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• W2473243602 abstract "Oncolytic vaccinia virus (VACV) therapy is an alternative treatment option for glioblastoma multiforme. Here, we used a comparison of different tumor locations and different immunologic and genetic backgrounds to determine the replication efficacy and oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models. With this approach, we expected to identify microenvironmental factors, which may be decisive for failure or success of oncolytic VACV therapy. We found that GL261 glioma cells implanted subcutaneously or orthotopically into Balb/c athymic, C57BL/6 athymic, or C57BL/6 wild-type mice formed individual tumors that respond to oncolytic VACV therapy with different outcomes. Surprisingly, only Balb/c athymic mice with subcutaneous tumors supported viral replication. We identified intratumoral IFN-γ expression levels that upregulate MHCII expression on GL261 cells in C57BL/6 wild-type mice associated with a non-permissive status of the tumor cells. Moreover, this IFN-γ-induced tumor cell phenotype was reversible. Oncolytic vaccinia virus (VACV) therapy is an alternative treatment option for glioblastoma multiforme. Here, we used a comparison of different tumor locations and different immunologic and genetic backgrounds to determine the replication efficacy and oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models. With this approach, we expected to identify microenvironmental factors, which may be decisive for failure or success of oncolytic VACV therapy. We found that GL261 glioma cells implanted subcutaneously or orthotopically into Balb/c athymic, C57BL/6 athymic, or C57BL/6 wild-type mice formed individual tumors that respond to oncolytic VACV therapy with different outcomes. Surprisingly, only Balb/c athymic mice with subcutaneous tumors supported viral replication. We identified intratumoral IFN-γ expression levels that upregulate MHCII expression on GL261 cells in C57BL/6 wild-type mice associated with a non-permissive status of the tumor cells. Moreover, this IFN-γ-induced tumor cell phenotype was reversible. Glioblastoma multiforme (GBM) is one of the most malignant forms of brain cancer (WHO grade IV) and also the most frequent type of glioma in adults.1Ohgaki H Kleihues P Epidemiology and etiology of gliomas.Acta Neuropathol. 2005; 109: 93-108Crossref PubMed Scopus (946) Google Scholar,2Wen PY Kesari S Malignant gliomas in adults.N Engl J Med. 2008; 359: 492-507Crossref PubMed Scopus (3208) Google Scholar The standard of care for GBM is surgical resection, followed by radiation and temozolomide chemotherapy.2Wen PY Kesari S Malignant gliomas in adults.N Engl J Med. 2008; 359: 492-507Crossref PubMed Scopus (3208) Google Scholar In spite of extensive research effort, the disease is still incurable and the prognosis is very poor with a median survival of less than 15 months.2Wen PY Kesari S Malignant gliomas in adults.N Engl J Med. 2008; 359: 492-507Crossref PubMed Scopus (3208) Google Scholar Difficulties associated with treatment of GBM are the highly aggressive and infiltrative nature of the tumor into the brain parenchyma. In addition, the histological heterogeneity of the tumor mass, the location of the neoplasm within the brain, the infiltration of the tumor with microglia/macrophages, and the function and morphology of the blood-brain barrier aggravate the therapy.2Wen PY Kesari S Malignant gliomas in adults.N Engl J Med. 2008; 359: 492-507Crossref PubMed Scopus (3208) Google Scholar, 3Patel MM Goyal BR Bhadada SV Bhatt JS Amin AF Getting into the brain: approaches to enhance brain drug delivery.CNS Drugs. 