Brain-Tumor-Regenerating 3D Scaffold-Based Primary Xenograft

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Brain-tumor regenerating 3D scaffold based primary xenograft models for glioma stem cell targeted drug screening Kottarapat Jeena, Cheripelil Abraham Manju, Koythatta Meethalveedu Sajesh, G Siddaramana Gowd, Thangalazhi Balakrishnan Sivanarayanan, Deepthi Mol C, Maneesh Manohar, Ajit Nambiar, Shantikumar V Nair, and Manzoor Koyakutty ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00249 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Biomaterials Science & Engineering

Brain-tumor regenerating 3D scaffold based primary xenograft models for glioma stem cell targeted drug screening

Kottarapat Jeena,1 Cheripelil Abraham Manju,1 Koythatta Meethalveedu Sajesh,1 G Siddaramana Gowd, 1 Thangalazhi Balakrishnan Sivanarayanan,2 Deepthi Mol C,1 Maneesh Manohar,1 Ajit Nambiar,3 Shantikumar V Nair,1 Manzoor Koyakutty 1*

1

Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Amrita Institute of Medical Sciences, Ponekkara, Kochi 682 041, India 2

Central lab animal facility, Amrita Vishwa Vidyapeetham, Amrita Institute of Medical Sciences, Ponekkara, Kochi 682 041, India

3

Department of Pathology, Amrita Vishwa Vidyapeetham, Amrita Institute of Medical Sciences, Kochi 682 041, India

KEYWORDS glioblastoma multiforme, glioma stem cells, 3D scaffold, patient derived xenograft

AUTHOR INFORMATION Corresponding Author *[email protected]

ACS Paragon Plus Environment

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

Glioma stem cells (GSC) present a critical therapeutic challenge for glioblastoma multiforme (GBM). Drug screening against GSC demands development of novel in vitro and in vivo platforms that can mimic brain microenvironment and support GSC maintenance and tumorigenesis. Here, we report, a 3-dimensionel (3D) biomimetic macro-porous scaffold developed by incorporating hyaluronic acid, porcine brain extra cellular matrix (ECM) and growth factors that facilitates regeneration of GBM from primary GSCs, ex vivo and in vivo. After characterizing with human and rat GBM cell lines and neurospheres, human GSCs expressing Notch1, Sox-2, Nestin and CD133 biomarkers were isolated from GBM patients, cultured in the 3D scaffold and implanted subcutaneously in nude mice to develop patient derived xenograft (PDX) models. Aggressive growth pattern of PDX with formation of intratumoral vascularization was monitored by magnetic resonance imaging (MRI). Histopathological and phenotypial features of the original tumors were retained in the PDX models. We used this regenerated GBM platform to screen novel siRNA nanotherapeutics targeting Notch, Sox-2, FAK signaling for its ability to inhibit the tumorigenic potential of GSCs. Current clinical drug, temozolomide and an anticancer phytochemical, nanocurcumin, were used as controls. The siRNA nanoparticles showed excellent efficacy in inhibiting tumorigenesis by GSCs in vivo. Our study suggests that the brain-ECM mimicking scaffold can regenerate primary gliomas from GSCs in vitro and in vivo and the same can be used as an effective platform for screening drugs against glioma stem cells.


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INTRODUCTION Recurrence of glioblastoma multiforme (GBM) is inevitable in ~ 90% of patients, despite administration of all the standards of care treatments including surgical resection, radiation and chemotherapy. The diffuse infiltration of glioma stem cells (GSC) into the brain parenchyma and their interaction with extra-cellular matrix (ECM) leads to adherence mediated drug-radiation resistance and recurrence.1 GSC functions within their specialized ecological niches, governed by the cues obtained from the microenvironment.2 One of the major challenges for developing drugs against GSCs was the lack of appropriate screening platforms that can support the maintenance of human GSC phenotype and tumorigenic potential in vitro as well as in vivo. Maintenance of GSCs in experimental models demands mimicking of the unique biochemical and cellular composition of tumor microenvironment.3 The GBM microenvironment includes the brain ECM, non-tumor cells such as astrocytes, macrophages, fibroblasts, growth factors and differentiation factors. The major components of brain-ECM are hyaluronic acid (HA), a nonsulphated glycoaminoglycan; and a family of aggregating proteoglycans called lecticans (neurocan, brevican, versican and aggrecan) and proteins such as tenascin and thrombospondin.4 HA is known to create an environment that facilitates the proliferation, invasion and survival of GBM cells. A number of studies have implicated that the interaction of HA with its cell receptor CD-44 and receptor for hyaluronan-mediated motility (RHAMM) promotes glioma progression and migration.5,6 The common ECM components (eg. fibronectin and collagen) are scarcely present in the brain.7 Therefore, the heterogeneity present in the complex ECM of brain tissue is an important consideration while designing substrates for neuronal culture, by maximizing the inclusion of ECM components from the specific tissue.8


