Preclinical Orthotopic Murine Xenograft Models in Glioblastoma

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Chapter 2

Abstract

Preclinical Orthotopic Murine Xenograft Models in Glioblastoma Jyothi Nair1,2 and Shilpee Dutt1,2* Shilpee Dutt Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, India 2 Homi Bhabha National Institute, Training School Complex, India 1

Corresponding Author: Shilpee Dutt, Shilpee Dutt Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Sector-22, Kharghar, Navi Mumbai 410210, India, Tel: +9122-27405000; Email: [email protected] *

First Published December 27, 2016 Copyright: © 2016 Jyothi Nair and Shilpee Dutt. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source. 2

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Glioblastoma (GBM), also known as grade IV astrocytoma, is the most malignant and lethal glial tumours, accounting for about 30% of the adult brain tumours. Due to its highly invasive nature and intrinsic resistance to the existing treatment modalities, the median survival time of GBM patients has not improved beyond 14.6 months. Animal models simulating human GBM have been the mainstay for comprehending the mechanisms underlying tumour initiation, progression, and therapy resistance; they also serve as a preclinical platform for testing novel therapeutic agents. Since microenvironment is a critical factor involved in modulating the behaviour of tumour cells, animal models are superior to the in vitro cultures of tumour cells. Of these, rodents have been the most commonly used animals for various preclinical studies. Xenotransplantation is a widely used method for generation of animal models of GBM, wherein the tumour tissues are engrafted into immunocompromised mice or rats. Orthotopic models are a type of xenograft models obtained by injecting the tumour material into the site of origin of the tumour, i.e. brain in the case of GBM models. In this chapter, we have discussed the importance of the development of murine orthotopic xenograft models of GBM. We have also outlined some of the major studies describing the use of orthotopic mouse models for recapitulating the clinical biology of GBM, along with preclinical testing of novel antineoplastic agents, predicting response to therapies, and examining therapy resistance, one of the most distressing features of GBM. www.avidscience.com

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Keywords Glioblastoma; Invasive; Resistance; Microenvironment; Xenotransplantation; Immunocompromised; Orthotopic Mouse Models

Introduction Neuroglia or glial cells occupy about half the volume of the central nervous system. Their name was derived from the Greek word for “glue”, as early histologists believed that these cells were the cementing material that holds the nervous tissue together. They are smaller in size, but are about 5 to 50 times more numerous than the neurons. In contrast to neurons, these cells do not generate or propagate nerve impulses, and can grow and multiply within the mature nervous system. On the basis of their size, cytoplasmic processes and intracellular organization, neuroglia found in the CNS are classified as astrocytes, oligodendrocytes, microglia and ependymal cells. Of these, astrocytes are star-shaped cells having multiple processes, are the largest and the most numerous among the neuroglia. They provide support to the neurons, maintain appropriate chemical environment for generation of nerve impulses, and during development, they secrete chemicals regulating the growth, migration and interconnection among the neurons [1,2].

Glioblastoma Brain tumours derived from glial cells are known as “gliomas”, most of which are highly malignant and grow 4

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rapidly [1]. On the basis of their growth rate, gliomas have been graded from I to IV. Thus, grade I gliomas are benign tumours, grade II gliomas are designated as “low-grade”, while grade III and grade IV gliomas are designated as “malignant or high-grade gliomas”. Astrocytoma is a type of glioma derived from astrocytes. Glioblastoma (GBM), also known as grade IV glioma is the most malignant form of astrocytoma [3], which can be distinguished from all the other grades on the basis of histological features such as presence of ischemic necrotic cells within tumours (known as pseudopalisading necrosis), activation of the hypoxia-inducible factor (HIF-1α) pathway, microvascular proliferation around the tumours, and infiltrating cells [4]. GBM accounts for 30% of all the brain tumours in adults. It can arise de novo or can evolve from low-grade astrocytomas. GBM patients have a dismal prognosis, with a median survival time of 14.6 months [5]. This is due to the highly infiltrative nature of GBM, which precludes its complete surgical resection [6] and its inherent resistance to conventional radio- and chemotherapeutic agents [7], resulting in tumour recurrence in more than 90% of the cases. In order to gain better insights into the mechanisms underlying tumour initiation, progression and therapy resistance, animal models have been widely used to simulate human GBM, [8,9], which also play an essential role in preclinical research, for testing novel antineoplastic agents. The importance of these in vivo models is further underlined by the role of microenvironment in modulating the behaviour of tumour cells, due to which www.avidscience.com

