngioma and determination of these cells' pathological significance.

OBJECTIVE: Nuclear ß-catenin, a hallmark of active canonical Wnt signaling, can be histologically detected in a subset of cells and cell clusters in up to 94% of adamantinomatous craniopharyngioma (ACP) samples. However, it is unclear whether nuclear ß-catenin-containing cells within human ACPs possess the characteristics of tumor stem cells, and it is unknown what role these cells have in ACP. METHODS: Primary ACP cells were cultured from 12 human ACP samples. Adamantinomatous CP stem cell-like cells (CSLCs) showing CD44 positivity were isolated from the cultured primary ACP cells by performing magnetic-activated cell sorting. The tumor sphere formation, cell cycle distribution, stemness marker expression, and multidifferentiation potential of the CD44- cells and the CSLCs were analyzed. RESULTS: Compared with the CD44- cells, the cultured human CSLCs formed tumor spheres and expressed CD44 and CD133; moreover, these cells demonstrated nuclear translocation of ß-catenin. In addition, the CSLCs demonstrated osteogenic and adipogenic differentiation capacities compared with the CD44- cells. The CSLCs also displayed the capacity for tumor initiation in human-mouse xenografts. CONCLUSIONS: These results indicate that CSLCs play an important role in ACP development, calcification, and cystic degeneration.

Pathological Relationship Between Adamantinomatous Craniopharyngioma and Adjacent Structures Based on QST Classification.

The aim of this study was to clarify pathological and anatomical relationships between adamantinomatous craniopharyngiomas (ACP) and their surrounding structures. We previously established a QST classification scheme based on the apparent anatomic origin of the tumors. According to this classification, 13 type Q tumors, 6 type S tumors, and 42 type T ACPs were analyzed. Type Q tumors, which are most likely to involve the pituitary gland, did not invade the area of contact with the adenohypophysis. Instead, tumor invasion was observed in areas where the tumor contacted the neurohypophysis. Type S tumors primarily involved the pituitary stalk; the arachnoid remained present between these tumors and normal structures. Type T tumors were located beneath the basal arachnoid membrane and outside the pia mater. The pia mater was disrupted and finger-like invasions were found in the neural layer of the third ventricle floor along the invasive front. Tumors were never observed to break through the ependymal layer of the third ventricle. The QST classification has important implications for understanding the growth pattern of tumors and can be used to guide surgical procedures.

[Primary culture of human malignant meningioma cells and its intracranial orthotopic transplantation in nude mice].

OBJECTIVE: To obtain stable primary cultures of human malignant meningioma cells and establish an intracranial in-situ tumor model in nude mice. METHODS: Ten surgical specimens of highly suspected malignant meningioma were obtained with postoperative pathological confirmation. Primary malignant meningioma cells were cultured from the tissues using a modified method and passaged. After identification with cell immunofluorescence, the cultured cells were inoculated into the right parietal lobe of 6 nude mice using stereotaxic apparatus and also transplanted subcutaneously in another 6 nude mice. The nude mice were executed after 6 weeks, and HE staining and immunohistochmistry were used to detect tumor growth and the invasion of the adjacent brain tissues. RESULTS: The primary malignant meningioma cells were cultured successfully, and postoperative pathology reported anaplastic malignant meningioma. Cell immunofluorescence revealed positivity for vimentin and EMA in the cells, which showed a S-shaped growth curve in culture. Flow cytometry revealed a cell percentage in the Q3 area of (95.99∓2.58)%. Six weeks after transplantation, tumor nodules occurred in the subcutaneous tumor group, and the nude mice bearing the in situ tumor showed obvious body weight loss. The xenografts in both groups contained a mean of (36∓5.35)% cells expressing Ki-67, and the intracranial in situ tumor showed obvious invasion of the adjacent peripheral brain tissues. CONCLUSION: We obtained stable primary cultures of malignant meningioma cells and successfully established a nude mouse model bearing in situ human malignant meningioma.

The purine receptor P2X7R regulates the release of pro-inflammatory cytokines in human craniopharyngioma.

Craniopharyngiomas (CPs) are usually benign, non-metastasizing embryonic malformations originating from the sellar area. They are, however, locally invasive and generate adherent interfaces with the surrounding brain parenchyma. Previous studies have shown the tumor microenvironment is characterized by a local abundance of adenosine triphosphate (ATP), infiltration of leukocytes and elevated levels of pro-inflammatory cytokines that are thought to be responsible, at least in part, for the local invasion. Here, we examine whether ATP, via the P2X7R, participates in the regulation of cytokine expression in CPs. The expression of P2X7R and pro-inflammatory cytokines were measured at the RNA and protein levels both in tumor samples and in primary cultured tumor cells. Furthermore, cytokine modulation was measured after manipulating P2X7R in cultured tumor cells by siRNA-mediated knockdown, as well as pharmacologically by using selective agonists and antagonists. The following results were observed. A number of cytokines, in particular IL-6, IL-8 and MCP-1, were elevated in patient plasma, tumor tissue and cultured tumor cells. P2X7R was expressed in tumor tissue as well as in cultured tumor cells. RNA expression as measured in 48 resected tumors was positively correlated with the RNA levels of IL-6, IL-8 and MCP-1 in tumors. Furthermore, knockdown of P2X7R in primary tumor cultures reduced, and stimulation of P2XR7 by a specific agonist enhanced the expression of these cytokines. This latter stimulation involved a Ca2+-dependent mechanism and could be counteracted by the addition of an antagonist. In conclusion, the results suggest that P2X7R may promote IL-6, IL-8 and MCP-1 production and secretion and contribute to the invasion and adhesion of CPs to the surrounding tissue.

TREM-1 expression in craniopharyngioma and Rathke's cleft cyst: its possible implication for controversial pathology.

Whether a mixed type of craniopharyngioma (CP) exists and whether papillary craniopharyngioma (pCP) is on a histopathological continuum with Rathke's cleft cyst (RCC) remain controversial. Herein, we examined the expression and localization of ß-catenin, BRAF p.V600E (V600E), and triggering receptor expressed on myeloid cells-1 (TREM-1) in 58 samples including 20 pCPs, 26 adamantinomatous craniopharyngiomas (aCP), and 12 RCCs. Five aCPs were diagnosed with mixed type CPs and the remaining 21 cases were pure aCPs. Four of the 12 RCCs presented with significant squamous epithelium (SE). V600E immunoreactivity was observed in all pCPs in the cytoplasm, but not in the nuclei. aCPs and RCCs, including mixed type CP, did not express V600E. Nuclear ß-catenin translocation was detected exclusively in aCPs. TREM-1 was expressed in pCPs. Additionally, TREM-1 expression was detected in the SE of 5 "mixed type" CPs, while it was absent in pure aCPs. TREM-1 was expressed in 4 RCCs with SE, but not in the remaining 8 RCCs. TREM-1 mRNA levels were compared in cultured pCP and aCP cells. TREM-1 mRNA level was significantly (p < 0.001; up to 4.045 fold) higher in pCPs than in aCPs. Western blotting revealed a significantly (p < 0.001; up to 7.19 fold) lower level of TREM-1 expression in aCP cells compared to that in pCP cells. Our findings further supported that RCC and pCP may represent two ends of a morphological spectrum. A variant showing overlapping histological features of aCP and pCP should not be considered as a mixed type.