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Earlier efforts to study glioma-ECM interaction in 3D environment were using hydrogels of collagen-I, laminin,9 synthetic HA,10 HA hydrogels,11 HA incorporated matrigels,12 and chitosan-alginate scaffold.13 Though these studies provided tremendous insight into the rigidity, mechanics, physio-chemical properties and glioma cell-ECM interactions, they do not fully support glioma stem cells based GBM regeneration which is critical for evaluating advanced drugs that can target the tumorigenic potential of GSCs.14-15 Although cell line based 3D culture and xenograft models have been reported,16-17 study on brain ECM mimicking scaffold that supports the growth of patient derived GSCs into regenerated GBM, which could be transplanted in to well vascularized PDX model for testing the tumorigenicity of GSC as a drug target has not been reported. Here, we have optimized a simple, 3D porous scaffold for GSC based primary GBM regeneration. HA loaded with appropriate concentrations of de-cellularized porcine brain-ECM and stem cell growth factors were used for the preparation. The scaffolds were formed by lyophilization and initial characterization for its ability to support neurosphere and tissue regeneration was done with human GBM cell lineT98G, and rat glioma cell line C6. Single-cell cultures of primary GSCs characterized by stem cell markers Notch, Sox-2, Nestin, CD133, derived from five human subjects were used for the final experiments. Further, the ex vivo expanded 3D tumor cultures were serially implanted in nude mice to develop patient-derivedxenograft models (PDX). In vivo vascularization and tumor tissue growth with concurrent degradation of the scaffold material were studied using MR imaging. Finally, the scaffold derived PDX models were used for screening the potential of three model drugs, temozolamide, nanocurcumin and nano-siRNA (FAK, Notch, Sox-2) for their ability to inhibit the tumorigenic potential of GSCs in vivo.


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MATERIALS AND METHODS Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Dulbecco’s Modified Eagle Medium-F12 (DMEM-F12), Pen-Strep, Antibiotic-antimycotic, Fetal bovine serum (FBS), Phosphate buffered saline (PBS), Minimum Essential Media (MEM), 0.05% trypsin-EDTA, and Alamar Blue reagent were purchased from Gibco, Invitrogen. Rat glioma C6 cells were purchased from National Centre for Cell Sciences, Pune, India and human glioma cell line T98G was purchased from American Type Culture Collection (ATCC). Cells were maintained in fully supplemented DMEM-F12 (rat glioma C6) or MEM (human glioma T98G) with 10% FBS and 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO2 in a fully humidified incubator. Glioma stem cells were grown in serum free DMEM-F12 media supplemented with human basic fibroblast growth factor (hbFGF)and human epidermal growth factor (hEGF) (20 ng/ml; R&D Systems) and B-27 (50 µg/ml; Invitrogen). Decellularization of porcine brain. Adult porcine brain was decellularized as per protocol.18 The dura mater was removed and chopped into finer pieces (~ 1 cm), and incubated in distilled water overnight at 4°C under constant stirring at 120 rpm. It was then incubated in the following supplemented with 1% antibiotic-antimycotic in agitated baths at 140 rpm: 0.05% trypsin-EDTA in phosphate-buffered saline (PBS) at 37°C for 1h, 3% triton X-100 in PBS for 1h, 1M sucrose solution for 15 min, distilled water for 15 min, 4% sodium deoxycholate in distilled water for 1h, 0.1% peracetic acid in 4% ethanol for 2h and finally, in distilled water overnight at 4°C. The decellularized brain tissues were then lyophilized and stored at -20°C.