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they behave differently from the in vitro culture conditions [10]. For various preclinical studies, rodents are the most commonly used models [11]. These animal models can be majorly divided into 3 categories: 1) chemical carcinogen or virus induced tumour models, 2) genetically engineered mouse (GEM) models, and 3) xenograft models [8,9]. Tumour induction by chemical carcinogens or viruses dates back to 1939, wherein implantation of polycyclic aromatic hydrocarbons such as methylcholanthrene into mouse brain was found to induce glioma and sarcoma. However, such induced brain tumours had different pathological types, genetic makeup, and histological characteristics as compared to the human tumours [8]. Increased understanding of tumour genomic alterations along with development of transgenic or gene knockout technologies has led to the development of genetically engineered mouse models of GBM, for e.g., by modulating the chief signalling pathways known to be deregulated in human gliomas such as PDGFR, EGFR, Rb, Ras and Akt. These models also reflect the histopathology and molecular etiology of human gliomas, providing insights into the molecular events and pathways contributing to tumour initiation, progression and metastasis. Furthermore, they can mimic the tumour-stroma interactions responsible for malignancy and angiogenesis. However, intratumoural heterogeneity, a characteristic of GBM, is poorly represented in these tumours, since they are composed of 6

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cells with specific and homogeneous genetic alterations. Moreover, tumour initiation cannot be controlled in these models, making therapeutic studies difficult [9]. This disadvantage of GEM models can be overcome in xenograft models, with a good reproducibility of engraftment rate, along with reliable growth and disease progression.

Murine Xenograft Models Xenograft models are generated by transplanting the tumour material, in the form of cell lines or patient tumour biopsies into immunocompromised mice. Based on the location of the implanted tumour material, there are two major types of human xenograft models – heterotopic and orthotopic. Heterotopic subcutaneous models are obtained by implanting the tumour material between the dermis and the underlying muscle within the flank or footpad of the mouse. Subcutaneous models are the most widely used preclinical mouse models for cancer research owing to their several advantages such as ease of establishment and tumour monitoring, rapid and economical process with good reproducibility, which can serve as a good platform to test novel antineoplastic drugs. However, more often, the drug regimens which show a promising outcome in these heterotopic preclinical models fail to produce any therapeutic benefit in humans. The primary reason for this has been attributed to the failure of subcutaneous microenvironment in modeling the appropriate

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conditions present in the organ/site of origin of the primary or metastatic tumour. These lacunae of heterotopic models have generated an increased interest in orthotopic tumour xenografts, which are further being explored for preclinical evaluation studies. Orthotopic models are generated by implanting or injecting the tumour material into the same organ in which the cancer originated or metastasized. The major advantages of orthotopic models are optimum tumour-microenvironment interactions, appearance of organ-specific metastases, and most importantly, recapitulating the clinical scenario of tumour progression, therapy response, and gene expression patterns. However, some of the inherent disadvantages of developing orthotopic models are the technical challenges, labour-intensive and expensive process and extended recovery time for the animals. In spite of these, orthotopic models are gaining preference over heterotopic models, on account of their clinical significance [12]. In GBM, the advantage of orthotopic models to the subcutaneous xenograft has been emphasized in a study which showed that the gene expression profiles of human GBM cell lines U251MG and U87MG varied significantly when grown in vitro, as subcutaneous xenografts or orthotopic intracranial xenografts. However, the gene expression profiles of intracranial tumours derived from both the cell lines were found to be similar to each other. These observations emphasize the role of microenvironment in modulating the gene expression and the resultant phenotype of tumour cells [13]. 8

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Mouse Strains Used for the Development of Orthotopic Xenograft Models A large number of strains and sub-strains of natural, transgenic, induced mutant or genetically engineered immunodeficient mouse models are available for tumour xenograft-based studies. These strains have defective Band/or T-cells, immunosuppression by knockdown of cytokine and cytokine receptor genes, TLR receptor genes, etc. These models consist of strains with a single-gene mutation such as nude-mice (nu) strains, strains with severe combined immunodeficiency (scid), non-obese diabetic (NOD) strain and strains with targeted deletion of recombination activating gene (RAG strains). Mice strains with additional deficiencies in the innate and adaptive immunity have been generated by crossing these mutant strains. Some of the most commonly used strains for orthotopic GBM models are:

Nude Strains In 1966, Flanagan described the first Balb/c nude mice strain, characterized by a mutation in the Foxn1 (HNF-3/ forkhead homolog 11 transcription factor) gene, which produced a hairless phenotype and a non-functional thymus in homozygotes. The hairless phenotype perpetuated the nickname “nude” for these strains. The homozygotes, with a rudimentary thymus generate few mature T cells, resulting in reduced T-cell response to antigens. Antibody response is mediated mainly by the IgM-class; however, www.avidscience.com

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the natural killer (NK) cells have a better potency as compared to normal Balb/c mice. As these homozygotes fail to mount a graft-rejection response, they can readily accept xenografts and have been used in imaging and examining novel treatments for tumours [14].

SCID Strains The first severe combined immunodeficiency mouse strain was found to harbor mutation in the Prkdcscid (protein kinase DNA activated), which is a DNA-dependent protein kinase (DNA-PK), necessary for non-homologous end joining (NHEJ) repair of DNA double strand breaks. Thus, the scid mutation disrupts the production of functional B and T cells, and hence, they can serve as appropriate hosts for transplantation of human tumours for imaging, novel therapies and metastasis studies. However, some of the strains such as C57BL/6J, Balb/c and CB-17 produce low levels of circulating T and B cells and IgM, IgGs and IgA during aging, a phenomenon known as “immunoglobulin leakiness”. These strains are also very sensitive to radiation, and hence cannot be used for examination of radiotherapy in tumours [14].

NOD-SCID Hybrid Strains Non-obese diabetic (NOD) mice are specially developed strains, used as a model for type 1 diabetes. These mice have a unique major histocompatibilty complex (MHC) haplotype, namely H-2g7, along with polymor-

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phisms in the TNF-α and CTLA-4 gene, which are responsible for the development of spontaneous, autoimmune, type-1 diabetes in these mice. NOD/SCID hybrids are generated by transferring the scid mutation into a NOD strain. Unlike NOD strains, the hybrids do not develop spontaneous diabetes, have decreased innate immunity and NK cell activity, and are more immunocompromised than the SCID or nude strains [14].

NOD-SCID IL2rg-/- Strains IL2rg-/- strains have homozygous targeted mutations at the interleukin-2 receptor (IL-2R) γ-locus. The absence of IL-2R γ-chain, an essential component of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors leads to severe impairments in the development and function of T- and Bcells and complete loss of NK-cell development. Crossing of IL2rg-/- strains with NOD-SCID mice have developed highly immunodeficient hybrid strains, with little chance of xenograft rejection [14].

Methods of Tumour Transplantation Most commonly, tumour cell lines grown in vitro, patient biopsy-derived tumour tissues or cell suspension from dissociated tumour tissues are used for transplantation [8]. Some of the major methods used for tumour transplantation are described below.

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Orthotopic Xenograft Models from GBM Cell Lines GBM cell lines have been generated by culturing and serial passaging of patient biopsy derived tumour tissues as monolayers in serum-supplemented media. These immortalized cell lines can be subcultured for an unlimited number of passages in vitro, to obtain enormous number of tumour cells for generating xenografts. Tumours generated by intracranial injections of these tumour cells show an extensive and circumscribed growth pattern, some levels of angiogenesis and a variable extent of invasion into the perivascular space. However, a major disadvantage of these cell-line based models is that they fail to represent the genotypic alterations present in the original tumours from which they were derived, because of the altered phenotype and gene expressions, clonal selection and genetic drift occurring during the adaptation of tumour cells to monolayer cultures [9]. Nevertheless, orthotopic GBM models established using human GBM cell line U251MG has been shown to display gene expression profile similar to that of the clinical GBM specimens [15]. These xenografts also recapitulated the prominent characteristics of human GBM such as invasion, presence of necrotic cells with activated HIF-1α pathway within the tumours, peripheral angiogenesis etc. Thus, orthotopic models established from U251MG has been proposed to be a reliable and predictive tool for use in preclinical studies [4].