2009; 23: 35-58Crossref PubMed Scopus (330) Google Scholar, 4Adamson C Kanu OO Mehta AI Di C Lin N Mattox AK et al.Glioblastoma multiforme: a review of where we have been and where we are going.Expert Opin Investig Drugs. 2009; 18: 1061-1083Crossref PubMed Scopus (387) Google Scholar There is a broad range of alternative treatment options presently studied in preclinical and also clinical trials for GBM.4Adamson C Kanu OO Mehta AI Di C Lin N Mattox AK et al.Glioblastoma multiforme: a review of where we have been and where we are going.Expert Opin Investig Drugs. 2009; 18: 1061-1083Crossref PubMed Scopus (387) Google Scholar, 5Selznick LA Shamji MF Fecci P Gromeier M Friedman AH Sampson J Molecular strategies for the treatment of malignant glioma-genes, viruses, and vaccines.Neurosurg Rev. 2008; 31: 141-155Crossref PubMed Scopus (34) Google Scholar, 6Lun X Chan J Zhou H Sun B Kelly JJ Stechishin OO et al.Efficacy and safety/toxicity study of recombinant vaccinia virus JX-594 in two immunocompetent animal models of glioma.Mol Ther. 2010; 18: 1927-1936Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar One of those is oncolytic virotherapy, defined as the use of replication-competent viruses that selectively infect, replicate in and destroy cancer cells while leaving healthy, nontransformed cells and tissues unharmed.7Chen NG Szalay AA Oncolytic virotherapy in cancer Minev BR.Cancer Manag. Man Chemother. Biol. Ther. Hyperth. Support. Meas. Springer, New York, New York, NY2011: 295-316Crossref Google Scholar Vaccinia virus (VACV) is a favorable candidate for oncolytic virotherapy due to its safety profile demonstrated during its use as a vaccine in the immunization against smallpox and as double-stranded DNA virus with the unique characteristic to replicate in the cytoplasm only, without integrating into the host genome.8Kirn DH Thorne SH Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer.Nat Rev Cancer. 2009; 9: 64-71Crossref PubMed Scopus (312) Google Scholar,9Chen NG Szalay AA Oncolytic vaccinia virus: a theranostic agent for cancer.Future Virol. 2010; 5: 763-784Crossref Scopus (23) Google Scholar The efficient killing of tumor cells by recombinant VACVs or VACV wild-type isolates was demonstrated in different tumor xenograft models including a GBM model.10Gentschev I Müller M Adelfinger M Weibel S Grummt F Zimmermann M et al.Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68.PLoS ONE. 2011; 6: e22069Crossref PubMed Scopus (35) Google Scholar, 11Weibel S Hofmann E Basse-Luesebrink TC Donat U Seubert C Adelfinger M et al.Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer.J Transl Med. 2013; 11: 106Crossref PubMed Scopus (13) Google Scholar, 12Donat U Rother J Schäfer S Hess M Härtl B Kober C et al.Characterization of metastasis formation and virotherapy in the human C33A cervical cancer model.PLoS ONE. 2014; 9: e98533Crossref PubMed Scopus (8) Google Scholar, 13Advani SJ Buckel L Chen NG Scanderbeg DJ Geissinger U Zhang Q et al.Preferential replication of systemically delivered oncolytic vaccinia virus in focally irradiated glioma xenografts.Clin Cancer Res. 2012; 18: 2579-2590Crossref PubMed Scopus (40) Google Scholar There is a number of oncolytic viruses tested against malignant gliomas in phase 1 and phase 1/2 clinical trials, e.g., Herpes simplex virus1Ohgaki H Kleihues P Epidemiology and etiology of gliomas.Acta Neuropathol. 