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Brain matrix solubilization. The decellularized brain ECM was solubilized by enzymatic digestion.19 Porcine pepsin was dissolved in 0.1M hydrochloric acid at 1mg/mL under agitation for 48 hrs and then sterile filtered through a 0.22 mm filter. Once solubilized, the brain matrix was mixed with basal media and neutralized with 1 M NaOH. The brain matrix was then stored at -20°C. Histological analysis. The brain samples (native and decellularized), were fixed for 24 hr in 10% neutral buffered formalin, sectioned in to 3 µm thickness and stained with hematoxylin and eosin. Scanning Electron Microscopy (SEM) analysis. The decellularized matrix and native brain tissue were fixed with 3% glutaraldehyde. After fixing, the specimens were dehydrated through a series of ethanol gradients (35-100%), critical point dried and sputter coated with gold before imaging with a JEOL JSM-6490LA Analytical SEM, Japan. Synthesis of 3D porous scaffold. Scaffolds were prepared by dissolving gelatin (2 wt%) and alginate (1 wt%) separately to 5 ml water and the solutions were mixed and allowed to completely dissolve before adding hyaluronic acid (2 wt%). The solutions were stirred together 2000 rpm for 5 hrs. The solution was then cast in tissue culture plates of 24 wells, frozen at -20 o

C overnight, and lyophilized for 24 h. The scaffolds obtained were then transferred to 2%

calcium chloride solution containing 50 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and 100 mM N-Hydroxysuccinimide and allowed to crosslink overnight. They were then washed 3 times with excess distilled water. The scaffolds were again lyophilized for over 24 h. The solubilised brain matrix was then coated on the scaffold and incubated overnight at 4°C.Growth


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factors hEGF and hbFGF were added in to the scaffold and again lyophilized for 2 h. Scaffolds were then sterilized with ethylene oxide gas for in vitro and in vivo trials. In vitro characterization of scaffold Scanning electron microscopy. Physical morphologies of scaffolds were studied using scanning electron microscopy SEM (JEOL JSM-6490LA Analytical SEM, Japan).Scaffolds were vacuum-dried overnight to remove moisture and then gold-coated using a sputter coater (HitachiE-1030). Porosity studies.

The pre-weighed scaffolds were immersed in dehydrated alcohol for 48 h,

until the scaffolds were fully saturated. Porosity was determined using the liquid displacement method, by the equation, Porosity =Wet weight − Dry weight /Density × Volume× 100. In vitro degradation studies. For in vitro degradation studies, the previously weighed scaffolds o

were immersed in PBS and lysozyme solution, incubated at 37 C for 28 days. At specific time intervals, the scaffolds were removed from the solution, washed with deionized water and dried. The percentage degradation was calculated using the formula, Initial weight−Dry weight/ Initial weight× 100.20

Cell seeding on scaffold.

The adhesion of stem cells formed from rat glioma C6 on the

scaffolds was examined using SEM analysis. The scaffolds were seeded with cells (1 x 103) and cultured for 7 days. Cells were then fixed using 2% glutaraldehyde for 1 h followed by dehydration, in graded ethanol series (50%, 75%, 95%, 100%) and observed under SEM. Samples were critical point dried and sputter coated with gold before imaging with a JEOL JSM-


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6490LA Analytical SEM, Japan. False colour was added to SEM images using Adobe Photoshop. In vitro stem cell growth on the scaffold. Scaffolds were sectioned into 3 mm circular discs and incubated overnight in PBS buffer (pH 7.4). They were then placed in a conditioned medium for 2 h before stem cells (1×103 cells per scaffold) were seeded in to the matrices. Stem cells were also seeded on scaffolds containing brain ECM alone without growth factors and kept as control. Stem cells grown from rat glioma C6 in 24 well plates were also kept. . The proliferation of cells was examined by Alamar Blue Assay using Beck-mann Coulter Elisa plate reader (BioTek Power Wave XS). All experiments were performed in triplicates. Viability was also assessed by using Live/Dead staining with calcein AM and ethidium homodimer (Molecular Probes, Invitrogen, USA). Primary tumor cell culture. Patients suspected of glioblastoma multiforme underwent a magnetic resonance imaging in GE3Tesla MR scan (Dept. of Radiology, Amrita Institute of Medical Sciences, Kochi). Resected brain tumor tissue of GBM patients was collected from the operating theatre (Amrita Institute of Medical Sciences, Kochi), with written consent from patients in accordance with institutional guidelines. The tumor tissues were graded pathologically according to the WHO criteria by the Department of Pathology, Amrita Institute of Medical Sciences, Kochi. The regions of clotted blood were removed and tumors cut in to smaller pieces, washed in PBS and dissociated using 2% collagenase IV (Gibco, Invitrogen). Digestion was stopped with double the volume of DMEM/F-12 complete media and the tissue was minced finely with Pasteur pipettes and filtrated through a 40 µm cell strainer (BD Falcon). Red blood cells were removed by density centrifugation in cell lysis buffer. For neurosphere cultures, cells were seeded at a density of 1 × 105 cells/ml in DMEM/F-12 serum free medium