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Orthotopic Xenograft Models Generated from GBM Tumour Cells Grown in Neurobasal Medium Neurobasal medium is a serum-free growth medium supplemented with epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin, wherein the tumour cells retain their stem-cell characteristics and propagate as spheres. These neurospheres have been shown to possess molecular and genetic features similar to the original parental tumours, which remain essentially unchanged in subsequent passages. Moreover, these spheres are known to be highly tumorigenic, which display extensive infiltration and variable angiogenesis. However, sphere cultures may not develop successfully from all the human tumours, and the success rates may vary from 10% to 100% in various laboratories [9]. A study showed that single cell suspensions prepared from freshly resected GBM tumours grown in neurobasal medium are highly tumorigenic in neonatal SCID mice (even with 1000 cells); tumorigenic potential was retained even after several in vitro passages under non-differentiating conditions. In contrast, patientderived cells cultured in serum conditions were found to be non-tumorigenic at early passages, although latepassage cells were able to form tumours. However, intracranial tumours generated from neurobasal medium cultured cells showed extensive infiltration into the cerebral cortex and migration along the white matter tracts such as corpus callosum, a critical histopathological facet of GBM www.avidscience.com

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tumours in patients, whereas tumours from late-passage cells showed delineated growth and little infiltration into the surrounding brain, a characteristic of tumours derived from established cell lines. Transcriptomic profiling also revealed that xenograft tumours derived from neurobasal medium cultured cells were closely similar to their parental tumours as well as other primary GBM tissues. Thus, the study emphasized that orthotopic models generated from tumour cells grown under conditions retaining stem-cell characteristics are superior systems for developing targeted therapies and personalized medicines [16].

Patient Biopsy-Derived Tumours Grown as Spheroids for Transplantation These models are derived by intracranial transplantation of spheroids cultured from patient biopsy-derived tumour tissues. Briefly, the biopsy tissue is minced with surgical blades and transferred into agar-coated culture dishes containing serum-supplemented tissue culture medium, so that the tissues form spheroids (multicellular aggregates) within a short time. These spheroids maintain the original tumour tissue architecture with extracellular matrix components, macrophages, ploidy levels and gene expression profiles as the parental tumours. Orthotopic xenografts established from these spheroids show diffuse single cell infiltration, preserve the invasive characteristics of the original tumour, and accrue other histological features of human GBM upon subsequent passages. However,

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xenografts generated from different patient biopsies differ in their morphologies and angiogenesis characteristic, which are most likely due to the inter-tumoral genetic heterogeneity. The advantages of this model over other model systems are that several clonal sub-populations within the original tumour are maintained over several passages in vivo, thereby recapitulating the genetic heterogeneity and aberrations of the parental tumour. However, with these models, standardization and experimental planning is tricky, due to higher variability [9].

Generation of Orthotopic Murine GBM Xenografts from Patient Biopsies by Tissue Dissociation The excised tumour tissue is initially minced with a sterile scalpel, followed by mechanical disruption through repeated pipetting to generate a cell aggregate suspension. This is subsequently passed through a 70 µm nylon mesh filter to generate single cell suspension for intracranial injections [17]. Alternatively, mechanical and enzymatic dissociation using Miltenyi gentleMACS™ system also generates single cell suspensions. This cell suspension is centrifuged at 1000 rpm for 10 minutes at 4°C, and the cell pellet is resuspended in a suitable volume of serum-free media or phosphate buffered saline (PBS) for intracranial injections. The advantages of xenografts generated using this method are similar to those using the neurosphere cultures in serum-free medium, with an added advantage of circumventing the need for in vitro culture conditions. www.avidscience.com

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should be applied to the eyes to prevent drying of the cornea. 3. Anaesthetized mouse is mounted appropriately on the stereotaxic frame with the help of nose clamps and the ear bars, to prevent head movements. Head of the mouse is maintained at a suitable level before initiating the surgery [18].

Surgical Procedure Figure 1: An overview of the methods used for tumour cell transplantation to generate orthotopic xenografts. Image adapted and modified from [9] and [17].

Procedure for Generation of Orthotopic Xenograft Models Preparing the Mouse 1. Prior to beginning the surgical procedure, the entire area should be disinfected using 2% chlorhexidine [17] or other suitable disinfectants. 2. Mouse is anaesthetized by intraperitoneal injection of a mixture of Ketamine (90 mg/kg) and Xylazine (10 mg/kg) using a sterile 1 ml syringe fitted with a 30G needle. Mouse reaches a sleeping phase within 15 min; the sedation and pain response are monitored using a gentle toe or tail pinch withdraw reflex. Neosporin eye ointment 16

