Patient-derived DIPG cells preserve stem-like characteristics and generate orthotopic tumors

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor, with a median survival of less than one year. Due to enormous difficulties in the acquisition of DIPG specimens and the sophisticated technique required to perform brainstem orthotopic injection, only a handful of DIPG pre-clinical models are available. In this study, we successfully established eight patient-derived DIPG cell lines, mostly derived from treatment-naïve surgery or biopsy specimens. These patient-derived cell lines can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities. In addition, these cells retained genomic hallmarks identical to original human DIPG tumors. Notably, expression of several neural stem cell lineage markers was observed in DIPG cell lines. Moreover, three out of eight cell lines can form orthotopic tumors in mouse brainstem by stereotactic injection and these tumors faithfully represented the characteristics of human DIPG by magnetic resonance imaging (MRI) and histopathological staining. Taken together, we established DIPG pre-clinical models resembling human DIPG and they provided a valuable resource for future biological and therapeutic studies.

Keywords: DIPG; orthotopic xenograft; patient-derived cell line; pediatric brain tumor; pre-clinical model.

Figure 1. Clinical information of the patients

Most of the DIPG patients were treatment naïve (except TT11111) when surgery or biopsy were performed. Histopathology showed that all patients were diagnosed as high-grade gliomas (3 cases of grade III anaplastic astrocytoma AA, 2 cases of grade III anaplastic oligodendroastrocytoma AOA, and 3 cases of grade IV glioblastoma GBM). MRI revealed the infiltrative tumors in pons and the invasion to midbrain, medulla oblongata, and cerebellum.

Figure 2. The morphology and immunophenotype of DIPG cell lines

(A) Cell lines derived from different DIPG samples displayed distinct morphologies (20X), including flattened or round, bipolar with long processes and multipolar shapes. (Scale bars: 400μm). (B) Neurosphere formations of representative DIPG cell lines (4X). (Scale bars: 1000μm). (C) Immunocytochemistry staining of representative DIPG cell lines. Positive staining of neural stem cell marker (Nestin), oligodendrocyte progenitor cell markers (Olig2 and PDGFRα) and astrocyte marker (GFAP) demonstrated neural progenitor status of established DIPG cells (20X). The positive staining of proliferation marker Ki-67 confirmed the proliferative property of these cells. (Scale bars: 200μm).

Figure 3. The genomic signature, proliferation capacity and chromosome abnormality of DIPG cell lines

(A) Sanger sequencing demonstrated thatestablished DIPG cell lines retained gene mutations in H3F3A, TP53 and PPM1D which were consistent with their matching human DIPG tumors. (B-C) Proliferation capacities of DIPG cell lines were measured by growth curves and doubling times. (D) Karyotype analysis of established DIPG cell lines. TT10630 and TT10714 manifested as significant structural abnormalities (red frames) with normal overall chromosome quantity, while TT10603, TT10728, TT10902, TT11111 and TT11201 represented severe numerical disorders such as haploid and polyploid (green frames). TT11118 demonstrated both structural and numerical abnormalities.

Figure 4. The establishment of DIPG xenograft models and their MRI manifestations

(A) The latency of forming DIPG xenografts by direct orthotopic injection of TT10603, TT10630 and TT10714 cells was 172, 186 and 155 days post injection, respectively. Third and fourth generation of serial transplantations of TT10603 cells after subcutaneous propagation showed a significant shorter latency of 67 and 40 days. (B-D) Representative MRI of orthotopic xenograft acquired from 7.0-T mouse MR machine. (B) T2-weighted sequence of orthotopic xenograft generated by direct injection of TT10603 cells (B1: axial; B2: coronal; B3: sagittal). (C) T2-weighted sequence of serial xenograft generated by subcutaneously propagated TT10603 cells (B1: axial; B2: coronal; B3: sagittal). (D) Axial T2-weighted sequence of TT10603 orthotopic xenografts with different cell numbers (D1: 1,000 cells. D2: 10,000 cells. D3: 100,000 cells) on the 70^th day after injection.

Figure 5. Histopathological characteristic of TT10603 patient specimen and mouse xenograft (20X)

H&E staining of all xenografts revealed high cell density similar to human DIPG tissues, representing the characteristic of high grade glioma. Immunohistochemistry showed that all xenografts as well as their human DIPG counterparts were positive for Nestin, Sox2, Olig2, PDGFRα and GFAP. Notably, serial xenograft demonstrated higher percent of immunoreactivity for these markers than direct orthotopic xenograft. Most tumor cells of xenografts were negative in H3K27me3 staining, indicating that they retained the loss of trimethylation at position 27 of histone H3 from human tumor tissues. (Scale bars: 200μm).