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containing pen-strep and growth supplements hbFGF and hEGF (20 ng/ml) and B-27 (50 µl/ml). After 3-4 days, neurospheres were formed. Flow cytometry analysis of CD133 positive cells. Glioma stem cells were stained with antiCD133 antibody (Milteny, Biotec), for 30 min and analyzed by flow cytometry FACS Calibur (Becton Dickinson, San Jose, CA). Stem cell characterization in scaffold Immunofluorescence imaging. Immunofluorescence analysis was performed on the 7th day after seeding the glioma stem cells on the scaffold matrix. The sections were fixed using 4% paraformaldehyde for 20 min, and then washed thrice with PBS. The sections were then washed with 1% triton- X for 5 min, washed thrice with PBS, blocked with 1% FBS in PBS for 30 min at room temperature. The sections were stained with neural stem or progenitor markers such as Nestin (Alexa Fluor 647 mouse anti-nestin, BD), Sox-2 (Alexa Fluor 488 mouse anti-Sox 2, BD), NOTCH-1(PE mouse anti-human Notch-1,BD), GFAP and CD133. Cell nuclei were counterstained with propidium iodide and immunofluorescence viewed by confocal microscopy. Establishment of patient derived xenograft model (PDX) in nude mice. Athymic nude female Balb/C mice (nu/nu) 6–8 weeks of age, were anesthetized with a solution of ketamine before the scaffolds containing glioma stem cells derived from patients were implanted subcutaneously. Tumors were measured using vernier calipers and the volume was calculated using the formula = π/4 (length × width × height). After 4 weeks of implantation, mice were sacrificed by CO2 inhalation, and the tumors were resected, fixed in 10% formalin solution, and histological analyses done using hematoxylin and eosin (H&E). Magnetic resonance imagings of the animals were performed on Biospec 7 Tesla MRI system (Bruker, Germany). Each resected tumor


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xenograft of the patient was examined, characterized and compared to H&E slides from the original patient tumors by neuropathologists. Preparation of therapeutics for anti glioma drug screening. Temozolomide (TMZ) was prepared by dissolving in DMSO (10mg/ml) and its aliquots (1 mg/ml) in serum free cell culture media. Nanocurcumin was formulated by using microemulsion technique. Briefly, PLGA and curcumin (9:1) was dissolved in acetone and stirred for 2 hours to get a uniform solution. The polymer solution was then added drop by drop into a 0.5% w/v pluronic F-68 water solution by stirring at 1000 rpm to produce nanoparticles. Acetone was evaporated at room temperature. The particles were centrifuged at 10000 rcf and supernatant was discarded. The resulting pellet was resuspended in ultrapure water and subsequently lyophilized using mannitol as cryoprotectant. It was then redispersed in serum free culture media (5 mg/ml) for cell culture studies. Nano siRNA cocktail was prepared by complexing the siRNAs (NOTCH, FAK and SOX-2) with protamine sulphate at an N/P ratio of 10 in aqueous phase. Nanotherapeutic drug screening using PDX models in vivo. Female Balb/c nu/nu mice were divided into four groups (n=5). Stem cells (2x104) isolated from GBM patient (Patient #1) were cultured in scaffold for 7 days before subcutaneously implanting these scaffolds into nude mice. Scaffolds were pretreated with different antiglioma nanotherapeutic (temozolomide-500 µM), nanophytochemical (nanocurcumin-300 µM), and nano siRNA cocktail (n-siRNA FAK + NOTCH+ SOX- 2 combination

-200 nM) to test the response of these drugs treatments in 3D PDX model in

vivo. Treatment efficacy was assessed in terms of effect of different anti-glioma therapeutics on the tumor volumes of treated mice relative to the tumor volumes of control mice. Tumor growth inhibition was calculated as the ratio of the median tumor volume in the treated group (T) versus