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1. Initially, the surface of the head is disinfected by 3 alternate applications each of povidone-iodine and alcohol, and a final application of 70% alcohol. 2. Using a sterile scalpel or scissors, a 10 mm to 15 mm long midline sagittal incision is made along the superior aspect of the cranium. The membrane between the skull and the skin is wiped off using a sterile Q-tip. 3. Bregma (junction of the coronal and sagittal sutures) and lambda (junction of the sagittal and lamboidal sutures), are used as landmarks for the stereotactic localization of target site for injection. Although different studies recommend various injection positions, the co-ordinates used in our laboratory is 2 mm right and 1 mm posterior of bregma, at a depth of 2.5 mm. 4. Using a Micro Drill with a sterile drill bit, a small www.avidscience.com

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burr hole is made as per the predetermined coordinates. 5. The optimum number of cells to be injected varies according to the type of cell lines; generally, about 3-5 × 105 cells are resuspended in 3-5 µL of serum-free media or PBS. Use of larger volumes may result in reflux of the tumour cells and formation of exophytic tumours [17]. 6. 3-5 µL of the cell suspension is mixed and loaded into a glass Hamilton syringe and is fitted into the needle holder of the stereotaxic apparatus to line up with the hole in the skull. 7. Slowly, the syringe is lowered till a depth of 3 mm below the surface of the skull. After waiting for 1 minute, the needle is retracted to 0.5 mm, and the cell suspension is injected at this position at a rate of 1 μl per minute for a total of 6-8 minutes, taking care to avoid any backflow. The needle is left in place for another 2 minutes to allow all the cells to settle in. 8. After slowly withdrawing the needle, the skull hole is sealed with bone wax and the incision on the skin is closed using a tissue adhesive (3M Vetbond). 9. The mouse is transferred to a heat pad maintained at 37°C, and is transferred to a cage upon regaining consciousness [18]. 18

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Figure 2: An overview of the procedure for generation of orthotopic xenografts. Image courtesy: [17].

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Applications of Orthotopic Mouse Models of GBM Orthotopic models remain the crux of fundamental and translational GBM research, with a broad scope of applications ranging from understanding tumorigenesis and tumour-biology, metastasis, preclinical testing of novel therapeutic agents and formulations, predicting therapy responses in patients, development of patient-specific therapies and studying therapy-resistance – a major detrimental characteristic of GBM. Some of the reported applications are described below:

Understanding the Biology of Human GBMs In order to recapitulate the biology of human GBM in preclinical settings, a library of orthotopic GBM models were established using surgical samples from GBM patients. The xenograft tumours were found to precisely replicate the morphological and pathological characteristics of their parental tumours, gene copy number alterations and mutations found in parental GBM. More importantly, the xenograft models were able to predict the therapy responses in the patients administered with conventional radiotherapy, chemotherapy using temozolomide – the commonly used drug in GBM, and targeted therapy using anti-angiogenic agents such as bevacizumab (VEGFneutralizing monoclonal antibody) [19]. Similarly, intracranial murine xenografts established using U251MG and U87MG were found to mimic several histopathological 20

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features of human GBM such as invasion into the brain parenchyma, neovascularization, immunostaining for vimentin, presence of infiltrating macrophages and T-cells [20]. Infiltrative growth pattern is a major characteristic of human GBM, which is poorly represented in subcutaneous xenografts, but is well-illustrated in orthotopic xenografts. An intracranial xenograft mouse model established using U1242MG cell line was found to display highly infiltrative growth pattern, geographic necrosis and tumour-induced vascular proliferation, which are the characteristic phenotypes of human GBM. Using this model authors could identify that the increased expression levels of MMP9 in these cell lines facilitates invasive growth by degrading the extracellular matrix components, inducing cell motility, cellular and vascular proliferation [21]. In another study GBM cell line developed from patient’s surgical sample was intracranially injected into immunocompetent mice, as opposed to the commonly used immunocompromised strains. These mice displayed tumour progression akin to those observed in GBM patients, such as induction of gliosis in surrounding brain tissue, high levels of angiogenesis, recruitment of microglia [22]. Understanding radio-chemo resistance is yet another aspect where studies have been done in orthotopic murine GBM model. To understand the role of EGFR amplification in mediating radioresistance in GBM, orthotopic xenografts were established from patient samples harbouring amplified EGFR. The study reports that EGFR amplification alone is inadequate in predicting the rewww.avidscience.com

