A Tumor-Targeted Replicating Oncolytic Adenovirus Ad-TD-nsIL12 as a Promising Therapeutic Agent for Human Esophageal Squamous Cell Carcinoma

Zifang Zhang, Chunyang Zhang, Jinxin Miao, Zhizhong Wang, Zhimin Wang, Zhenguo Cheng, Pengju Wang, Louisa S Chard Dunmall, Nicholas R Lemoine, Yaohe Wang, Zifang Zhang, Chunyang Zhang, Jinxin Miao, Zhizhong Wang, Zhimin Wang, Zhenguo Cheng, Pengju Wang, Louisa S Chard Dunmall, Nicholas R Lemoine, Yaohe Wang

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers in China and existing therapies have been unable to significantly improve prognosis. Oncolytic adenoviruses (OAds) are novel promising anti-tumor drugs and have been evaluated in several cancers including ESCC. However, the antitumour efficacy of the first generation OAds (H101) as single agent is limited. Therefore, more effective OAds are needed. Our previous studies demonstrated that the novel oncolytic adenovirus Ad-TD-nsIL12 (human adenovirus type 5 with E1ACR2, E1B19K, E3gp19K-triple deletions)harboring human non-secretory IL-12 had significant anti-tumor effect, with no toxicity, in a Syrian hamster pancreatic cancer model. In this study, we evaluated the anti-tumor effect of Ad-TD-nsIL12 in human ESCC. The cytotoxicity of Ad-TD-nsIL12, H101 and cisplatin were investigated in two newly established patient-derived tumor cells (PDCs) and a panel of ESCC cell lines in vitro. A novel adenovirus-permissive, immune-deficient Syrian hamster model of PDCs subcutaneous xenograft was established for in vivo analysis of efficacy. The results showed that Ad-TD-nsIL12 was more cytotixic to and replicated more effectively in human ESCC cell lines than H101. Compared with cisplatin and H101, Ad-TD-nsIL12 could significantly inhibit tumor growth and tumor angiogenesis as well as enhance survival rate of animals with no side effects. These findings suggest that Ad-TD-nsIL12 has superior anti-tumor potency against human ESCC with a good safety profile.

Keywords: Ad-TD-nsIL12; ESCC; Syrian hamster model; antitumour efficacy; oncolytic adenovirus.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SBRC-EC01 and SBRC-EC02 mimic the biological characteristics of the primary tumors. (A) Phase contrast images of SBRC-EC01 and SBRC-EC02 at different passages are presented. Polygonal epithelial-like cells were surrounded by long fusiform cells at passage 1. At passage 15, cells of different shapes and sizes were observed; (B) Histopathological examination of the original tumors and the derived cell populations. Immunostaining for Pan Cytokeratin (AE1/AE3), Ki67, p63 are shown. Scale bar: 50 μm.
Figure 2
Figure 2
Cell Viability assays display the effect of 5-fluorouracil and cisplatin in ESCC cell lines and two patient-derived tumor cells. (A–C) Cytotoxicity assay of cisplatin in ESCC cell lines and two patient-derived tumor cells. (D–F) Cytotoxicity assay of 5-fluorouracil in ESCC cell lines and two patient-derived tumor cells. Patient-derived tumor cells include SBRC-EC01 and SBRC-EC02. Tumor cells were treated with increasing concentrations of drugs for 72 h. Cell viability was measured using MTS assay, and IC50 value (half maximal inhibitory concentration) was calculated. Data are shown as the mean ± SD of 3 independent experiments.