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the median tumor volume in the control group (C): T/C% = (median tumor volume of treated group on day Y/median tumor volume of control group on day Y) x 100. Statistical analysis. Acquired data are expressed as mean ± SD. Statistical significance was determined by one way analysis of variance (ANOVA) and Student’s t test. Values of P < 0.01 were considered significant. Results and Discussion 1. Decellularization of brain ECM The brain-ECM derived through decellularization of porcine brain was stained with hematoxylin and eosin to confirm the absence of cells and nuclear material. The images (Figure 1a&1b) showed complete absence of nuclei and cellular components in the decellularized brain matrix compared to native brain tissue, indicating that total removal of cells without loss of brain matrix, was achieved through the decellularization protocol. This was also validated by the SEM images of the native brain tissue (Figure 2a) presented in comparison with decellularized porcine brain matrix (Figure 2b).


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Figure 1. (a) Hematoxylin and eosin stained sections of porcine brain matrix. (b) decellularized brain porcine brain matrix showing complete absence of cells. Scale bars are 100 mm. 2. 3D porous scaffold synthesis and characterization The biomimetic scaffolds were prepared by lyophilization and cross linking HA, alginate and gelatin with calcium chloride, EDC and NHS (Figure 2c). Alginate was used for better mechanical integrity while retaining the biocompatibility and low immunogenicity,21 and gelatin was used for the presence of Arg-Gly-Asp (RGD) sequences which promotes cell adhesion.22 Brain ECM and growth factors, Epidermal Growth Factor (hEGF) and Fibroblast Growth Factor (hbFGF) were coated on to the scaffold and and subjected to freeze-drying to encapsulate them inside the pore walls of the already formed scaffold. hEGF is a key factor in GBM cellular proliferation, tumor angiogenesis and increased vascular permeability.23 hbFGF also plays a significant role in the progression of GBM, contributing to proliferation, angiogenesis, and survival.24 Decellularized tissue will preserve the original organization of ECM with all the bioactive molecules necessary for stem cell maturation. Synthesis of the scaffold was done using these critical components. The scaffold morphology was analysed by SEM (Figure 2d), which showed a highly porous internal structure that allows the cells to penetrate throughout the scaffold and provide a large area for cell attachment and proliferation. Porosity studies indicated that the scaffold (Figure 2e) had an average pore size of 85 µm. The in vitro degradation of the scaffold was evaluated by placing the scaffold in lysozyme for 28 days. The degradation was found to be 25% at the end of 28 days. This denotes the stability of the scaffold in physiological conditions (Figure 2f).


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Figure 2. SEM imaging of (a) native porcine brain tissue (b) decellularized porcine brain matrix (c) 3D porous scaffold (d) 3 D porous scaffold with ECM coating (e) photograph of 3D porous scaffold (e) photograph of 3D porous scaffold (f) in vitro degradation study of 3D porous scaffold.


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3. Cell morphology, proliferation and viability in scaffold Cell attachment, morphology and distribution was analysed through SEM analysis. The scaffold was seeded with rat glioma C6 and cultured in vitro for 14 days. Rat glioma C6 were seeded in serum free media with growth supplements (hEGF, hbFGF, and B27) in 24 well plates for 2D culture (Figure 3a). Stem cells grown from rat glioma C6 or neurospheres on the 3D scaffold showed a rounded and spheroid appearance which resembles the structure of in vivo tumor cells (Figure 3b). SEM observations show that the scaffold promoted aggregations of cells on day 4 (Figure 3c), which resulted in the formation of multilayered cellular spheroids or tumor clusters by day 7 (Figure 3d) that is typical of an in vivo malignant phenotype. Thus, the scaffold provides the appropriate cell-ECM interactions needed to form tumor spheroids. The presence of neural progenitor cells in the neurospheres was identified using nestin. The neurospheres were stained positive for nestin indicating the presence of neural precursor cells (Figure 3e). The proliferation rate of stem cells in scaffolds was assessed by Alamar Blue Assay. A differentially increased proliferation was observed (from day 3) in the scaffolds due to the presence of ECM but a significantly elevated growth pattern was seen in the scaffold loaded with both the brain ECM and growth factors. This indicates that ECM with additional growth factors promoted aggressive growth of glioma cells in the scaffold. (Figure 4a).

