The effect of cytosine deaminase-expressing mesenchymal stem cells in orthotopic glioma model of immune competent mice

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The effect of cytosine

deaminase-expressing mesenchymal stem cells in

orthotopic glioma model of immune

competent mice

By

Ha Vu Hai

Major in Neuroscience

Neuroscience Graduate Program

The Graduate School, Ajou University

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The effect of cytosine

deaminase-expressing mesenchymal stem cells in

orthotopic glioma model of immune

competent mice

By

Ha Vu Hai

A dissertation Submitted to The Graduate School of

Ajou University for Degree of Master in Neuroscience

Supervised by

Haeyoung Suh-Kim, Prof.

Major in Neuroscience

Neuroscience Graduate Program

The Graduate School, Ajou University

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This certifies that the dissertation of Ha Vu Hai

is approved.

SUPERVISORY COMMITTEE

_______________________________

Haeyoung Suh-Kim

_______________________________

Sung-Soo Kim

_______________________________

Young-Don Lee

The Graduate School, Ajou University

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The effect of cytosine deaminase-expressing

mesenchymal stem cells in orthotopic glioma model of

immune competent mice

ABSTRACT

Glioblastoma multiforme (GBM) is the tumor common deriving from glia and aggressive primary brain tumor. Current standard therapy provides only modest improvements in progression-free and overall survival of patients. Most of the preclinical studies for glioma utilize immune deficient animals, which limits the assessment of role of immune system in glioma progression and therapy. However, the contribution of innate immune system in glioma progression cannot be excluded in clinical settings.

In this study, an orthotopic glioma mouse model were established using a syngeneic mouse glioma cell line, GL261 in immune competent wild type C57BL/6 mice. GL261 cells (3x104 cells) were transplanted to the striatum and tumor growth kinetics was assessed by MRI imaging and histological analyses. The therapeutic effects of mesenchymal stem cells (MSC) that was previously engineered to express a suicide gene, cytosine deaminase (MSC/CD) was evaluated in this model. MSC/CD with administration of a

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prodrug 5-florocytosine (5-FC) was effective to suppress tumor growth. Therapeutic effect of MSC/CD with 5-FC was further enhanced when followed by treatment of temozolomide. Compared to nude mice, the overall survival of the animals treated with MSC/CD and 5-FC was higher in C57BL/6 mice, suggesting contribution of systemic immune system. Histological analysis revealed that more immune cells including macrophage/microglia, T lymphocytes were infiltrated to the tumor sites. The result indicate that combination therapy of MSC/CD with 5-FC and TMZ possess potent anticancer effects by direct chemoablation and subsequent activation of innate immune system.

Keywords: Mesenchymal stem cells, Temozolomide, Cytosine deaminase,

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LIST OF FIGURES

Figure 1. Cytosine deaminase and metabolism of 5-FU and 5-FC ...4 Figure 2. Migratory property of MSC toward tumor site ...5 Figure 3. Cytotoxic effect of TMZ in cancer cells ...7 Figure 4. Characteristics of GL261 cells were not altered by GFP labeling.14 Figure 5. Suicide effect of MSC/CD...15 Figure 6. Bystander effect of MSC/CD on GL261/GFP glioma cells. ...17 Figure 7. Synergistic effects of 5-FU and TMZ on GL261/GFP glioma cells ...18 Figure 8. Synergistic interaction of MSC/CD with 5-FC and TMZ on GL261 glioma cells ...19 Figure 9. Kinetics of tumor growth: ...21 Figure 10. Synergistic efﬁcacy of MSC/CD with TMZ in syngeneic glioma model...23 Figure 11. Inflammatory response of immune system in preclinical model.25 Figure 12. Critical function of immune system synergistically contributed the combination of TMZ and MSC/CD therapy to prolong life-span of glioma-bearing mice ...28

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ABBREVIATION

MTT 3,4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide 5-FC 5-Fluorocytosine 5-FU 5-Fluorouracil CD Cytosine deaminase CI Combination index DMSO Dimethyl sulfoxide FBS Fetal bovine serum GBM Glioblastoma multiforme GFP Green fluorescence protein HSA Human serum albumin I.P Intraperitoneal injection

IC50 The half maximal inhibitory concentration M.S. Median survival

MRI Magnetic resonance imaging MSCs Mesenchymal stem cells O.S. Overall survival

P.O Oral administration TMZ Temozolomide

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INTRODUCTION

Glioblastoma multiforme (GBM)

Glioblastoma multiforme (GBM) is the most common tumor of malignant brain tumor in the central nervous system. GBM has a poor prognosis and high incidence of recurrence because it infiltrates the brain parenchyma due to suboptimal resection, innate resistance of tumor cells to therapeutic agents, and impermeability of the blood-brain barrier (BBB) to most chemotherapeutic agents. Brain resection followed by a concurrent treatment of chemotherapy and radiotherapy remains the most effective therapy and adopted as a standard of care. However, such standard of care extends the median survival period for glioma patients by 7 to 9 months(Stupp R et al. 2009).