2005; 109: 93-108Crossref PubMed Scopus (946) Google Scholar, adenovirus, reovirus, Newcastle disease virus, and measles virus.14Zemp FJ Corredor JC Lun X Muruve DA Forsyth PA Oncolytic viruses as experimental treatments for malignant gliomas: using a scourge to treat a devil.Cytokine Growth Factor Rev. 2010; 21: 103-117Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,15Wollmann G Ozduman K van den Pol AN Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates.Cancer J. 2012; 18: 69-81Crossref PubMed Scopus (132) Google Scholar The application of those replication-competent viruses was generally safe, however, their antitumor effects observed in the preclinical studies need to be confirmed in human patients.14Zemp FJ Corredor JC Lun X Muruve DA Forsyth PA Oncolytic viruses as experimental treatments for malignant gliomas: using a scourge to treat a devil.Cytokine Growth Factor Rev. 2010; 21: 103-117Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,15Wollmann G Ozduman K van den Pol AN Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates.Cancer J. 2012; 18: 69-81Crossref PubMed Scopus (132) Google Scholar One successful approach for oncolytic virotherapy may be the concept of personalized medicine, the screening of cancer patients for best treatment options and thereby maximizing therapeutic outcome.16Kaur B Chiocca EA Personalizing oncolytic virotherapy?.Mol Ther. 2007; 15: 6-7Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar,17Ogino S Galon J Fuchs CS Dranoff G Cancer immunology - analysis of host and tumor factors for personalized medicine.Nat Rev Clin Oncol. 2012; 8: 617-632Google Scholar Therefore, the identification of biomarkers to locate patients that respond to a particular therapy is paramount.18Galon J Angell HK Bedognetti D Marincola FM The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures.Immunity. 2013; 39: 11-26Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar,19Angell H Galon J From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer.Curr Opin Immunol. 2013; 25: 261-267Crossref PubMed Scopus (344) Google Scholar Cancer formation is associated with a close interaction of malignant tumor cells and the tumor microenvironment.17Ogino S Galon J Fuchs CS Dranoff G Cancer immunology - analysis of host and tumor factors for personalized medicine.Nat Rev Clin Oncol. 2012; 8: 617-632Google Scholar,19Angell H Galon J From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer.Curr Opin Immunol. 2013; 25: 261-267Crossref PubMed Scopus (344) Google Scholar The amount and composition of infiltrating immune cells varies in different cancer types and in individual patients and is investigated as biomarker in several studies, highlighting the link between the immune status of a tumor and the clinical outcome.19Angell H Galon J From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer.Curr Opin Immunol. 2013; 25: 261-267Crossref PubMed Scopus (344) Google Scholar,20Fridman WH Pagès F Sautès-Fridman C Galon J The immune contexture in human tumours: impact on clinical outcome.Nat Rev Cancer. 2012; 12: 298-306Crossref PubMed Scopus (3033) Google Scholar In addition to cellular interactions, soluble factors released from cells in the micromilieu or from tumor cells control tumor-host interactions.17Ogino S Galon J Fuchs CS Dranoff G Cancer immunology - analysis of host and tumor factors for personalized medicine.Nat Rev Clin Oncol. 2012; 8: 617-632Google Scholar,21Allen M Louise Jones J Jekyll and Hyde: the role of the microenvironment on the progression of cancer.J Pathol. 2011; 223: 162-176Crossref PubMed Scopus (135) Google Scholar Possible mechanisms resulting in a diverse immune cell infiltration and activation of a particular tumor microenvironment are reviewed by Ascierto et al.22Ascierto ML De Giorgi V Liu Q Bedognetti D Spivey TL Murtas D et al.An immunologic portrait of cancer.J Transl Med. 2011; 9: 146Crossref PubMed Scopus (69) Google Scholar and include the genetic background of the host, genetic polymorphism of cytokine receptors, and the genetics of the tumor itself as potential factors. It is known, that one of the most important determinants in the regulation of immune responses in humans and mice is their genetic background.23Kuroda E Noguchi J Doi T Uematsu S Akira S Yamashita U IL-3 is an important differentiation factor for the development of prostaglandin E2-producing macrophages between C57BL/6 and BALB/c mice.Eur J Immunol. 