Figure 3
Figure 3
AD-TD-LUC, Ad-TD-nsIL12 and H101 can replicate in and cause oncolysis of human ESCC cell lines and PDCs. (A) To assess virus cytotoxicity, cell proliferation assays were carried out in two PDCs and two ESCC cell lines (KYSE180, KYSE510). Cells were infected with AD-TD-LUC, Ad-TD-nsIL12 or H101 separately. The cell viability was determined by the MTS assay 6 d after infection. EC50 value for each virus is shown as a measure of the cytopathic effect; (B–E) Replication assays were carried out in two ESCC cell lines (KYSE180, KYSE510), SBRC-EC01 and SBRC-EC02 at a MOI of 5 PFU/cell. Data are expressed as mean ± SD (n = 6) and analyzed by one way ANOVA. * p < 0.05, ** p< 0.01, *** p < 0.001, **** p < 0.0001. NS, no statistical significance.
Figure 4
Figure 4
Ad-TD-nsIL12 can effectively control tumor growth in SBRC-EC01 subcutaneous xenograft tumor model. (A–E) SBRC-EC01 cells were used to establish a xenograft model in ZZU001 hamsters. 1 × 107 cells were seeded into the right flank of ZZU001. When the tumors reached 300 mm3, Ad-TD-nsIL12 (5 × 108 PFU), H101 (5 × 108 PFU) or PBS were each injected intratumorally on days 0, 2, 4. 6, 8 and 10; 3 mg/kg cisplatin was intraperitonealy injected once a week for 4 weeks. Ad-TD-nsIL12 could significantly inhibit the tumor growth compared with other groups. The arrow shows the start time of treatment. Tumor sizes were expressed as the mean ± SD in each group. Statistical significance was determined using a two-way ANOVA and a Student’s t-test for the comparison between groups; (F) The body weight of virus treatment group and control group increased slowly. But in cisplatin group, the body weight of all hamsters decreased; (G) Kaplan–Meier survival curves were generated and significance was assessed using the log-rank (Mantel–Cox) test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5
Figure 5
Ad-TD-nsIL12 persists in tumor tissue for a longer duration than H101. Tumors growing in ZZU001 were directly injected with Ad-TD-nsIL12 or H101 (5 × 108 PFU) on days 0, 2, 4, 6, 8, and 10. On days 11, 21 and 31 after the first viral injection, three tumors harvested from each treatment group were analyzed for expression of E1A. The data are calculated as mean ± SD and analyzed by one way ANOVA. ND, undetectable.
Figure 6
Figure 6
Ad-TD-nsIL12 can inhibit the proliferation activity of tumor cells and reduce the microvessel density of tumor tissue. (A) Ki67 staining of tumor cells in xenografts. The percentage of Ki67 positive cells was determined in five high-power fields of tumor section; (B) CD31 staining of xenografts in each group. The mean microvascular density was determined by counting CD31-positive microvascular structures in five high-power fields of tumor section. The data are calculated as mean ± SD and analyzed by one way ANOVA. Scale bar: 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

References

    1. Zeng H., Zheng R., Zhang S., Zuo T., Xia C., Zou X., Chen W. Esophageal cancer statistics in China, 2011: Estimates based on 177 cancer registries. Thorac. Cancer. 2015;7:232–237. doi: 10.1111/1759-7714.12322.
    1. He L., Jin-Hu F., You-Lin Q. Epidemiology, etiology, and prevention of esophageal squamous cell carcinoma in China. Cancer Biol. Med. 2017;14:33–41. doi: 10.20892/j.issn.2095-3941.2016.0093.
    1. Chang J., Tan W., Ling Z., Xi R., Shao M., Chen M., Luo Y., Zhao Y., Liu Y., Huang X., et al. Genomic analysis of oesophageal squamous-cell carcinoma identifies alcohol drinking-related mutation signature and genomic alterations. Nat. Commun. 2017;8:15290. doi: 10.1038/ncomms15290.
    1. Russell L., Peng K.-W. The emerging role of oncolytic virus therapy against cancer. Chin. Clin. Oncol. 2018;7:16. doi: 10.21037/cco.2018.04.04.
    1. Dolgin E. Oncolytic viruses get a boost with first FDA-approval recommendation. Nat. Rev. Drug Discov. 2015;14:369–371. doi: 10.1038/nrd4643.