Anti-cancer therapies for GBM patients Immunotherapy

Adoptive therapeutics is a strikingly specialized anticancer therapy that concern both immune cell administration and direct anticancer activity to the cancer-bearing host. Cytokine induced killer (CIK) cells are major histocompatibility (MHC)-unrestricted cytotoxic lymphocytes that can be generated in vitro from peripheral blood mononuclear cells and cultured with the addition of interferon (IFN)-γ, interleukin (IL)-2, and CD3 monoclonal antibody (CD3mAb). Recently, the application of CIK cells has evolved from experimental studies to phase II clinical studies. Since the first clinical trial of adoptive immunotherapy using CIK cells in 1999, a growing number of trials have suggested that CIK therapy is associated with a significantly prolonged mean survival and control of disease progression. Recent studies demonstrated

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that immunotherapy with CIK cells improved clinical outcome and promoted the quality of life in cancer patients.

These studies supported the contention that immune responses could actually contribute to the success of anticancer therapies and possibly eliminate residual tumor cells resistant to the action of cytotoxic drugs. Results from the group led by Kroemer and Zitvogel (Apetoh L et al. 2007) further supported an essential role for immune responses in the efficacy of some anticancer therapies. Upon dissecting the molecular bases accounting for the remarkable ability of these cytotoxic agents to promote anticancer immune responses(Logg CR et al. 2012; Ghiringhelli FApetoh L 2013). The aforementioned findings may suggest that future anticancer therapies will most probably rely on combinatorial treatments associating chemotherapy with immunomodulation.

Suicide gene therapies

Herpes simplex virus thymidine kinase (HSV-tk) with ganciclovir (GCV)

The suicide gene/prodrug system applied to prostate cancer, ovarian cancer and GBM patients in clinical trials is HSV-tk with GCV (Alvarez RD et al. 2000; Immonen A et al. 2004; Nasu Y et al. 2007). Thymidine kinase can convert GCV to its cytotoxic metabolite, GCV-triphosphate that subsequently inhibits activity of DNA polymerase by utilizing its cytotoxic effects. However, the bystander effects of HSV-tk on cancer cells are restrained to permeate into cellular membrane because GCV-triphosphate could only be transported through gap junction (Portsmouth D et al. 2007).

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5-FC anti-cancer drug and Cytosine Deaminase (CD) gene

5-Fluorouracil is one of the most commonly used chemotherapy agents for auxiliary therapy of patients with solid tumor such as colon, breast, prostate and liver cancer more than 40 years. 5-FU is conceded to be an effective anticancer drugs on gastric cancer as either mono-therapy or relying on a combinatorial treatment (Ohtsu A et al. 2000; Longley DB et al. 2003). However, 5-FU has shown limited delivery to brain blood barrier which lead to the least effective response in clinical tests of malignant glioma patients. However, the irregular structure of vascular vessel in glioma, eliminating the possibility of poor delivery but rather indicating the heterogeneity of glioma (Schneider et al. 2004). Therapeutic reception of glioma patients relies on individuals even treated same drug. Even for GI-tumors, only 10-30% of patients are positive response to 5-FU, further implying the inherent heterogeneity.

Cytosine deaminase (CD) enzyme converts the prodrug, 5-fluorocytosine (5-FC) into the 5-fluorouracil (5-FU), leading to direct cytotoxicity of the cancer cells. This treatment modality has immense pertinence in various tumor types (Ghiringhelli FApetoh L 2013).

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Figure 1. Cytosine deaminase and metabolism of 5-FU and 5-FC Stem cells as cellular vehicle of therapeutic genes

MSCs have recently been derived from various sources, including bone marrow, adipose tissue, and umbilical cord blood. The strong tropism toward brain tumors of MSCs were explained by exhibiting that are attributed to receptors for chemokines and growth factors including SDF-1, MCP-1, HGF, IL-8, NT3, TGF-β and VEGF. The migratory properties make MSCs as a powerfully potential cellular vehicle for delivering therapeutic genes to brain tumors (Chang DY et al. 2010; Imitola J et al. 2004; Nakamizo A et al. 2005; Kuratsu J-i et al. 1993; Kendall SE et al. 2008).

Neural stem cells (NSCs) have essential tumor-tropic properties that can be utilized for targeted delivery of therapeutic agents to invasive and metastatic tumors. NSCs can conquer the major restrictions to achieve the high efficacy in treatments by their capability penetrating the glioma region regardless BBB. For use as delivery vehicles, NSCs have been modified to express a ectopic anti-cancer genes, including prodrug-activating enzymes, apoptosis-inducing agents, antibodies, and oncolytic viruses (Aboody K et al.