2007; 37: 2185-2195Crossref PubMed Scopus (16) Google Scholar Two inbred mouse strains are used in this study which are defined as prototypical type 1 T helper (Th1) mouse strain in case of C57BL/6 and type 2 T helper (Th2) mouse strain in case of Balb/c mice.23Kuroda E Noguchi J Doi T Uematsu S Akira S Yamashita U IL-3 is an important differentiation factor for the development of prostaglandin E2-producing macrophages between C57BL/6 and BALB/c mice.Eur J Immunol. 2007; 37: 2185-2195Crossref PubMed Scopus (16) Google Scholar, 24Karupiah G Type 1 and type 2 cytokines in antiviral defense.Vet Immunol Immunopathol. 1998; 63: 105-109Crossref PubMed Scopus (55) Google Scholar, 25Kuroda E Kito T Yamashita U Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice.J Immunol. 2002; 168: 5477-5482Crossref PubMed Scopus (57) Google Scholar, 26Watanabe H Numata K Ito T Takagi K Matsukawa A Innate immune response in Th1- and Th2-dominant mouse strains.Shock. 2004; 22: 460-466Crossref PubMed Scopus (327) Google Scholar Adaptive immune response is divided into Th1/Th2 immune response based on a distinct cytokine secretion pattern: Classical Th1 cytokines are, e.g., interleukin 2 (IL-2), IL-12, interferon-γ (IFN-γ), and tumor necrosis factor alpha (TNF-α) whereas IL-4, 5, 6, 10 are Th2 cytokines.24Karupiah G Type 1 and type 2 cytokines in antiviral defense.Vet Immunol Immunopathol. 1998; 63: 105-109Crossref PubMed Scopus (55) Google Scholar In addition to T-cell response, the innate immune cells, primarily macrophages, also show different characteristics in the two mouse strains used in response to pathogenic stimuli like lipopolysaccharide (LPS).23Kuroda E Noguchi J Doi T Uematsu S Akira S Yamashita U IL-3 is an important differentiation factor for the development of prostaglandin E2-producing macrophages between C57BL/6 and BALB/c mice.Eur J Immunol. 2007; 37: 2185-2195Crossref PubMed Scopus (16) Google Scholar,25Kuroda E Kito T Yamashita U Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice.J Immunol. 2002; 168: 5477-5482Crossref PubMed Scopus (57) Google Scholar, 26Watanabe H Numata K Ito T Takagi K Matsukawa A Innate immune response in Th1- and Th2-dominant mouse strains.Shock. 2004; 22: 460-466Crossref PubMed Scopus (327) Google Scholar, 27Mills CD Kincaid K Alt JM Heilman MJ Hill AM M-1/M-2 macrophages and the Th1/Th2 paradigm.J Immunol. 2000; 164: 6166-6173Crossref PubMed Scopus (1920) Google Scholar It is well known, that the outcome of diseases in mice infected with several intracellular pathogens is dependent on the genetic background and the Th1/Th2 balance with IFN-γ as one major factor.24Karupiah G Type 1 and type 2 cytokines in antiviral defense.Vet Immunol Immunopathol. 1998; 63: 105-109Crossref PubMed Scopus (55) Google Scholar,26Watanabe H Numata K Ito T Takagi K Matsukawa A Innate immune response in Th1- and Th2-dominant mouse strains.Shock. 2004; 22: 460-466Crossref PubMed Scopus (327) Google Scholar,28Szulc L Gierynska M Winnicka A Martyniszyn L Boratynska-Jasinska A Niemialtowski M T cell cytokine synthesis at the single-cell level in BALB/c and C57BL/6 mice infected with ectromelia virus.Postepy Hig Med Dosw (Online). 