    1. Marelli G., Howells A., Lemoine N.R., Wang Y. Oncolytic Viral Therapy and the Immune System: A Double-Edged Sword Against Cancer. Front. Immunol. 2018;9:866. doi: 10.3389/fimmu.2018.00866.
    1. Lei J., Li Q.-H., Yang J.-L., Liu F., Wang L., Xu W.-M., Zhao W.-X. The antitumor effects of oncolytic adenovirus H101 against lung cancer. Int. J. Oncol. 2015;47:555–562. doi: 10.3892/ijo.2015.3045.
    1. Lin X., Li Q.-J., Lao X.-M., Yang H., Li S. Transarterial injection of recombinant human type-5 adenovirus H101 in combination with transarterial chemoembolization (TACE) improves overall and progressive-free survival in unresectable hepatocellular carcinoma (HCC) BMC Cancer. 2015;15:707. doi: 10.1186/s12885-015-1715-x.
    1. Liang M. Oncorine, the World First Oncolytic Virus Medicine and its Update in China. Curr. Cancer Drug Targets. 2018;18:171–176. doi: 10.2174/1568009618666171129221503.
    1. Wang Y., Hallden G., Hill R.P., Anand A., Liu T.-C., Francis J., A Brooks G., Lemoine N.R., Kirn D.H. E3 gene manipulations affect oncolytic adenovirus activity in immunocompetent tumor models. Nat. Biotechnol. 2003;21:1328–1335. doi: 10.1038/nbt887.
    1. Ma J., Li N., Zhao J., Lu J., Ma Y., Zhu Q., Dong Z., Liu K., Ming L. Histone deacetylase inhibitor trichostatin A enhances the antitumor effect of the oncolytic adenovirus H101 on esophageal squamous cell carcinoma in vitro and in vivo. Oncol. Lett. 2017;13:4868–4874. doi: 10.3892/ol.2017.6069.
    1. Wang P., Li X., Wang J., Gao D., Li Y., Li H., Chu Y., Zhang Z., Liu H., Jiang G., et al. Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent. Nat. Commun. 2017;8:1395. doi: 10.1038/s41467-017-01385-8.
    1. Mansurov A., Ishihara J., Hosseinchi P., Potin L., Marchell T.M., Ishihara A., Williford J.-M., Alpar A.T., Raczy M.M., Gray L.T., et al. Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours. Nat. Biomed. Eng. 2020;4:531–543. doi: 10.1038/s41551-020-0549-2.
    1. Nguyen H.-M., Guz-Montgomery K., Saha D. Oncolytic Virus Encoding a Master Pro-Inflammatory Cytokine Interleukin 12 in Cancer Immunotherapy. Cells. 2020;9:400. doi: 10.3390/cells9020400.
    1. Song H.-N., Lee C., Kim S.T., Kim S.Y., Kim N.K., Jang J., Kang M., Jang H., Ahn S., Kim S.H., et al. Molecular characterization of colorectal cancer patients and concomitant patient-derived tumor cell establishment. Oncotarget. 2016;7:19610–19619. doi: 10.18632/oncotarget.7526.
    1. Lee J.-K., Liu Z., Sa J.K., Shin S., Wang J., Bordyuh M., Cho H.J., Elliott O., Chu T., Choi S.W., et al. Pharmacogenomic landscape of patient-derived tumor cells informs precision oncology therapy. Nat. Genet. 2018;50:1399–1411. doi: 10.1038/s41588-018-0209-6.
    1. Miao J.X., Wang J.Y., Li H.Z., Guo H.R., Dunmall L.S.C., Zhang Z.X., Cheng Z.G., Gao D.L., Dong J.Z., Wang Z.D. Promising xenograft animal model recapitulating the features of human pancreatic cancer. World J. Gastroenterol. 2020;26:4802–4816. doi: 10.3748/wjg.v26.i32.4802.
    1. Toth K., Lee S.R., Ying B., Spencer J.F., Tollefson A.E., Sagartz J.E., Kong I.-K., Wang Z., Wold W.S.M. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control. PLOS Pathog. 2015;11:e1005084. doi: 10.1371/journal.ppat.1005084.