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2008; Frank RT et al. 2010). The engineered NSCs were injected intra-cerebrally to exert robust therapeutic efficacy in orthotopic glioma model (Ahmed AU et al. 2011), medulloblastoma (Gutova M et al. 2013), melanoma brain metastases (Aboody KS et al. 2006), neuroblastoma (Danks MK et al. 2007) and primary and metastatic breast cancer (Seol HJ et al. 2011).

Previously, we demonstrated that mesenchymal stem cells (MSCs) have a high tropism for brain tumors, and engineered MSCs to express a bacterial CD gene (MSC/CD) that successfully suppressed tumor growth in a rat glioma model (Chang DY et al. 2010). Using the tumor-tropic MSC as a cellular vehicle for delivering the CD gene would enable effective conversion of 5-FC to 5-FU close to tumor sites, thereby localizing the cytotoxicity. Therefore, this stem cell-mediated suicide gene-prodrug therapy may resolve the challenges of delivering 5-FU while keeping the therapeutic index high.

(Chang et al. Int J Cancer 2010; 97:12846)

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Temozolomide

Temozolomide (TMZ) is a prodrug that is extraordinarily stable at an acidic pH, resulting in 100% oral bioavailability. In slightly basic pH environment of the blood and tissue, TMZ is spontaneously hydrolyzed into its active metabolite, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, which sequentially methylate DNA at the N7 and O6 positions of guanine, and the N3 position of adenine (Nagasawa DT et al. 2012) . The guanine-thymine mismatches created by O6-methylguanine causes DNA breaks if the lesions are not repaired by O6-methylguanine DNA methyltransferase (MGMT), a DNA methylation repairing enzyme. TMZ crosses the BBB with tolerable systemic toxicity, to halt or slow brain cancer cell growth. The standard of care for GBM currently includes surgery followed by fractionated radiotherapy (RT) with adjuvant daily TMZ administration. In a phase III trial, combination of chemotherapy and radiotherapy significantly increased median survival rate and overall survival rate compared to that observed with radiotherapy single treatment (Stupp R et al. 2005). Although the survival advantage of the combined treatment lasted for up to 5 years in the long-term follow-up study, in most patients, the tumors recurred and eventually patients died because of tumor burden. The antitumor effect of TMZ is less efficient by high level of MGMT expression in cancer cells (Damia GD’Incalci M 1998; Stupp R et al. 2006). Inversely, the methylation in promoter region of MGMT gene induces epigenetic silencing that prevents synthesis of MGMT, leading to cancer cells sensitize to TMZ (Hegi ME et al. 2005). The correlation of MGMT expression levels and TMZ sensitivity are essential to develop alternative, multi-modal therapies for cancer patients who cannot receive

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remarkable efficacy from TMZ treatment because of intrinsic resistance to MGMT.

Figure 3. Cytotoxic effect of TMZ in cancer cells

(L. Liu et al. Clin Cancer Res. 2006, 12(2):328-331.) It is worth of studying whether MSC/CD therapy or combination of TMZ and MSC/CD therapy exert better therapeutic effects when combined system immune surveillance. For example, the tumor antigen can be exposed after cell death induced by MSC/CD+ 5FC and intrinsic immune cells such as dendritic cells better recognize tumor antigen, and boost cytotoxic T cells that subsequently eradicate tumors. This study can be done in an immune-competent mouse model. Here, we will find the contribution of micro-environmental factors surrounding a nest of neoplastic tumor cells including blood vessel, immune cells infiltration. We will also determine the effects of those factors with respect to the lifespan of the GBM animals.

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MATERIALS AND METHODS

Cultivation of syngeneic murine glioma cell lines

GL261 cells were obtained from The Jackson Laboratory#000664 and tested for the absence of mycoplasma infection. GL261 cells were cultured in RPMI medium, supplemented with 10% fetal bovine serum (FBS, Australia, Cat# SH30084.03) 1% L-glutamine (Gibco, Cat#) and 1% pen/strep (Gibco, Cat# 15140-122). All experimental protocols using human MSCs were approved by the Institutional Review Board of the Ajou University Medical Center (Suwon, South Korea). GL261 cells were transduced with lentiviral vector that was produced with pGFP-C-shLenti in 293T cells using a standard method. Briefly, 293T cells were plated in a density of 2x106 cells in 100-mm culture dish. Next day, the cells were transfected with plasmids including: pGFP-C-shLenti (10 µg), pΔ8.9 (8 µg) and pVSV-G (5 µg) by a standard calcium phosphate precipitation method. Eight hours later the medium was replaced with fresh one and the viral supernatant was collected twice at 48 hours and 72 hours after transfection. Viral supernatant was filtered through a 0.45 µm filter and used for transducing GL261 cells with 8 µg/ml polybrene for 8hrs. Viral transduction was repeated one more and the GFP-positive GL261 cells were FACS sorted to enhance the purity.