2012; 66: 222-230Crossref PubMed Scopus (3) Google Scholar In this study, we set out to identify microenvironmental differences or factors that decisively influence treatment outcome of oncolytic virotherapy and which may be relevant to affect therapeutic success in human patients. We investigated the oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models in a comparative approach: Specifically, we used immunocompetent C57BL/6 wild-type (wt) mice and immunodeficient mouse strains of different genetic background (C57BL/6 athymic nude and Balb/c athymic nude mice) and also studied the effect of different tumor locations (subcutaneous and orthotopic). Infection of GL261 glioma cells with LIVP 1.1.1 (multiplicity of infection (MOI) 0.1) in cell culture revealed replication capacity of 612 ± 244% within 24 hpi, and 6,272 ± 3,821% at 72 hpi referred to the initial infection dose of the cells which was set 100% (Figure 1a). In addition, LIVP 1.1.1-mediated cell death could be demonstrated in a MTT-cell survival assay with only 20 ± 11% surviving cells (MOI 1.0) or 55 ± 12% (MOI 0.1) at 96 hpi (Figure 1b). Using a comparative approach with different tumor locations, immunocompetent and immunodeficient mouse strains as well as different genetic backgrounds, we set out to analyze the replication efficacy of the VACV LIVP 1.1.1 upon intratumoral injection in murine GL261 glioma models. Viral replication was assumed as an increase in viral titer compared to the initial injection dose (5 × 106 pfu/mouse) delivered i.t. The results revealed viral replication exclusively in Balb/c athymic mice bearing subcutaneous tumors with 5 × 107 ± 3 × 107 pfu/g tissue 1 dpi and 8 × 107 ± 2 × 107 pfu/g tissue 7 dpi (Figure 1c). All other mouse models and tumor locations led to a decrease in the LIVP 1.1.1 titers during the observation time course. The replication capacity was 15-fold increased in subcutaneous tumors of Balb/c athymic mice at 7 dpi compared to a 28- and 8-fold decrease of the viral load in C57BL/6 wt mice and C57BL/6 athymic mice tumors, respectively (Figure 1c). Replication was not detected either in Balb/c athymic mice (1 × 106 ± 8 × 105 pfu/g tissue 7dpi) or in the C57BL/6 wt mice (6 × 104 ± 6 × 104 pfu/g tissue 7dpi) with orthotopic brain tumors (Figure 1d). These findings coincide with a 4-fold decrease of the viral load in Balb/c athymic and 100-fold decrease in C57BL/6 wt mice at 7 dpi (Figure 1d). Since major differences in virus replication in the subcutaneous tumors in different mouse strains were detected, we analyzed tumor growth kinetics in response to oncolytic virotherapy of these tumor models. In the LIVP 1.1.1 groups of all three models there was a slight tumor growth delay (Figure 1e–g) but only in Balb/c athymic mice a significant tumor growth delay from 4 dpi to the end of the study was detected (Figure 1e). All tumor models showed a very fast and aggressive tumor growth as mice reached a tumor size of 4,000 mm3 in 9–14 days. Replication analysis affirmed that the mouse genetic background had a major impact on viral replication in the subcutaneous GL261 tumor models, as both C57BL/6 mouse models (athymic and wt) did not support viral replication, whereas the BALB/c background does. This led us to the question, which additional immunological factors in the tumor microenvironment of the subcutaneous tumor model of Balb/c athymic mice enabled virus replication on the one hand and prevented replication in mice with C57BL/6 background on the other side. We found that the virus titer differed significantly at 1 dpi (Figure 1c) implicating that the differences seem to exist in tumors before virus infection. Therefore, we performed a detailed characterization of tumor microenvironments of the subcutaneous tumor models already on d0. In a first step, a subset of 59 biomarkers was tested in