Quantitativein vivobioluminescence imaging of orthotopic patient-derived glioblastoma xenografts

Abstract

25Despite extensive research, little progress has been made in glioblastoma therapy, 26 owing in part to a lack of adequate preclinical in vivo models to study this disease. To 27 mitigate this, primary patient-derived cell lines, which maintain their specific stem-like 28 phenotypes, have replaced established glioblastoma cell lines. However, due to 29 heterogenous tumour growth inherent in glioblastoma, the use of primary cells for 30 orthotopic in vivo studies often requires large experimental group sizes. Therefore, 31 when using intracranial patient-derived xenograft (PDX) approaches, it is 32 advantageous to deploy imaging techniques to monitor tumour growth and allow 33 stratification of mice. Here we show that stable expression of near-infrared 34 fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid direct 35 non-invasive monitoring of tumour development without compromising tumour 36 stemness or tumorigenicity. Moreover, as this approach does not depend on the use 37 of agents like luciferin, which can cause variability due to changing bioavailability, it 38 can be used for quantitative longitudinal monitoring of tumour growth. Notably, we 39 show that this technique also allows quantitative assessment of tumour burden in 40 highly invasive models spreading throughout the brain. Thus, iRFP transduction of 41 primary patient-derived glioblastoma cells is a reliable, cost-and time-effective way 42 to monitor heterogenous orthotopic PDX growth.68 xenografts (PDX) 7 . However, due to their intrinsic heterogeneity, orthotopic PDXs 69 display a much greater range of growth rates between animal subjects compared to 70 established glioblastoma cell lines 4,10 . Although this variability is a good reflection of 71 the heterogeneity of the parental tumour, differences in growth characteristics, and 72 therefore PDX size and the degree of dissemination at defined time points, lead to 73 difficulties in quantifying and interpreting animal studies. Thus, large cohorts of 74 animals are needed to achieve the necessary statistical power in these preclinical 75 studies. 76 77 Currently, intra-vital bioluminescent imaging (BLI) with luciferase is commonly 78 used to monitor xenograft growth 11 . Yet, in vivo luciferase assays are limited to 79 descriptive analysis since factors such as fluctuating substrate access to the target 128 129 Cell proliferation assay 130 GSC were seeded in three wells of a 6-well plate at a density of 5x10 4 cells 131 per well. Cell number for each well was assessed using a haemocytometer 1, 3 and 132 6 7 days after seeding. A median was calculated for each time point. Cell count was 133 normalised to day 1 (n=3 independent repeats per cell line). 135Neurosphere formation assay 136 GSC were seeded in uncoated 96-well plates at a density of 10 cells per well.was first described in 2011, it has been shown to be a powerful and reliable tool to 193 measure cell proliferation in vitro and in vivo 11,14 . We deployed a retroviral 194 transduction approach to express iRFP in...

Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness

“…Therefore, to minimise any bias that might result from inadvertent focus on white matter track-rich regions we tracked ≥60 cells per condition across the entire brain slice. Surprisingly, both the G7 and E2 cell lines migrated similarly (and poorly) on brain slices (Figure S1A) despite their markedly different invasive behaviour in vivo (Koessinger et al, 2020). We hypothesised that factors released by the GBM cells may induce changes in the brain microenvironment which promote tumour cell invasion, and that this may take some time to establish.…”

Increased apoptotic priming of glioblastoma enables therapeutic targeting by BH3-mimetics

“…We next investigated whether MCL-1 was required for tumourigenesis in vivo. iRFP-labelled vector CRISPR and MCL-1 CRISPR G7 GSC were orthotopically injected in CD-1 nude mice and tumour growth was monitored with cranial magnetic resonance imaging (MRI) and iRFP signal detection (36). We observed a substantial impairment of tumour growth in MCL-1 deleted tumours (Figure 2B,C; Supplementary Figure 2C) that was reflected in the significantly prolonged survival of these mice (Figure 2D).…”

“…To examine intravital, intracranial tumour growth in animals bearing iRFP-positive G7 GSC, mice were monitored by PEARL imaging (Li-Cor) as previously described (36). MRI scans performed on brain tumour bearing mice using a nanoScan PET/MRI scanner (Mediso Medical Imaging Systems, Hungary).…”

“…Two days following infection, cells were selected by growth in puromycin (E2: 1 g/ml, G7: 0.5 g/ml; Sigma Aldrich) or blasticidin (G7, R15 and R24: 10 g/ml; InvivoGen) containing medium. As described previously, plasmids encoding iRFP IRES puro have been inserted into a pBABE vector (36).…”

Longitudinal monitoring of disease burden and response using ctDNA from dried blood spots in xenograft models

“…These measures are often poorly reproducible and can be biased by treatment-induced tissue necrosis and oedema. Using imaging as an alternative to estimate response in PDXs is more time-consuming, requires general anaesthesia and may also need the introduction of in-vivo reporter genes 24,25 .…”