Characterization of GL261 cell line

GL261 cells were plated 1x104 cells/well in 24 well-plate. Twenty-four hours later, 5FU and TMZ were individually added to culture medium with serial concentration (0; 0.3; 1; 3; 10; 30 µM of 5-FU and 0; 10; 30; 100; 300; 1000 µM of TMZ)The medium was replaced every 2 days with same concentration of 5-FC. Five days later, the medium was aspirated and subject to MTT assays. Briefly, the remaining cells were incubated in PBS containing 0.5 mg/ml 3,4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide

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(MTT) for 2 hours at 37oC. After removal of the supernatant, dimethyl sulfoxide was added to the cells and allowed 5 min to dissolve the purple

formazan product. The formazan in the DMSO solvent was transferred to 96 well plates and absorbance was measured at 540 nm. The OD540nm was used to represent the mitochondrial NADPH activity and indirectly surviving cells.

In vitro suicide effect of MSC/CD

Cryopreserved MSC/CD cells were thawed and were plated in a density of 1x104 cells/well in 12 well plates. Twenty-four hours later, 5-FC was added to the culture to a final concentration of 1~1,000 µM. The medium was replaced every 2 days with same concentration of 5-FC. Seven days later, the medium was aspirated and subject to MTT assays. Briefly, the remaining cells were incubated in PBS containing 0.5 mg/ml 3,4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) for 2 hours at 37oC. After removal of the supernatant, dimethyl sulfoxide was added to the cells and allowed 5 min to dissolve the purple formazan product. The formazan in the DMSO solvent was transferred to 96 well plates and absorbance was measured at 540 nm. The OD540nm was used to represent the mitochondrial NADPH activity and indirectly surviving cells.

In vitro bystander effects

Cryopreserved MSC/CD cells with indicated numbers were simultaneously plated with 1x104 GL261/GFP glioma cells per well in 12 well plates. Twenty-four hours later, various concentrations of 5-FC was added to the culture and medium was replaced every 2 days. Seven days later, remaining cells were rinsed with PBS and lysed in 200 µl 1x Lysis buffer (Promega, Madison, WI, USA) for 10 min at 4o_{C. The cell lysates were} transferred to the centrifuge tubes and centrifuged at 10,000 x g for 10 min at

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4oC. The supernatant was transferred to 96 cell plates and the fluorescence was measured using fluorometry (Molecular Devices, Sunnyvale, CA, USA)

Isobologram analysis

To assess the interaction of 5-FU and TMZ, the cells were incubated in the presence of various concentrations of 5-FU and TMZ. Five days later, the cells were subject to MTT assays and isobologram analysis. The IC50 value of each drug was determined using dose response assays. The synergism between 5-FU and TMZ was quantified by determining the combination index (CI), which is a quantitative evaluation of a two-drug pharmacological interaction:

CI = C1/S1 + C2/S2

S1, S2 indicated the concentration of 5-FU or TMZ, whereas C1 and C2 were doses of the respective drugs in the combination required to produce the same effect that S1 or S2, the dose of each drug would produce. The combined effect of both drugs can be summarized as, CI = 1, < 1, and > 1 indicate summation (additive with no interaction), synergism, and antagonism, respectively. The CI was calculated using the CompuSyn ver 1.0 program (ComboSyn Inc, Paramus, NJ, USA) for each isobologram point. For the isobologram analysis of combined bystander effect of MSC/CD with 5-FC and TMZ, the IC50 of the each test drug was first determined by evaluating its individual dose-dependent cytotoxicity, and then each value was further evaluated using a fixed concentration of another test drug. The interaction between 5-FC and TMZ was then quantified by determining the CI as described above. The IC50 values for all the assays were plotted, and a hypothetical additive line was drawn between two IC50 values from either drug alone. Above and below the additive line are the hyper- and hypo-additive regions, indicating antagonistic and synergistic interactions, respectively (Zhao L et al. 2004).