subcutaneous tumors of five mice of each mouse strain using a mouse immune related antigen profiling. Nineteen biomarkers that showed significant differences in the mouse strains tested are listed in Table 1. The significant differences of the proinflammatory signature were all detectable in the C57BL/6 wt mice compared to both athymic mouse strains (C57BL/6 and Balb/c) but not in the C57BL/6 athymic in comparison to Balb/c athymic mice. In most cases, highest biomarker concentrations were detectable in C57BL/6 wt mice followed by C57BL/6 athymic and Balb/c athymic mice.Table 1Mouse immune-related protein antigen profiling of C57BL/6 wt, C57BL/6 athymic, and Balb/c athymic mice in GL261 tumors at the day of infection (d0)aShown are the mean values and standard deviation (n = 5; C57BL/6 and Balb/c athymic and n = 4 C57BL/6 wt). Differences between the mouse strains were analyzed using two-sided t-test with unequal variances, *P < 0. 05 (yellow), **P < 0. 01 (orange), ***P < 0. 001 (dark orange). Proinflammatory cytokines and chemoattractants are marked in green; modulators of tissue homeostasis are marked in blue.Mouse strainsP valueBiomarkerAbbr.UnitC57BL/6 wtC57BL/ 6 athymicBalb/ c athymicC57BL/6 wt: Balb/c athymicC57BL/6 wt: C57BL/6 athymicC57BL/6 athymic: Balb/c athymicGranulocyte chemotactic protein-2GCP-2ng/ml0. 2 ± 0. 030. 1 ± 0. 020. 1 ± 0. 010. 0050. 0040. 220Interferon-γ -induced protein 10IP-10pg/ml2940. 0 ± 602. 2921. 0 ± 1123. 9423. 0 ± 154. 20. 0030. 0070. 323Interleukin-1αIL-1 αpg/ml1035. 5 +/- 123. 5572. 5 ± 158. 6438. 8 ± 218. 30. 0020. 0040. 266Interleukin-11IL-11pg/ml101. 3 ± 17. 970. 8 ± 6. 2n. d.—0. 036—Leukemia inhibitory factorLIFpg/ml775. 0 ± 112. 9533. 8 ± 167. 1452. 3 ± 71. 50. 0060. 0370. 378Lymphotactinpg/ml879. 5 ± 201. 4516. 2 ± 267. 6188. 0 ± 32. 00. 0060. 0530. 051Macrophage inflammatory protein-1βMIP-1 βpg/ml1457. 5 ± 281. 2294. 2 ± 164. 5171. 0 ± 15. 90. 0030. 0010. 170Macrophage inflammatory protein-2MIP-2pg/ml34. 0 ± 6. 617.6 ± 4.915. 3 ± 2. 80. 0060. 0070. 399Monocyte chemotactic protein 1MCP-1pg/ml436. 8 ± 122. 1160. 3 ± 51. 3115.8 ± 44.00. 0100. 0140. 183Monocyte chemotactic protein 3MCP-3pg/ml320. 5 ± 120. 9143. 4 ± 64. 296. 8 ± 25. 50. 0320. 0530. 189Monocyte chemotactic protein-5MCP-5pg/ml184. 5 ± 38. 994. 8 ± 38. 287. 6 ± 33. 00. 0070. 0120. 758T-cell-specific protein RANTESRANTESpg/ml0. 3 ± 0. 10. 1 ± 0. 10. 03 ± 0. 010. 0170. 0200. 226Vascular cell adhesion molecule-1VCAM-1ng/ml47. 0 ± 7. 527. 0 ± 4. 026. 6 ± 3. 90. 0070. 0070. 876Vascular endothelial growth factor AVEGF-Apg/ml9105. 0 ± 2539. 61684. 2 ± 829. 32230. 0 ± 600. 30. 0110. 0070. 271von Willebrand factorvWFng/ml12. 0 ± 2. 28. 6 ± 1. 58. 3 ± 2. 30. 0450. 0430. 805Stem cell factorSCFpg/ml1380. 0 ± 175. 7791. 6 ± 237. 3542. 8 ± 195. 90. 00030. 0040. 110CD40CD40pg/ml724. 8 ± 361. 3138. 0 ± 51. 7154. 6 ± 29. 90. 0510. 0460. 556Tissue inhibitor of metalloproteinases 1TIMP-1ng/ml10. 1 ± 1. 39. 5 ± 1. 95. 9 ± 1. 60. 0030. 6540. 011Matrix metalloproteinase-9MMP-9ng/ml20. 8 ± 5. 911.0 ± 4.16. 7 ± 1. 70. 0140. 0370. 075a Shown are the mean values and standard deviation (n = 5; C57BL/6 and Balb/c athymic and n = 4 C57BL/6 wt). Differences between the mouse strains were analyzed using two-sided t-test with unequal variances, *P < 0. 05 (yellow), **P < 0. 01 (orange), ***P < 0. 