The

cytocompatiblity of the scaffold was also assessed by Live/Dead staining assay with calcein and ethidium homodimer after 72 h using confocal microscopy. The image suggests that more than 95% of cells adhering to scaffold were viable and metabolically active as indicated by the intensely green flourescent stain in live cells (Figure 4b). The cell attachment and infiltration of cells in to the scaffold studied by DAPI staining using confocal microscopy (Figure 4c) showed a visual cell attachment in all layers indicating successful cell migration throughout the scaffold.


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Figure 3. (a) Phase contrast image of C6 spheroid formation in 2D culture. SEM imaging of stem cell spheroid formation of rat glioma C6 in scaffold on (b) 2nd day (c) 4th day (d) 7th day (e) Confocal imaging showing nestin positive staining of C6 stem cells indicating neural progenitor cells


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4. In vivo tumorigenesis and angiogenesis of tumor formed from 3D porous scaffold Human glioma T98G cell line is an experimentally important cell line as it expresses MGMT (O6-methylguanine–DNA methyltransferase), which is responsible for the temozolamide resistance of GBM. But human glioma T98G has a relatively weak tumorigenic potential in vivo even in severely immunocompromised mice when injected with Matrigel. To test if our scaffold supports the human glioma T98G tumor growth, 1x104 cells were seeded into scaffold and cultured. SEM analysis revealed spheroid formation of cells inside the scaffold (Figure 5a). To further evaluate in vivo, the scaffolds with neuropsheres were subcutaneously implanted in 5 Balb/c nu/nu mice and tumor growth was monitored for 30 days (Figure 5b). All the five animals showed significant tumor growth, indicating that the scaffolds were able to successfully establish human glioblastoma in mice. (Figure 7a). MR imaging of the animal enabled clear visualization of the concurrent biodegradation of the scaffold with the development of tumor mass (Figure 5c ). The xenograft tumors formed were explanted 30 days after implantation. Visible blood vessel formation and angiogenesis were seen in all the tumors which were closely examined under a stereomicroscope (Figure 5d). The high tumor growth rate and proliferation under in vivo condition may correlate with recruitment of blood vessels that provided necessary, nutrients and fluid exchange needed for tumor growth. Immunohistochemical staining for Ki-67, a marker of cell division, showed areas of active tumor proliferation (Figure 5e). Histological analysis of the glioma tumor grown in athymic nude mice clearly shows the blood vessel vasculature and nuclear staining in cells (Figure 5f). Thus,

the scaffolds could be used to develop otherwise tough to establish

T98G human glioma tumors in vivo for drug screening purpose.


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Figure 4. (a) Proliferation pattern of stem cells from rat glioma C6 in 2D culture on 24 well plates, scaffold with brain ECM alone, scaffold with brain ECM and growth factors (hEGF, hbFGF on days 3, 7 and 14 of cell culture as determined by Alamar Blue assay (b) Image of


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Live/Dead staining after 72 h in 3D porous scaffold (c) DAPI staining showing viability of cells in scaffold. **P < 0.01; *** P < 0.001, by one-way ANOVA (n=3) compared to scaffold with brain ECM alone 5. Propagation and evaluation of glioma stem cells isolated from patient samples Numerous studies have demonstrated that glioma stem cells or neurospheres isolated from primary brain tumors have the capacity to differentiate in to cell types present within the parental tumor.25-26 The neurosphere formation can be used as an independent

predictor of

clinical outcome of malignant glioma.27 These cells can be used to establish xenograft tumors with all the features of human GBM. The in vivo models of glioma orthotopic transplantation are established by intracranial injection of cell suspension with craniotomy. This procedure is complicated involving challenging issues. Establishing a patient derived subcutaneous xenograft in a 3D scaffold with the microenvironment for glioma stem cells to propagate is convenient not only to observe the tumor volume by direct measurement, but also to evaluate the effects of anticancer drugs.28 We were able to establish human glioma T98G tumors in nude mice. Further, we extended this work to create clinically relevant predictive model for GBM by isolating stem cells from GBM patients and propagating them in the scaffold. As a preliminary study, we had taken resected tumor tissue samples from five GBM patients with consent. Clinical data of these patients are summarized in Table1. The MRI of the patient whose tumor tissue was used for the study is shown in Figure 6a. The MR image shows a T2 flair hyperintense lesion on the right tempero-parietal lobe of the patient. Glioma stem cells were isolated from the tumor samples of patients as per protocol , cultured in DMEM F-12 media supplemented with hEGF (20 ng/ml), hbFGF (20 ng/ml) and B-27 (50 µl/ml). The stem


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cell marker, CD133, was examined on all primary samples and represented 0.1-15% of the total population examined by flow cytometry (Figure 6b). Culturing in serum-free supplemented media allows amplified proliferation of the small subpopulation of GBM stem cells while maintaining their cell characteristics. Neurospheres were formed within 24-48 h of culturing (Figure 6c).