    1. Ayyoob K., Masoud K., Vahideh K., Asadi J. Authentication of newly established human esophageal squamous cell carcinoma cell line (YM-1) using short tandem repeat (STR) profiling method. Tumor Biol. 2015;37:3197–3204. doi: 10.1007/s13277-015-4133-4.
    1. Wen J., Zheng B., Hu Y., Zhang X., Yang H., Luo K.J., Zhang X., Li Y.F., Fu J.H. Establishment and biological analysis of the EC109/CDDP multidrug-resistant esophageal squamous cell carcinoma cell line. Oncol. Rep. 2009;22:65–71.
    1. Hagihara Y., Sakamoto A., Tokuda T., Yamashita T., Ikemoto S., Kimura A., Haruta M., Sasagawa K., Ohta J., Takayama K., et al. Photoactivatable oncolytic adenovirus for optogenetic cancer therapy. Cell Death Dis. 2020;11:1–9. doi: 10.1038/s41419-020-02782-6.
    1. Martin D.K., Uckermann O., Bertram A., Liebner C., Hendruschk S., Sitoci-Ficici K.H., Schackert G., Lord E.M., Temme A., Kirsch M. Differential growth inhibition of cerebral metastases by anti-angiogenic compounds. Anticancer. Res. 2014;34:3293–3302.
    1. Xu J., Bai Y., Xu N., Li E., Wang B., Wang J., Li X., Wang X., Yuan X. Tislelizumab Plus Chemotherapy as First-line Treatment for Advanced Esophageal Squamous Cell Carcinoma and Gastric/Gastroesophageal Junction Adenocarcinoma. Clin. Cancer Res. 2020;26:4542–4550. doi: 10.1158/1078-0432.CCR-19-3561.
    1. Wold W.S., Toth K. Chapter three--Syrian hamster as an animal model to study oncolytic adenoviruses and to evaluate the efficacy of antiviral compounds. Adv. Cancer Res. 2012;115:69–92.
    1. Yang C., Zhang J., Ding M., Xu K., Li L., Mao L., Zheng J. Ki67 targeted strategies for cancer therapy. Clin. Transl. Oncol. 2017;20:570–575. doi: 10.1007/s12094-017-1774-3.
    1. Wong R.J., Chan M.-K., Yu Z., Ghossein R.A., Ngai I., Adusumilli P.S., Stiles B.M., Shah J., Singh B., Fong Y. Angiogenesis Inhibition by an Oncolytic Herpes Virus Expressing Interleukin 12. Clin. Cancer Res. 2004;10:4509–4516. doi: 10.1158/1078-0432.CCR-04-0081.
    1. Wei D., Xu J., Liu X.-Y., Chen Z., Bian H. Fighting Cancer with Viruses: Oncolytic Virus Therapy in China. Hum. Gene Ther. 2018;29:151–159. doi: 10.1089/hum.2017.212.
    1. Shaw A.R., Suzuki M. Immunology of Adenoviral Vectors in Cancer Therapy. Mol. Ther. - Methods Clin. Dev. 2019;15:418–429. doi: 10.1016/j.omtm.2019.11.001.
    1. Ma G., Kawamura K., Li Q., Okamoto S., Suzuki N., Kobayashi H., Liang M., Tada Y., Tatsumi K., Hiroshima K., et al. Combinatory cytotoxic effects produced by E1B-55kDa-deleted adenoviruses and chemotherapeutic agents are dependent on the agents in esophageal carcinoma. Cancer Gene Ther. 2010;17:803–813. doi: 10.1038/cgt.2010.37.
    1. Cheng P.-H., Wechman S.L., McMasters K.M., Zhou H.S. Oncolytic Replication of E1b-Deleted Adenoviruses. Viruses. 2015;7:5767–5779. doi: 10.3390/v7112905.