001 (dark orange). Proinflammatory cytokines and chemoattractants are marked in green; modulators of tissue homeostasis are marked in blue. Open table in a new tab The profiling revealed that there was a significant upregulation of proinflammatory cytokines such as interferon-γ-induced protein 10 (IP-10), IL-1α, IL-11 and macrophage inflammatory protein-1β (MIP-1β), and chemoattractants such as monocyte chemotactic protein (MCP)-1, MCP-3, MCP-5 recruiting mainly monocytes, macrophages, lymphocytes, eosinophils, MIP-2, and granulocyte chemoattractant protein-2 (GCP-2) attracting neutrophils and lymphotactin and IP-10 recruiting lymphocytes.29http://www.rbm.myriad.com/products-services/biomarker-detail/, 2014Google Scholar Factors responsible for the proinflammatory signature (GCP-2, MIP-1β, MIP-2, MCP-3, and MCP-5) are mainly produced by macrophages. Another factor which was almost fivefold upregulated in the wild-type mice was vascular endothelial growth factor A (VEGF-A) as well as CD40. In a second experiment, we performed an immune cell profiling of the subcutaneous GL261 tumors at d0 to determine, whether there were major qualitative and/or quantitative differences in the immune cell populations within these tumor models (Table 2). Single cell suspensions of the tumors were prepared and flow cytometry analysis was performed. In the C57BL/6 wt mice besides CD3+/CD4+ T-lymphocytes, immune cell subsets of the innate immune system such as CD49+ natural killer (NK) cells, CD11b+/CD11c+ immature myeloid cells and cells of the monocyte/macrophage lineage (CD45+/CD11b+, CD11b+/MHCII+, F4/80+/MHCII+) were significantly upregulated compared to the C57BL/6 athymic and Balb/c athymic mice (Table 2). Flow cytometry analysis of the blood in these mice (Table 2) showed that besides the expected CD3+/CD4+ and CD3+/CD8+ T-lymphocytes present exclusively in the wild-type mice, significantly more CD19+ B-lymphocytes, CD49+ NK cells, CD11b+/Ly6c+ dendritic cells, CD11b+/CD11c+ immature myeloid cells, and CD11b+/Gr1+ myeloid-derived suppressor cells (MDSCs) were detected in the C57BL/6 athymic mice compared to the Balb/c athymic mice. In case of CD49+ NK cells and CD11b+/CD11c+ immature myeloid cells, the differences were also significant between C57BL/6 wt and C57BL/6 athymic mice.Table 2Immune cell profiling and comparison of single cell suspensions isolated from subcutaneous GL261 tumors and blood of C57BL/6 wt, C57BL/6 athymic, and Balb/c athymic mice on d0aShown are mean and standard deviation of three mice per group in percentages (%). Differences in marker expression between mouse strains were analyzed using two-sided t-test with unequal variances *P < 0. 05 (yellow), **P < 0. 01 (orange), ***P < 0. 001 (dark orange). Highlighted in red is the mouse strain showing the highest percentage of a particular marker combination. 10, 000 events/sample and staining were measured. Percentages below 1% are described as < 1.TumorMarkerC57BL/6 wtC57BL/6 athymicBalb/c athymicC57BL/6 wt: C57BL/6 athymicC57BL/6 wt: Balb/c athymicC57BL/6 athymic: Balb/c athymicLymphocytesCD3+/CD4+2. 4 ± 0. 7< 1< 10. 0300. 039-CD3+/CD8+< 1< 1< 1---CD19+2. 4 ± 2. 01. 1 ± 0. 12. 5 ± 0. 70. 260. 710. 05NK cellsCD49b+5. 5 ± 0. 31. 9 ± 0. 04. 1 ± 0. 60. 0010. 040. 01Dendritic cellsCD11b+/Ly6c+5. 7 ± 1. 71. 9 ± 0. 8< 10. 160. 080. 09Immature myeloid cellsCD11b+/CD11c+4. 7 ± 0. 93. 3 ± 1. 11. 5 ± 0. 40. 040. 030. 08MDSCCD11b+/Gr1+< 11. 7 ± 0. 5< 10. 03-0. 12NeutrophilsCD11b+/Ly6G+< 1< 1< 1---Monocytes/CD45+/CD11b+8. 8 ± 1. 13. 5 ± 0. 12. 3 ± 0. 50. 010. 0040. 04MacrophagesCD11b+/MHCII+6. 78 ± 0. 01. 9 ± 0. 4<10. 00020. 00040. 03CD68+/MHCII+3. 5 ± 2. 01. 6 ± 0. 81. 0 ± 0. 30. 220. 160. 37F4/80+/MHCII+2. 5 ± 0. 5< 1< 10. 020. 01-BloodMarkerC57BL/6 wtC57BL/6 athymicBalb/c athymicC57BL/6 wt: C57BL/6 athymicC57BL/6 wt: Balb/c athymicC57BL/6 athymic: Balb/c athymicCD3+/CD4+16. 9 ± 3. 0< 1< 10. 010. 01-LymphocytesCD3+/CD8+16. 9 ± 2. 0< 1< 10. 0060. 006-CD19+52. 2 ± 5. 061. 2 ± 1. 944. 6 ± 3. 10. 100. 010. 02NK cellsCD49+10. 0 ± 2. 429. 1 ± 3. 013. 1 ± 0. 70. 0010. 030. 002Dendritic cellsCD11b+/Ly6c+10. 