Conditional reprogramming culture conditions facilitate growth of lower-grade glioma models

Abstract

Background: The conditional reprogramming cell culture method was developed to facilitate growth of senescence-prone normal and neoplastic epithelial cells, and involves co-culture with irradiated fibroblasts and the addition of a small molecule Rho kinase (ROCK) inhibitor. The aim of this study was to determine whether this approach would facilitate the culture of compact low-grade gliomas.

Methods: We attempted to culture 4 pilocytic astrocytomas, 2 gangliogliomas, 2 myxopapillary ependymomas, 2 anaplastic gliomas, 2 difficult-to-classify low-grade neuroepithelial tumors, a desmoplastic infantile ganglioglioma, and an anaplastic pleomorphic xanthoastrocytoma using a modified conditional reprogramming cell culture approach.

Results: Conditional reprogramming resulted in robust increases in growth for a majority of these tumors, with fibroblast conditioned media and ROCK inhibition both required. Switching cultures to standard serum containing media, or serum-free neurosphere conditions, with or without ROCK inhibition, resulted in decreased proliferation and induction of senescence markers. Rho kinase inhibition and conditioned media both promoted Akt and Erk1/2 activation. Several cultures, including one derived from a NF1-associated pilocytic astrocytoma (JHH-NF1-PA1) and one from a BRAF p.V600E mutant anaplastic pleomorphic xanthoastrocytoma (JHH-PXA1), exhibited growth sufficient for preclinical testing in vitro. In addition, JHH-NF1-PA1 cells survived and migrated in larval zebrafish orthotopic xenografts, while JHH-PXA1 formed orthotopic xenografts in mice histopathologically similar to the tumor from which it was derived.

Conclusions: These studies highlight the potential for the conditional reprogramming cell culture method to promote the growth of glial and glioneuronal tumors in vitro, in some cases enabling the establishment of long-term culture and in vivo models.

Keywords: BRAFV600E; NF1; conditional reprogramming; enescence; low-grade glioma.

Fig. 1

Culturing of primary pilocytic astrocytoma cells. (A) Magnetic resonance imaging of tumor from which JHH-NF1-PA1 cells were derived. (B) Representative H&E image of JHH-NF1-PA1 primary tumor (×200, scale bar 50 microns). (C) Phase contrast image of JHH-NF1-PA1 cultures. (D) Growth of JHH-NF1-PA1 in CRC, FBS, or FBS + Y conditions. (E) Expression of nestin (left) and vimentin (right) in JHH-NF1-PA1. (F) Focal staining of SMA in JHH-NF1-PA1. (G) Increased GFAP in JHH-NF1-PA1 cultured in astrocyte condition media, and immunofluorescent staining of these cells. (H) Glial fibrillary acidic protein (green) and nestin (red) positive cells in JHH-CRC43. (I) NG2-positive cells in JHH-NF1-PA1. (J) Rare CD68-positive microglia in JHH-NF1-PA1. (K) Western blots showing loss of NF1 in CRC cultures. CRC, conditionally reprogrammed cells; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; PA, pilocytic astrocytoma.

Fig. 2

Xenografts of JHH-CRC cultures. (A) Representative H&E-stained image of JHH-CRC18 primary tumor (×400, scale bar 25 microns). (B) Phase contrast image of JHH-PXA1 (CRC18) culture. (C, D) Representative H&E-stained images of JHH-PXA1 mouse xenografts (×100, ×400, scale bars 100 microns, 25 microns). (E) Confocal images showing migration of JHH-NF1-PA1 cells (green) in zebrafish xenografts, taken at ×15 on days 1, 4, and 6 postinjection. CRC, conditionally reprogrammed cells; PA, pilocytic astrocytoma.

Fig. 3

Culturing of JHH-273 in CRC conditions. (A) Phase contrast image of JHH-273 CRC cells. (B) The IDH1 mutation was present in cultured cells. (C) Induction of senescence in FBS evidenced by acidic β-galactosidase staining of JHH-273 (arrows), as well as increased p21 expression in (D). CRC, conditionally reprogrammed cells; FBS, fetal bovine serum; IDH, isocitrate dehydrogenase.

Fig. 4

Switching JHH-NF1-PA1 cultures to FBS condition induced apoptosis, senescence and inhibited cell proliferation. (A, B) Slow growth of JHH-NF1-PA1 cells switched to FBS conditions. (C) After 7 days in FBS conditions, JHH-NF1-PA1 cells accumulated in G1 phase. (D, E) Decreased proliferation (BrdU+ cells) and increased apoptosis (Annexin V assay) after the switch to FBS conditions. (F, G) Levels of p27 protein and mRNA also increased when JHH-NF1-PA1 was cultured in FBS. (H) Induction of acidic β-galactosidase-positive cells in FBS (*P < .05, **P < .01). BrdU, bromodeoxyuridine; FBS, fetal bovine serum; PA, pilocytic astrocytoma.

Fig. 5

Chemosensitivity of CRC cultures. (A, B) Survival fraction of JHH-NF1-PA1 and other pLGG cell lines (Res186, Res259, and BT66) after 5-d treatment with carboplatin or vinblastine. (C) JHH-NF1-PA1 cells treated with vinblastine for 6 h accumulate in G2/M. (D) Immunofluorescent staining of α-tubulin in JHH-NF1-PA1 cells treated with vinblastine for 6 h shows abnormal spindles. (E) Minimal increase in Annexin V+ apoptotic cells after vinblastine treatment. (F) Decreased numbers of JHH-NF1-PA1, JHH-PXA1, and JHH-CRC32 cells after 5-d treatment with MEK162. (G) Inhibition of Erk1/2 phosphorylation by MEK162. CRC, conditionally reprogrammed cells; PA, pilocytic astrocytoma; pLGG, pediatric low-grade glioma. *P < .05.