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In vivo therapeutic effects

Wild-type C57BL/6 mice, athymic nude BALB/c mice (6 weeks age) were anesthetized with intra-peritoneal injection by 75 mg/kg ketamine and postured in a stereotaxic device (Stoelting, Wood Dale, IL, USA) for right cerebral hemisphere. GL261/GFP cells were harvested by trypsinization, washed three times with PBS, and finally resuspended in PBS with a density of 1x104_{cell/µl and inoculate 3x10}4_{cells/mouse (3 µl).Frozen MSC/CD cells} were thawed in 37°C water-bath and absolutely removed DMSO by 5% HSA. And then, mixture of MSC/CD and GL261/GFP were resuspended in PBS with a density of 3x104 cells in 3µl. These cells were inoculated into the striatum (anteroposterior, +0.05 cm; mediolateral, -0.18 cm; dorsoventral, -0.3 cm) using a Hamilton micro-syringe. After surgery, the skin was closed with a clip. The animals were orally administered with 5-FC (1,000 mg/kg/day twice a day)for 7 consecutive days and TMZ was intraperitoneally injected with 5 mg/kg/day for 5 days. The body weights were measured twice a week.

Histological analysis

Mouse brains were removed and fixed for 24 h in 4 % paraformaldehyde in PBS. The next day, these samples were processed in paraffin embedding assay. For immunohistochemistry studies, primary antibodies used were: microglia-specific anti-Iba1 (1:2000; Wako cat.# 019-19741); anti-CD3 (1:100 abcam cat.# ab5690); anti-CD8 (1:100, eBioscience 14-0808); anti-CD4 (1:100, eBioscience 14-9766). After the first incubation, these section were incubated with secondary antibody anti-rat (1:250,#) and anti-rabbit (1:250,#), followed by development using 3,3′-diaminobenzidine (DAB) horseradish peroxidase (HRP) substrate (Vector Laboratories).

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MRI analysis of tumor growth

Glioma-bearing mice were imaged in a 15.2T MR scanner. Thirty slices of brain at 0.3 mm slice thickness were consecutively taken per each mice. Images were analysis using MicroDicom (Escott EJRubinstein D 2003).

Tumor Area (mm3) = Number of pixels of tumor area

Number of pixels per unit area x Depth

Statistics analysis

Results from at least three independent in vitro experiments were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post-hoc test. For animal studies, the results were analyzed using Student’s t-test for single comparisons or ANOVA, followed by Tukey’s HSD post-hoc test for multiple comparisons. Values are expressed as the mean S.E

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RESULTS

5-FU Sensitivity of glioma cells was preserved after reporter gene labeling

Morphology of GL261/GFP glioma cells were similar to those found in naïve GL261. Subsequently, the 5-FU sensitivity of GL261/GFP and naïve GL261 also were assessed whether GFP expression might altered cell characteristics. MTT assays measuring the mitochondrial activity of surviving cells showed IC50 values of GL261/GFP and naïve GL261 were 2.4 µM and 2.7 µM, respectively. The date indicated that negligible changes were made by transduction of reporter genes in primitive morphology and 5-FU sensitivity.

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(A)

(B)

Figure 4. Characteristics of GL261 cells were not altered by GFP labeling. (A) The morphology of naïve GL261 and GL261/GFP. (B) The

sensitivity to 5-FU of naïve GL261 (black circle) and GL261/GFP (white circle). Cells were plated 1x104 cells/well (24 well plate) and then incubated with different concentration of 5-FU for 5 days. MTT assays measuring the mitochondrial activity of surviving GL261 and GL261/GFP cells showed IC50 values of 2.4  0.3 µM and 2.7  0.1 µM, respectively. The data indicated that negligible changes were made by transduction of reporter genes in morphology and 5-FU sensitivity. (*p <0.05, use T-test).

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In vitro Suicide effects of MSC/CD

To test the effect of the suicide CD gene on the MSC/CD, they were cultured with 5-FC for 7 days. While the survival rate of the MSC remained consistent, the MSC/CD underwent cell death with an IC50 value of 67.3μM 5-FC.

(A)

(B)

(C)

Figure 5. Suicide effect of MSC/CD

(A).CD-expressing retroviral vector was produced in packaging cell

line, FLYRD18. MSCs were transduced with a retroviral-vector and selected in puromycin for 2 weeks. (B).MSC/CD express the CD gene by RT-PCR analysis. (C).MSC/CD was similar to proliferation and morphology of native MSCs. However MSC/CD was showed suicide effect in the culture medium containing 5-FC. The IC50 value of MSC/CD was 67.3 M.

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In vitro Bystander effects of MSC/CD against GL261/GFP cells

To confirm the bystander effect of MSC/CD with 5-FC against glioma cell lines,GL261/GFP cells were plated with MSCs or MSC/CDs in 1:1 ratio and incubated in growth medium containing the indicated concentrations of 5-FC for seven days. Fluorometric analysis revealed that the IC50 for GL261/GFP cells was 84 μM. However, when less MSC/CD (3x103_{or 1x10}3 cells) were used in the co-culture with 1x104 Gl261/GFP cells, the bystander effect of 5-FC with IC50 values of 173 and 433.2 µM, respectively. The parental MSCs did not exert any cytotoxic effects on glioma cells even in the presence of this highest 5-FC concentration. Fluorometric assays indicated that the bystander effect of MSC/CD and 5-FC was dependent not only the concentration of 5-FC, but also on the ratio of therapeutic MSCs to glioma cells.