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Figure 5. (a) 2D culture of human glioma T98G cell line (b) Balb/c nu/nu mice bearing GBM tumor (c) coronal view of MRI images of a mouse bearing scaffold with GBM.(d) stereomicroscopic view of tumor showing angiogenesis (e) IHC staining of anti- Ki67 staining showing active nuclear proliferation (f) H&E of human glioma T98G cells 6. Stem cell characterization in scaffold Stem cells isolated from patients (1x104) were seeded in to the scaffold in supplemented media. Cells were allowed to attach and proliferate in the scaffold for seven days. The stemness of the isolated cells in the scaffold were assessed by staining for stem cell markers; Notch, Sox2, Nestin, CD133 and GFAP. Nestin is a prognostic marker for GBM stem cell malignancy. GFAP a class-III intermediate filament, is a cell-specific marker that distinguishes astrocytes from other glial cells and Sox-2 is required for maintaining the embryonic stem cells, pleuropotency and self renewal.29 Notch plays an important role in the proliferation, differentiation and regulation of cancer stem cell population.30 CD133 is an important cancer stem marker as it initiates neurosphere growth and helps in the formation of tumors when transplanted in to immunocompromised mice.31 Cells grown in scaffold showed expression of all the above markers, suggesting that scaffold maintains the stemness of GBM cells by providing an appropriate microenvironment inside the scaffold (Figure 6d).


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Table 1: Patient characterization table Patient

Gender

Age

Pathology

Primary/Recurrent

1

F

61

2

F

20

Central neurocytoma (grade II)

Primary

3

M

21

Low grade glioma (grade II)

Primary

4

M

53

Glioblastoma multiforme

Primary

5

M

41

Oligo astocytoma grade II

Recurrent

Left temporal glioma

Primary


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Figure 6. Representative figures of patient sample characterization. (a) MR imaging of patient showing GBM (b) FACS of patient sample with GBM showing presence of CD133 (c) Primary cultures of adult GBM forming neurospheres in serum free culture supplemented with hEGF, hbFGF (20 ng/ml) and B27 (50 µl/ml) (d) Expression of Sox-2, Nestin, Notch, CD133, and GFAP measured by immunofluorescence in confocal microscopy. Nuclei was counterstained red with PI (e) Balb/c nu/nu mice bearing patient derived GBM tumor (f) Single slice from a series of transaxial, T2-weighted MRI images of a mouse bearing PDX (g) FACS analysis of xenograft tumor from nude mice showing presence of CD133 and nestin. (h) H&E of PDX tumor grown in nude mice.

7. In vivo PDX model development and characterization The GSC populated scaffolds (5 different patients) were implanted subcutaneously in to the nude mice. Interestingly, all the implanted scaffolds formed tumors (100%) of approximately 1-4 cm3 (Figure 6E).This clearly shows that the scaffold supported tumorigenicity of GSCs in vivo. MR imaging of the animal showed the presence of a visible tumor mass (Figure 6f). The tumor formed in vivo was characterized after resection on day 30, using flow cytometry (Figure 6g). The isolated cells were assessed for CD133 and nestin expression, which showed a very similar pattern as that obtained of the corresponding GBM patient. Although, detailed genomic and proteomic analysis is required for thorough characterization, we believe that, the phenotypical characteristics of original tumor is broadly maintained in the PDX. A hallmark of tumor progression in GBM is high vascularisation and angiogenesis. The PDX showed visible blood vessel and angiogenesis in all resected tumors, which was also confirmed in the histopathology


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(Figure 6h). An accelerated tumor growth pattern was observed in all cases (Figure 7A). The recruitment of new blood vessels and angiogenesis may have accelerated the growth potential as supply of blood and nutrients are essential for tumor growth. The recapitulation of patient tumor heterogeneity is important in establishing PDX models. Histopathological characterization of PDX models revealed that the primary glioma patient tissues propagated in nude mice conserved key morphological characteristics of original parental tumors (Figure 7b). Thus, we were successfully able to establish PDX models from GBM stem cells from patient samples using the scaffold. This offers a robust 3D platform for testing anti-glioma drugs in vitro and in vivo.