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sponse to radiotherapy, although the use of EGFR inhibitors during and after radiotherapy may provide an added benefit in the treatment of some GBM tumours [23]. In yet another study an examination of CpG methylation within the MGMT promoter and temozolomide responsiveness in orthotopic patient-derived xenografts showed that the prediction of methylation status by standard MS–PCR (methylation-specific PCR) and/or qMS–PCR (quantitative MS-PCR) alone is not sufficient to predict therapy outcome with temozolomide [24]. Anti-angiogenic therapy such as bevacizumab (VEGFinhibitor) has been widely used in the treatment of GBM, with the aim of “normalizing” the abnormal tumour vasculature and decreasing vascular permeability, thereby improving the transport of oxygen and drugs to the tumours. However, decreased vascular permeability is not sufficient for providing long-term survival benefits to the patients, as tumour cells can adapt rapidly and “escape” from the anti-angiogenic therapy. Using orthotopic xenograft models established using U87MG cell lines, prolonged use of VEGF-inhibitor was found to cause increased tumour cell invasion, similar to those seen in clinical scenario, in patients who received anti-VEGF therapy [25]. These kinds of studies are only possible in an orthotopic mouse model.

Examining Tumour Progression in GBM Using Non-Invasive Imaging Modalities Monitoring disease burden has been a major hindrance in the orthotopic xenograft models, owing to in22

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accessibility of the tumour tissues for visual inspection. Thus, animals had to be sacrificed at various time points for inspecting the gross morphology and histopathology of tumours, which required the use of a large number of animals for a single study. The advent of non-invasive small animal imaging methods such as ultrasound (US), magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) have aided in overcoming the major shortcoming of monitoring disease burden in orthotopic models [26]. MicroPET/ CT imaging was used for in vivo monitoring of tumour growth in orthotopic models established using U87MG cells, and also for assessment of treatment response with standard 2 Gy fractionated radiation versus pulsed-low dose radiation, administered as 0.2 Gy pulses for 10 fractions with interpulse intervals of 3 mins. Pulsed low-dose radiation was found to be more effective than the standard treatment in delaying tumour growth, along with reduced damage to the normal neuronal tissues [27]. Recently, bioluminescence imaging has gained popularity as a noninvasive, semi-quantitative approach for localizing small tumours, metastasis, and for longitudinal examination of therapy response in tumour-bearing mice. The major advantage of bioluminescence imaging lies in its extreme sensitivity, which can be useful in detecting a minimal number of tumour cells present very early during initiation of a primary tumour or a metastatic disease, before they can be observed by other imaging methods. The unwww.avidscience.com

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derlying principle of bioluminescence imaging is the production of light by enzymatic reaction between luciferase and its substrate luciferin, in the presence of ATP and O2. This light is captured by a super-cooled charged coupled device (CCD) camera within a light-proof chamber. For bioluminescence imaging, genetically engineered tumour cells expressing luciferase are used for establishing orthotopic tumours [26]. MicroCT and bioluminescence imaging was performed for monitoring GBM progression and response to radiotherapy in intracranial GBM xenograft models established using U87MG cell lines expressing luciferase. Bioluminescence intensity measurements were found to significantly correlate with the tumour volumes measured by microCT in these models, underpinning the significance of these non-invasive imaging modalities in translational GBM research. Combined use of both bioluminescence imaging and contrast-enhanced microCT aids in early stage monitoring of tumour growth and measurement of tumour volumes, respectively, to accomplish small animal treatment-planning using dedicated softwares that rely on CT data for dose calculations, as performed in clinical settings [28].

Monitoring Therapy Response and Testing of Novel Therapies Since GBM patients as yet exhibit poor prognosis, a large number of studies have been carried out with the aim of testing new therapies and response to therapies in 24

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GBM, using murine orthotopic xenografts as a preclinical model system. Some of these studies are highlighted in the table below: Mouse strain Nude athymic mice