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(A)

(B)

Figure 6. Bystander effect of MSC/CD on GL261/GFP glioma cells. (A)GL261/GFP cells were plated with MSCs or MSC/CDs in various

ratio and incubated in growth medium containing the indicated concentrations of 5-FC for seven days. (B) Fluorometric analysis revealed that the IC50 for GL261/GFP cells was 84  4.1 μM. However, when less MSC/CD (3×103_or 1x103_{cells) were used in the co-culture with 1x10}4_{Gl261/GFP cells, the} bystander effect of 5-FC became evident at concentrations >100 µM with IC50 values of 173  25 and 496  85.5 µM (*p <0.05, ANOVA single factor). The IC50 values were described above (*p<0.05; ANOVA)

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Combination effect of TMZ and 5FU in GL261/GFP cells in vitro

To investigate the mechanism of the synergistic effects of MSC/CD with 5-FC and TMZ, we used 5-FU which is metabolite product of 5FC with present of CD gene. The IC50 values of 5-FU alone were 3.0 μM, and 90.8 μM for TMZ alone. The MTT assay used various TMZ concentration combined with different fixed 5-FU concentrations. Using the dose-response viability data, a normalized isobologram was constructed, and IC50 values from were plotted using the Chou-Talalay method. The CIs were <1, which corresponded to hypo-additive points and verified that 5-FU and TMZ interacted synergistically.

Figure 7. Synergistic effects of 5-FU and TMZ on GL261/GFP glioma cells

The GL261/GFP were cultured in the presence of the indicated concentration of 5-FU and TMZ for 5 days. Viability of cells was measured by MTT assay. Combination of 5-FU and TMZ showed synergistic effect. Values are presented as the mean ± S.E at least three independent experiments.

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Combination effect of MSC/CD+5-FC and TMZ in GL261/GFP cells

Figure 8. Synergistic interaction of MSC/CD with 5-FC and TMZ on GL261 glioma cells

To evaluate the combinatorial interaction of MSC/CD with 5-FC and TMZ, MSC/CD and GL261/GFP were co-cultured with 5-FC and TMZ. The IC50 values of 5-FC and TMZ alone were 71.3 and 20 μM, respectively. Importantly, combined treatment lowered the drug’s individual IC50 values interactively. To evaluate the combinatorial interactions, the IC50 values shown in plotted using the Chou-Talalay method and the combination indices were < 1, which corresponding to hypo-additive points and indicated 5-FC and TMZ acted synergistically.

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Figure 9. Kinetics of tumor growth:

(A) 3x104 GL261/GFP cells resuspended in 3 µl PBS were intracranially inoculated into striatum as described in method part. Mice were sacrificed at indicated day (1d, 7d, 10d, 19d, n=3). (B) Representative MR images showed tumor growth with 15.2 Tesla. Tumor volume were measured using MicroDcom softwave (4d, 11d, 18d) (n=3).

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In vivo therapeutic effect

TMZ were cytotoxic to both cancer and normal cells when administered systemically. To avert TMZ’s cytotoxicity on the MSC/CD and retain the synergy, we sequentially treated the mouse glioma model with MSC/CD followed by 5-FC first and then TMZ. Next day, 5-FC and TMZ were administrated by oral administration with 1000 mg/kg twice in a day, during 7 days and intraperitoneal injection with 5 mg/kg, one time in a day, during 5 days, respectively. A similar TMZ dose finding study as for the. Concentration of TMZ (5 mg/kg) in combination with a fixed dose of 5-FC (1000 mg/kg) equal with GBM patients which is the dose used during first-line chemotherapy in clinical trials were administered. All mice were sacrificed on day 20 after glioma cell inoculation. Mice with large tumor burden, regardless of treatment group, showed weight loss. Importantly, the tumor areas decreased more dramatically with the combination treatments. Treatment of single MSC/CD-5-FC regimen and TMZ or combination resulted in statistically reduced tumor areas compared with the control group. These results demonstrate a synergistic efficacy when combining MSC/CD with TMZ treatment in syngeneic orthotopic glioma model. The Kaplan-Meier graph indicated that the median survival of the control group alone was 19 days, similarly, MSC/CD treated tardy tumor state group (D10), while that of the MSC/CD at tumor initial state groups (D0 and D1, respectively) showed a slight increase of 7 days.