    1. Öberg D., Yanover E., Adam V., Sweeney K., Costas C., Lemoine N.R., Halldén G. Improved potency and selectivity of an oncolytic E1ACR2 and E1B19K deleted adenoviral mutant in prostate and pancreatic cancers. Clin. Cancer Res. 2010;16:541–543. doi: 10.1158/1078-0432.CCR-09-1960.
    1. Mao L.-J., Kan Y., Li B.-H., Ma S., Liu Y., Yang D.-L., Yang C. Combination Therapy of Prostate Cancer by Oncolytic Adenovirus Harboring Interleukin 24 and Ionizing Radiation. Front. Oncol. 2020;10:421. doi: 10.3389/fonc.2020.00421.
    1. Cicchelero L., Denies S., Haers H., Vanderperren K., Stock E., Van Brantegem L., De Rooster H., Sanders N.N. Intratumoural interleukin 12 gene therapy stimulates the immune system and decreases angiogenesis in dogs with spontaneous cancer. Vet. Comp. Oncol. 2016;15:1187–1205. doi: 10.1111/vco.12255.
    1. Lu X. Impact of IL-12 in Cancer. Curr. Cancer Drug targets. 2017;17:682–697. doi: 10.2174/1568009617666170427102729.
    1. Lasek W., Zagozdzon R., Jakobisiak M. Interleukin 12: Still a promising candidate for tumor immunotherapy? Cancer Immunol. Immunother. 2014;63:419–435. doi: 10.1007/s00262-014-1523-1.
    1. Freytag S.O., Barton K.N., Zhang Y. Efficacy of oncolytic adenovirus expressing suicide genes and interleukin-12 in preclinical model of prostate cancer. Gene Ther. 2013;20:1131–1139. doi: 10.1038/gt.2013.40.
    1. O Freytag S., Zhang Y., Siddiqui F. Preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for prostate cancer. Mol. Ther. Oncolytics. 2015;2:15006. doi: 10.1038/mto.2015.6.
    1. Fredebohm J., Boettcher M., Eisen C., Gaida M.M., Heller A., Keleg S., Tost J., Greulich-Bode K.M., Hotz-Wagenblatt A., Lathrop M., et al. Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System. PLoS ONE. 2012;7:e48503. doi: 10.1371/journal.pone.0048503.
    1. Ohkura Y., Ueno M., Udagawa H. Risk factors for febrile neutropenia and effectiveness of primary prophylaxis with pegfilgrastim in patients with esophageal cancer treated with docetaxel, cisplatin, and 5-fluorouracil. World J. Surg. Oncol. 2019;17:125. doi: 10.1186/s12957-019-1665-x.
    1. Bortolanza S., Alzuguren P., Buñuales M., Qian C., Prieto J., Hernandez-Alcoceba R. Human adenovirus replicates in immunocompetent models of pancreatic cancer in Syrian hamsters. Hum. Gene Ther. 2007;18:681–690. doi: 10.1089/hum.2007.017.
    1. Li X., Wang P., Li H., Du X., Liu M., Huang Q., Wang Y., Wang S. The Efficacy of Oncolytic Adenovirus Is Mediated by T-cell Responses against Virus and Tumor in Syrian Hamster Model. Clin. Cancer Res. 2016;23:239–249. doi: 10.1158/1078-0432.CCR-16-0477.
    1. Milanovic M., Fan D.N.Y., Belenki D., Däbritz J.H.M., Zhao Z., Yu Y., Dörr J.R., Dimitrova L., Lenze D., Barbosa I.A.M., et al. Senescence-associated reprogramming promotes cancer stemness. Nat. Cell Biol. 2018;553:96–100. doi: 10.1038/nature25167.
    1. Saunders N.A., Simpson F., Thompson E.W., Hill M.M., Endo-Munoz L., Leggatt G., Minchin R.F., Guminski A. Role of intratumoural heterogeneity in cancer drug resistance: Molecular and clinical perspectives. EMBO Mol. Med. 2012;4:675–684. doi: 10.1002/emmm.201101131.

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