Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma

Abstract

To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133⁺ (<1.5%) and CD15⁺ (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP^-/Vimentin⁺ cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP^-/Vimentin⁺ cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.

Keywords: BRAF V600E; CDKN2A; cancer stem cell; low grade glioma; orthotopic xenograft.

Figure 1. Establishment of in vivo and in vitro models of PLGGs

(A) H&E and IHC staining of mouse brain show scars of surgical implantation without tumor formation, disturbed granular layer neurons (b-c, circle), and reactive mouse astrocytes (monoclonal antibodies against GFAP) (e-f). Human tumor cells detected with human-specific antibodies against mitochondria (MT) (h-i). Magnification (x10: a, b, d, e, g, h and x40: c, f, i). (B) H&E stained cross section of IC-3635PXA (top left image). Log-rank analysis of animal survival times during serial sub-transplantation from passage I (P-I) to IV (P-IV) (top right panel). IHC of tumor cells with human-specific MT antibodies (lower panel). (C) Histopathological features of IC-3635PXA xenograft tumors (at passage I and III) compared with patient tumor (magnification: x20). (D) Morphology of cultured PDOX cells in FBS-based media (BXD-3635PXA-mono) and serum-free media supplemented with EGF/bFGF (BXD-3635PXA-NS) from passages 1 (p-1) to 20 (p-20) (magnification: x10).

Figure 2. Analysis of CD133⁺ and CD15⁺ cells

(A) Representative graph showing successful double staining of CD133 and CD15 in GBM (left 2 panels) and PLGGs with high (middle panel) and low (right panel) CD15⁺ cells. (B) Summary graph showing relative abundance of mono- and dual-positive (CD133 and CD15) cells related to tumor cell growth, PDOX formation, pathological grade, years of follow up, and status of progression. Due to limited viable tumor cell yields, not all samples were tested with all assays. “-”: not tested; N = did not grow/no progression, Y = grew in culture or in vivo or progressed.

Figure 3. Quantitative analysis of BRAF V600E mutation allele frequency using pyrosequencing

(A) Representative pyrograms indicating A (wild type) to T (mutated) substitution in percent for 3635PXA patient tumor, xenograft passage I as well as in vitro cultured 3635PXA cells (monolayer and neurospheres) are shown. Pyrosquencing was control by including a positive control (HT-29) as well as negative controls with either no mutation (Tonsil) or no DNA. Percent (%) A/T is calculated using pyromark analysis software. (B) Allele frequency of BRAF V600E mutation in patient tumor, xenografts and cultures cells of 3635PXA.

Figure 4. FISH validation of CDKN2A deletion

(A) Location and the coverage of CDKN2A by spectral orange (R = red) on chromosome 9. The alpha satellite on the centromere of chromosome 9 is labeled with SpectrumGreen (G = green) and used as the reference control. (B) Images of FISH hybridization in paraffin sections (patient tumor and xenograft) and in cultured cells (monolayer and neurospheres). Normal cells with two copies of chromosome 9 centromere (green) and two copies of CDKN2A (red) highlighted in circle and labelled as 2R2G. Loss of CDKN2A (no red = 0R) were found in disomy (0R2G), trisomy (0R3G) and quadrosomy (0R4G) tumor cells (arrows). (C) Graph showing the relative percentage of cells with or without CDKN2A deletion. For each sample, 200 cells were counted under high magnification (10 × 60). Compared with the patient tumor, in which CDKN2A were still present in a small fraction of cells (mostly 2R2G), xenograft tumors and both the cultured cells were enriched with homozygous deletion of CDKN2A (0R1G to 0R5G). Note the gradual decrease of disomy chromosome 9 (0R2G) and increase of trisomy 9 (0R3G) (arrows) over in vivo sub-transplantations of IC-3635PXA.

Figure 5. In vivo tumor invasion and host responses detected with IHC

(A) Modes of IC-3635PXA invasion in mouse brains. Tumor cells positively stained with Vimentin (VIM) (arrows, a-c), and blood vessels (bv) with vWF (d). Same area in two consecutive sections was included (c and d, dotted line with dual arrow heads). (B) Images showing long-range perivascular invasion (left panel) and quantitative analysis perivascular migration (right panel) (^** P< 0.01). Tumor cells positively stained with VIM (arrow heads). (C) IHC showing mutually exclusive positivity between GFAP (marker for mature glial cells) (red arrowheads, e-g) and VIM in tumor mass (Tum) and in invasive satellites and single cells (blue arrowheads, h and i). Note presence of reactive astrocytes in normal brain tissues (g) without presence of tumor cells (j). Matched areas in consecutive sections were used (dotted line with dual arrow heads) for GFAP and VIM staining (magnification x 20). (D) IHC of paired primary and recurrent PA confirming invasive cells to be GFAP- (red arrow, l and m) and VIM⁺ (blue arrowheads, o and p) Magnification (x40: k and n; x20: l, m, o, p).

Figure 6. In vitro drug testing

Cultured 3635PXA cells were exposed to small molecule inhibitors (0.01 to 10 μM) for 7 days and plotted as the fraction of cell killing. (A) Two of the 7 Inhibitors targeting BRAF V600E (left panel) and 2 of the 9 inhibitors against BRAF wild-type and RAF (right panel) were active. Proliferation of the 3635PXA cells were not affected by the remaining agents. (B) Chemotherapy agent Vincristine was included as reference.