2 ± 4. 515. 4 ± 1. 78. 7 ± 2. 30. 050. 680. 02Immature myeloid cellsCD11b+/CD11c+5. 6 ± 1. 119. 3 ± 2. 24. 6 ± 0. 80. 0020. 290. 003MDSCCD11b+/Gr1 +5. 5 ± 3. 612. 7 ± 2. 16. 4 ± 2. 40. 050. 720. 03NeutrophilsCD11b+/Ly6G+6. 2 ± 3. 613. 0 ± 1. 88. 2 ± 2. 90. 070. 510. 08MonocytesCD11b+/MHCII+4. 2 ± 1. 35. 6 ± 1. 67. 0 ± 0. 80. 290. 060. 28MacrophagesF4/80+/MHCII+1. 0 ± 0. 21. 5 ± 0. 33. 3 ± 0. 20. 040. 0020. 004a Shown are mean and standard deviation of three mice per group in percentages (%). Differences in marker expression between mouse strains were analyzed using two-sided t-test with unequal variances *P < 0. 05 (yellow), **P < 0. 01 (orange), ***P < 0. 001 (dark orange). Highlighted in red is the mouse strain showing the highest percentage of a particular marker combination. 10, 000 events/sample and staining were measured. Percentages below 1% are described as < 1. Open table in a new tab This study highlighted that implantation of the same tumor cells in three different mouse strains resulted in a completely different tumor microenvironment with highest differences between immunodeficient and immunocompetent mice. It also demonstrated the influence of the adaptive immune system as main microenvironmental modulator. The comparison of these three mouse strains further revealed a tendency that the C57BL/6 athymic mice might be in an “intermediate state” between C57BL/6 wt and Balb/c athymic mice. After characterization of the subcutaneous GL261 tumor model in different mouse strains, we tried to elucidate whether therapeutic efficiency in the “non-responder” C57BL/6 wt mice can be improved. As the proinflammatory signature was mainly produced by macrophages (Table 1) which were significantly recruited to subcutaneous GL261 tumors in C57BL/6 wt mice in comparison to athymic mice, we depleted macrophages in C57BL/6 wt mice with clodronate liposomes prior to virus infection. The depletion efficiency was confirmed by flow cytometry analysis with different macrophage marker combinations 1 dpi (Figure 2a–d). Standard viral plaque assay revealed that the virus titer in both groups (PBS and clodronate liposomes) did not differ significantly either on day 1 or on day 7 post infection (Figure 2e). Therefore, macrophages were excluded as factor responsible for the pronounced reduction of viral particles in this model detected already 1 dpi. The immunohistochemical analysis of subcutaneous GL261 tumor sections of C57BL/6 wt and athymic as well as Balb/c athymic mice at d0 revealed a significant upregulation of MHCII in the C57BL/6 wt mice (Figure 3a). The upregulation of MHCII in C57BL/6 athymic mice compared to Balb/c athymic mice was not significant. Additionally, amounts of CD68+ immune cells were not significantly different in the three mouse strains (Figure 3b). Surprisingly, the expression of the marker MHCII did not colocalize with the immune cell marker CD68 (Figure 3d,e), implicating the existence of a MHCII+ nonmacrophage population. The expression of MHCII was distributed homogenously throughout the tumor center and at the tumor rim (quantification not shown). An upregulation of MHCII on CD68- cells was observed in C57BL/6 athymic mice with patchy d" @default.
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• W2473243602 date "2015-01-01" @default.
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• W2473243602 title "Intratumoral INF-γ triggers an antiviral state in GL261 tumor cells: a major hurdle to overcome for oncolytic vaccinia virus therapy of cancer" @default.
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• W2473243602 doi "https://doi.org/10.1038/mto.2015.9" @default.
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