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(A)

(B)

(C) (D)

Group M.S. O.S. Mean

PBS ₁₈ ₂₆ _20.3

MSC/CD 26 29 27.1

TMZ 31 35 31.6

MSC/CD + TMZ 43 49 42.5

Figure 10. Synergistic efﬁcacy of MSC/CD with TMZ in syngeneic glioma model

(A) Schematic diagram to evaluate antitumor effect of MSC/CD + TMZ in

syngeneic mice GL261/GFP glioma cells and MSC/CD cells were transplanted with 3x104_{cells and administrated 5-FC and TMZ at indicated}

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time points with 1000 mg/kg/day P.O. of 5-FC and 5 mg/kg/day of TMZ in C57BL/6 mice, respectively. (B) At 19d, glioma-bearing mice were taken MRI to measure tumor mass. The tumor volume (mm3) showed synergistical antitumor effect of MSC/CD and TMZ therapy in syngeneic glioma mouse model (n=5) (C).The Kaplan-Meier graph indicated that the survival of mice (n=8).

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Contribution of immune-component factors in preclinical model

(A)

(B)

Figure 11. Inflammatory response of immune system in preclinical model (A) Immunohistochemistry analysis in tumor including CD3+pan T cells, CD4+Th cells, CD8+Tc cells, Iba1+microglia/macrophage. (B) Quantification of CD3+pan T cells, CD4+Th cells, CD8+Tc cells (n=3) and

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percentage of CD3+/CD4+Th cells, CD3/CD8+Tc cells, CD11b+macrophage, B220+B cells in blood by FACS analysis (n=5)

Population of CD3+pan-T cells, CD4+Th cells significantly increased in MSC/CD, TMZ single treated mice and combination of MSC/CD and TMZ treated mice compared with PBS group, furthermore, CD8+_{Tc cells population} in therapy treated mice were higher than non-treat mice. On these other hands, the number of CD3+_/CD4+_{Th, CD3}+_/CD8+_{Tc and CD11b}+_{macrophage were} increased in the blood in treated groups compared to PBS groups. However, the number of B220+B cells were not altered between treated groups and PBS groups. These observation suggested that CD3+pan-T cells and CD4+Th cells play important role in prevent glioma growth in MSC/CD + 5-FC and TMZ therapy. Microglia and macrophages population, are also known antigen-presenting cells in brain, infiltrate into tumor site particularly therapy treated mice, however their function within this study were still unclear. These results further support a mechanism of action for the combination of MSC/CD and TMZ that involves activation of an antitumor immune response.

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Critical function of immune system influent to tumor rejection in preclinical model

(A)

(B)

(C)

Group C57BL/6 Athymic Balb/c M.S. O.S. Mean M.S. O.S. Mean

PBS 23 26 23 23 25 23.4

MSC/CD 28 35 31 27 28 26.4

TMZ 35 37 34.6 30 32 29.8

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Figure 12. Critical function of immune system synergistically contributed the combination of TMZ and MSC/CD therapy to prolong life-span of glioma-bearing mice

(A) Schematic diagram to examine function of immune system

contributed the combination of TMZ and MSC/CD therapy. (B) Immunocompetent mice (C57BL/6) were inoculated with GL261/GFP glioma cells and treated with MSC/CD and TMZ. (C)Immunocompromised (Athymic Balb/c mice)were inoculated with GL261/GFP glioma cells and treated with MSC/CD and TMZ.The survival rates of animals were shown in Kaplan-Meier graph. Treatment with MSC/CD + TMZ elicited higher therapeutic effects in immune-competent mice compared immunocompromised mice. The results indicate that immune components play an important role in suppressing tumor growth in a preclinical model (n=5).

To evaluate effect of the role of immune components which contribute MSC/CD with TMZ therapy, immunocompetent mice and immunocompromised mice were inoculated GL261/GFP glioma cells and single treated with MSC/CD, TMZ or combination of MSC/CD and TMZ with same scheme and results were shown survival rate of mice by Kaplan-Meier graph.In the immune-compromised mouse model, mice that received MSC/CD, TMZ or combination of MSC/CDand TMZ showed poor short-term survival compared with PBS group. On the other hand, in similar experiments in immune-competent mice, glioma-bearing mice treated therapy led to statistically signiﬁcant efﬁcacy and long-term survivors.Therefore, immune components play an important role in eradicating tumor in our preclinical model.

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DISCUSSION

Effective suicide gene therapies rely on strong bystander cytotoxicity and successful delivery of therapeutic genes to tumor sites. In this study, I showed that MSCs can overcome physical barriers to serve as efficient vehicles for delivering suicide gene to brain tumors.