Source of tumour xenograft U87MG and U251MG cell lines

Nude athymic mice NMRI Nude mice

SNB-19 and LN18 cell lines

NODSCID and Nude mice

T98G cells in NOD-SCID mice and SNB19 cells in nude mice

NODSCID mice

A172 cells and 19 human GBM explants

Nude athymic mice

U87MG cells overexpressing or knocking-down PP6c

Balb/c nude mice

U87MG, U373MG cells and primary GBM578 cells

R28 brain cancer stem cell (CSC) line

Findings

Reference

Elevated expression of the endoplasmic reticulum (ER) chaperone GRP78, a key pro-survival component of the ER stress response system has been implicated in chemoresistance to chemotherapy. This study showed that inclusion of the green tea component EGCG (epigallocatechin 3-gallate), which inhibits GRP78 function, along with temozolomide significantly increased survival in orthotopic GBM models, as compared to temozolomide alone. Combination treatment by intrathecal injections of hTERT siRNA and IFN-γ at the site of tumour implantation was found to inhibit angiogenesis and tumour formation in nude mice. YB-1 is a multi-functional protein regulating transcription and translation, and is one of the downstream phosphorylation substrate of both PI3K/AKT and RAS/MAPK pathway. This protein is highly expressed in brain CSCs, and is responsible for drug resistance. Ad-Delo3-RGD is an adenoviral vector, dependent on YB-1 for its replication. Intratumoral injection of AdDelo3-RGD into orthotopic models developed from temozolomide resistant GBM cell line was found to significantly improve survival than mice treated with temozolomide alone. TWIST1 is a transcription factor which mediates cancer cell metastasis through regulation of EMT. Overexpression of TWIST1 in T98G and SNB19 GBM cell lines was found to increase invasion in orthotopic xenografts, by increased expression of genes associated with cell adhesion, cell motility, migration, actin cytoskeletal organization and extracellular matrix proteins. O6-methylguanine–DNA methyltransferase (MGMT) repairs O6-methylguanine in DNA, generated by the chemotherapeutic agent Temozolomide, and hence, tumours with methylated MGMT promoter show increased sensitivity to temozolomide. In this study, the base excision repair enzyme alkylpurine–DNA–N-glycosylase (APNG), which repairs N3-methyladenine and N7-methylguanine has been found to mediate temozolomide resistance. Upon treatment with radiation and temozolomide or temozolomide alone, median survival was found to be significantly enhanced in ANPG-negative than ANPG-positive orthotopic xenografts established from human GBM explants; survival was further increased in MGMT promoter methylated (MGMT negative) tumours. Similar results were obtained upon administration of temozolomide therapy to orthotopic xenograft models established using A172 cell lines expressing ANPG, MGMT or both PP6 is a serine/threonine protein phosphatase, which plays a role in IR-induced phosphorylation of histone H2AX, is a component of the DNA-PK complex that mediates non-homologous end joining repair of DNA double strand breaks. The catalytic subunit of PP6 (PP6c) has been shown to be upregulated in 44.7% of GBM patients; its level was found to correlate with increased radioresistance and poor survival. Overexpression of PP6c was found to be associated with increased radioresistance and reduced survival in orthotopic xenograft models, whereas PP6c knockdown correlated with enhanced radiosensitization and improved survival, thereby emphasizing the role of PP6 as a regulator of radiosensitivity in GBM cells. Studies aimed at understanding molecular mechanisms of radioresistance in GBM are imperative for improving median survival of GBM patients. In this study, activation of stem cell-associated pathways, including Wnt signalling was found to be critical mediators of radioresistance in GBM.

[29]

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[30]

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Conclusion and Future Perspectives On the whole, orthotopic mouse models have opened up a whole new arena for replicating the biological and clinical characteristics of human GBM, along with setting up a preclinical platform for testing novel therapeutics. Though cell lines do not adequately represent the genetic aberrations present in the original tumour from which they were derived, they have remained as a preferred source of tumour cells for intracranial implantations, on account of their availability and reproducibility in terms of engraftment rate, which makes standardization less complicated. However, development of targeted therapies, patient-specific treatments and prediction of therapy response in patients require the use of tumour biopsies, for which sphere cultures in neurobasal medium or spheroids grown in agar-containing medium are preferable. Thus, the choice of the tumour tissue for transplantation should be decided on the basis of prime application of the xenograft models. As outlined in this chapter, the ranges of application of these xenograft models are enormous. Prediction of therapy response is the most important application of orthotopic models derived from patient-specific tumours, which will help in distinguishing therapy responders from the non-responders. Multiple longitudinal tumour biopsies can be performed on these xenograft models, to monitor and characterize the mechanisms of differential response in responders versus non-responders, a practice which is impossible to be performed in patients. Therapyresistance, an inevitable facet of human GBM can also be 26

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addressed at the molecular level using orthotopic mouse models, which will help open new avenues for novel therapies aimed at pre-sensitization of tumour cells to the current therapies or elimination of resistant cells. Overall, it can be expected that the preclinical knowledge generated from these orthotopic models have immense potential for translation into clinical practice, ultimately improving patient life and providing them with survival benefits.

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