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Figure 7. (a) In vivo growth rates of tumors formed from implants of human glioma T98G and GBM patients as determined by caliper measurements (n=5) (b) Tumor biopsy samples from GBM patients and samples from PDX models implanted with the respective neurosphere cultures. Tumors were formalin fixed, paraffin embedded, sectioned and stained with H&E. Representative images are shown for primary tumors biopsy and corresponding xenograft.


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8. In vivo screening of anticancer drugs targeted to glioma stem cells in PDX model One of the main applications of 3D scaffold mediated ex vivo GBM tissue culture and PDX model is to study the efficiency of chemotherapeutics and nanomedicines towards the inhibition of tumorigenesis. Here, we have tested the efficiency of three different anti-glioma therapeutics to study their ability to inhibit the tumorigenicity of GSCs. For a model experiment, we have isolated GSCs from patients, formed neurospheres in the scaffold and then treated with the drugs under testing; temozolomide, nanocurcumin, and nano-siRNA targeted to key stem cell signaling- FAK, Notch and Sox2 (n-siRNAFAK+NOTCH+SOX-2). After seven days of treatment, the scaffolds were implanted in to nude mice. The median volume of tumor on day 30 in untreated control was 3.3 cm3 whereas, all the treated groups exhibited significantly reduced tumor growth compared to the control. The tumor to control (T/C) volume fraction for TMZ or nanocurcumin treated group was 38% and 44.6%, respectively. In contrast, treatment with stem cell targeted combinatorial siRNA nano-therapeutics showed significant reduction in the tumor volume (T/C= 2%) (Figure 8). This clearly shows that the nano-siRNA formulation was most efficient in inhibiting aggressive tumor growth compared to TMZ or nanocurcumin. In effect, we showed that 3D scaffold based tissue engineered PDX is an excellent platform for testing and optimizing the tumorigenic-inhibitory capacity of new therapeutics against GSC.


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Figure.8. Tumor growth pattern of patient derived xenograft (PDX, n=5) mice pretreated for 7 days with temozolomide(500 µM), nanocurcumin-(300 µM), and nano siRNA cocktail (n-siRNA FAK + NOTCH+ SOX- 2 combination

-200 nM) compared to control. **P < 0.01; *** P < 0.001, by

one-way ANOVA.

Conclusions We have developed a 3D brain-tumor regenerating scaffold mimicking brain ECM and supporting the growth of primary glioma stem cells in vitro and in vivo. The scaffold facilitated proliferation of multiple glioma cell lines, primary tumor cells and GSC based neurospheres derived from GBM patients. Further, PDX models could be established using the tumor regenerating scaffold and the xenografts showed pathophysiological characteristics similar to


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that of original tumor tissue. We used this system for the preclinical screening of three model drugs such as temozolamide, nanocurcumin and glioma stem cell targeted nano-siRNA therapeutics in vitro as well as in vivo. Compared to the clinical drug TMZ, and phytochemical, the siRNA nanoformulation showed excellent efficiency in inhibiting the tumorogenic potential of GSC in vivo. In effect, we show that brain ECM mimicking 3D scaffold based regeneration of glioma using GSCs from patients can facilitate screening and optimization of novel therapeutics against glioma stem cells.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors are grateful to the Department of Biotechnology (DBT), Government of India for the financial support under the project ‘Targeted Silencing of Cancer Stem Cell Signalling Using Novel Nano- siRNA conjugates’(BT /PR/2413/AGR/36/699/2011) for the financial support and also thankful to Amrita Vishwa Vidyapeetham University for the excellent infrastructure for research. ACKNOWLEDGMENT We also thank Mr. Sajin P. Ravi and Mr. Sarath S for the technical assistance in characterization using SEM and confocal microscopy.


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For Table of contents graphic Brain-tumor regenerating 3D scaffold based primary xenograft models for glioma stem cell targeted drug screening Kottarapat Jeena, Cheripelil Abraham Manju, Koythatta Meethalveedu Sajesh, G Siddaramana Gowd, Thangalazhi Balakrishnan Sivanarayanan, Deepthi Mol C, Maneesh Manohar, Ajit Nambiar, Shantikumar V Nair, Manzoor Koyakutty *


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Brain-Tumor-Regenerating 3D Scaffold-Based Primary Xenograft

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