Several recent studies have shown that MSCs can be utilized to deliver therapeutic genes to brain tumors. Genetically modified MSCs that express tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Kim SM et al. 2008), interleukin-2 (IL-2) (Nakamura K et al. 2004) or interferon  (Nakamizo A et al. 2005) have been shown to inhibit tumor growth and prolong the lifespan of tumor-bearing animals. However, these therapies may not be effective for all types of brain tumors, since some tumors can escape from TRAIL-mediated cell death (Malhi HGores GJ 2006) or immune surveillance (Watters JJ et al. 2005). In our study, MSC/CD originated from human displayed robust antitumor effect in fully immunosurveillance model- immunocompetent mice model. The glioma-bearing mice treated with combination MSC/CD (D0) and TMZ (D10) exhibited a more significant improvement in median survival of mice compared with control groups. These results demonstrate a synergistic efﬁcacy when combining MSC/CD with TMZ treatment in syngeneic orthotopic glioma model.In this study, I demonstrated a human-applicable, ex vivo therapeutic modality utilizing bone marrow-derived MSCs as cellular vehicles to deliver therapeutic CD gene into brain tumors. My outcomes underscore the potential advantages of auto-graft MSCs over allograft NSCs for the treatment of established tumors.

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Of note, Bracci et al highlight that tumor cell death after treatment with select chemotherapeutic agents can reveal tumor antigens and promote epitope spread through ICD. Additionally, several chemotherapeutic agents have specificity for immunosuppressive cells in the tumor microenvironment which, upon clearance, leads to reactivation of immune effector function. (Ghiringhelli FApetoh L 2013; Bracci L et al. 2014). The antineoplastic agent 5-FU has been reported to promote ICD, deplete immunosuppressive cells (specifically MDSCs) from the tumor microenvironment, and activate the adaptive arm of the immune system (Bracci L et al. 2014). Both CD4+Th and CD8+Tc cells were significantly increased in tumors of treated animals. An increase in the ratio of effector T cells to tumor targets is favorable during promotion of an antitumor immune response, especially when Tregs remain unchanged as seen here. In fact, the work described herein showed that while 5-FC was detectable throughout the circulation, 5-FU was only present at detectable levels within the tumor tissue.Hiraoka et al assessed specific lymphocyte subsets that contribute to tumor eradication upon tumor re-challenge in antibody-mediated depletion of CD4+Th, CD8+Tc, and nature killer (NK) cells. The tumors rejection were observed in all animals treated CD8 or NK antibody, however, CD4 or combination of anti-CD4 and anti-CD8 antibody treated groups prevented tumor rejection in previously cured animals (Hiraoka K et al. 2017). These data indicate the importance of CD4+Th cells in antitumor and antiviral immunity, andCD4+ cells may be even more efficient as cytolytic effectors than CD8+_{Tc cells} (Perez-Diez A et al. 2007).In our study, population of CD3+pan-T cells, CD4+_{Th cells significantly increased in therapy treated groups compared with} PBS group, furthermore, CD8+_{Tc cells population in therapy treated mice were} higher than non-treat mice. These observation suggested that CD3+pan-T cells and CD4+_{Th cells play important role in prevent glioma growth in our therapy.}

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Microglia and macrophages population, are also known antigen-presenting cells in brain, infiltrate into tumor site particularly therapy treated mice, however their function within this study were still unclear.

In the immune-compromised mouse model, mice that received MSC/CD, TMZ or combination of MSC/CDand TMZ showed poor short-term survival compared with PBS group. On the other hand, in similar experiments in immune-competent mice, glioma-bearing mice treated therapy led to statistically signiﬁcant efﬁcacy and long-term survivors.Therefore, immune components play an important role in eradicating tumor in preclinical model.We previously showed that combination of MSC/CD and TMZ resulted in long-term survival in intracranial tumor-bearing mice. In this current study, we have demonstrated that comparable long-term survival can also be achieved with MSC/CD and TMZ in the immune-competent mouse model. These results further support a dual mechanism of action for the combination of MSC/CD and TMZ that involves both direct tumor chemoablation and the consequent activation of an antitumor immune response.

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CONCLUSIONS

Dual mechanism of action for the combination of TMZ and MSC/CD + 5-FC that involves both direct tumor chemoablation and the consequent activation of an antitumor immune response.

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Supplementary data

(A)

(B)

Group M.S. O.S. Mean

TMZ 26 29 25.7

MSC/CD 18 23 19.4

TMZ + MSC/CD 25 30 26

S1. Efﬁcacy of MSC/CD with TMZ in syngeneic glioma model: (A) Schematic diagram to evaluate antitumor effect of MSC/CD + TMZ in

syngeneic mice GL261/GFP glioma cells and MSC/CD cells were transplanted with 3x104 cells and administrated 5-FC and TMZ at indicated time points with 1000 mg/kg/day P.O. of 5-FC and 5 mg/kg/day of TMZ in C57BL/6 mice, respectively. (B) The Kaplan-Meier graph indicated that the survival of mice (n=8).

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