Optimized creation of glioblastoma patient derived xenografts for use in preclinical studies

Abstract

BackgroundGlioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults, highlighting the need for novel treatment strategies. Patient derived xenografts (PDX) represent a valuable tool to accomplish this task.MethodsPDX were established by implanting GBM tissue subcutaneously. Engraftment success was compared between NMRI Foxn1nu and NOD/SCID as well as between fresh and cryopreserved tissue. Established PDX were analyzed histologically and molecularly. Five PDX were experimentally treated with different drugs to assess their potential for preclinical drug testing.ResultsEstablishment of PDX was attempted for 36 consecutive GBM cases with an overall success rate of 22.2% in NMRI Foxn1nu mice. No difference was observed between fresh or cryopreserved (20–1057 days) tissue in direct comparison (n = 10 cases). Additionally, engraftment was better in NOD/SCID mice (38.8%) directly compared to NMRI Foxn1nu mice (27.7%) (n = 18 cases). Molecular data and histology of the PDX compare well to the primary GBM. The experimental treatment revealed individual differences in the sensitivity towards several clinically relevant drugs.ConclusionsThe use of vitally frozen GBM tissue allows a more convenient workflow without efficiency loss. NOD/SCID mice appear to be better suited for initial engraftment of tumor tissue compared to NMRI Foxn1nu mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1128-5) contains supplementary material, which is available to authorized users.

Establishment and Characterisation of Heterotopic Patient-Derived Xenografts for Glioblastoma

“…In the present study, we describe the successful engraftment of glioblastoma samples to nude mice, to generate heterotopic PDXs. Other studies reported successful take rates of 22-26% for glioblastoma in nude mice [25,26]. In our study, the subcutaneous heterotopic transplantation of tumour chunks on NMRI nude mice yielded 46% success.…”

“…Microsatellite analysis corroborated that the PDXs originated from the transplanted material, although minor changes, such as loss of heterozygosity and imbalance could be observed. Other studies described genetic modifications (e.g., gene amplification, gain/loss of mutation) in early passage PDXs in comparison to the patients' samples [26,43], despite a high similarity. Some changes might be occurring de novo, or they might reflect selection of pre-existing subpopulations during transplantation.…”

“…Besides the nature of cryoprotectant, different factors can influence the recovery of tumour growth after cryopreservation, such as the time lapse between removal of the sample from blood supply and cryopreservation, presence of necrotic tissue in the sample, the size of the frozen sample and its preservation, and the washing off of cryoprotectant before re-engraftment [32]. Studies about the transplantation of tumour samples after cryopreservation report different recovery rate of PDXs after cryopreservation (22% [26] (glioblastoma); 50% [33] (lung cancer), 100% [34] (liver metastases of uveal melanoma)). Additionally, Kageyama et al showed that PDXs derived from frozen tissue maintained copy number variation (CNV) and proteomic profiles, but presented slower growth compared to the transplantation of fresh tissues.…”

Amplification of the EGFR gene can be maintained and modulated by variation of EGF concentrations in in vitro models of glioblastoma multiforme

“…Cell lines were established from GBM tissue of three different heterotopic patient derived xenograft (PDX) tumors with varying EGFR copy numbers [ 22 ]. All PDX models were established by implanting GBM tissue received from the operation theater at the department of neurosurgery at the university medicine of Rostock subcutaneously into the flanks of immunodeficient NMRI Foxn 1 nu mice [ 22 ].…”

“…However, substances targeting EGFR or its downstream targets have yet to prove effective in clinical GBM trials [ 19 – 21 ]. Although genomic EGFR amplification can be well maintained in in vivo models of glioblastoma [ 22 , 23 ], research in a preclinical setting in vitro is hampered by the fact that common cell culture models of GBM lack EGFR gene amplification [ 24 , 25 ].…”