Volume 24, Issue 12, Pages 2109-2117 (December 2016) Robust Oncolytic Virotherapy Induces Tumor Lysis Syndrome and Associated Toxicities in the MPC-11 Plasmacytoma Model  Lianwen Zhang, Michael B Steele, Nathan Jenks, Jacquelyn Grell, Marshall Behrens, Rebecca Nace, Shruthi Naik, Mark J Federspiel, Stephen J Russell, Kah-Whye Peng  Molecular Therapy  Volume 24, Issue 12, Pages 2109-2117 (December 2016) DOI: 10.1038/mt.2016.167 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Syngeneic MPC-11 tumors in Balb/c mice are highly responsive to systemic VSV-mIFNβ-NIS therapy. Mice were given saline or one intravenous dose of VSV at 2 × 106, 2 × 107, or 2 × 108 TCID50. (a) Tumor volume, (b) percent change in body weight, and (c) survival curves after virus treatment. (d) Photograph showing hemorrhagic VSV-treated tumor. Molecular Therapy 2016 24, 2109-2117DOI: (10.1038/mt.2016.167) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Robust vesicular stomatitis viruses (VSV) replication in MPC-11 tumor results in secondary viremia. (a) Immunohistochemical staining for VSV antigens (red) in tumor sections harvested at necropsy (day 3). (b) Titers of infectious virus recovered from explanted tumors of mice treated IV with 107 TCID50 VSV-mIFNβ-NIS. (c) Quantitative RT-PCR for VSV-N RNA indicate VSV replication in the tumors at days 2 and 4 (P = 0.17 between days 2 and 4), (d) Secondary viremia as a result of intratumoral replication of VSV, (e) Increase and decline in serum levels of IFNβ in mice, reflecting the numbers of VSV infected cells between day 2 to 7. ***P ≤ 0.001, Student's t-test. Mean ± SEM values are shown. Molecular Therapy 2016 24, 2109-2117DOI: (10.1038/mt.2016.167) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Biochemical characterization of the blood or serum from nontumor or MPC-11 tumor bearing mice treated IV with 107 TCID50 VSV-mIFNβ-NIS. (a) Potassium, (b) phosphate, (c) calcium, (d) cell-free DNA, (e) uric acid, (f) aspartate aminotransferase, (g) alanine aminotransferase, (h) alkaline phosphatase. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, Student's t-test. Mean ± SEM values are shown. Molecular Therapy 2016 24, 2109-2117DOI: (10.1038/mt.2016.167) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Histological analysis of blood and tissues from vesicular stomatitis viruses (VSV)-treated MPC-11 bearing mice suggest intravascular coagulation and consumptive coagulopathy. Photographs of VSV treated mice taken at necropsy showed cases of (a) abdominal wall bleeding, and (b) pale ischemic liver. (c) A normal liver is shown for comparison. H&E stained liver sections from (d) saline-treated or (e) VSV-treated MPC-11 mice showing fibrin deposits and trapped erythrocytes in vessels. (f) Erythrocytes and platelets were seen in May-Grünwald-Giemsa stained blood smears from saline-treated mice. (g) Platelets were noticeably reduced in blood smears of VSV-treated mice at day 4 post-treatment. Immunohistochemical staining for platelets (CD41) and lymphocytes (CD45) in tumors harvested from (h, i) VSV-treated and (j, k) saline-treated mice. Aggregates of platelets and lymphocytes, as a result of intravascular coagulation, can be seen in the blood vessels of VSV-treated animals at day 4. Molecular Therapy 2016 24, 2109-2117DOI: (10.1038/mt.2016.167) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Systemically administered vesicular stomatitis viruses (VSV) replication is highly tumor selective with minimal infection of the liver. Immunohistochemical staining for VSV antigen (brown DAB (3,3′-Diaminobenzidine) stain) reflect robust VSV expression in (a) tumors at day 4 post-virus, and (b) minimal VSV expression in the liver of these VSV-treated mouse. (c) VSV staining of a liver section from a saline-treated animal. (d) QRT-PCR for VSV-N RNA and (e) infectious virus recovered from the liver of VSV-treated nontumor or MPC-11 bearing animals at day 2 and 4 post-virotherapy. ND, not detectable. (f) QRT-PCR showed minimal VSV replication in liver as indicated by declining VSV-N RNA in nontumor bearing Balb/c mice. Limit of detectable of VSV-N RNA by qRT-PCR is ≥1,000 copies/µg RNA, and infectious virus is >190 TCID50/ml. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Student's t-test. Molecular Therapy 2016 24, 2109-2117DOI: (10.1038/mt.2016.167) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Early death of vesicular stomatitis viruses (VSV)-treated MPC-11 mice can be mitigated by modulating tumor size or using VSV with different replication profiles. (a) Lymphocyte, (b) platelet count, (c) Alkaline phosphatase, and (d) Aspartate aminotransferase levels from mice with small or larger tumors, that were treated IV with 107 TCID50 VSV-mIFNβ-NIS. (e) Survival of VSV-treated mice with smaller tumors is significantly prolonged (P = 0.035) compared to those with larger (but responding) tumors. (f) Tumor volumes in mice treated with the respective viruses. (g) Recombinant VSV-M51R viruses with slower tumor regression resulted in prolonged survival of mice compared to VSV-mIFNβ viruses (P < 0.05). (h) VSV-NIS, a noninterferon inducing virus with wild-type M protein, is equally lethal to MPC-11 mice as the interferon expressing viruses. VSV-M51-NIS is the only group that is significantly different from saline (P = 0.0002). Survival curves were compared using Mantel Cox Log rank test. Mean ± SEM values are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Student's t-test. Molecular Therapy 2016 24, 2109-2117DOI: (10.1038/mt.2016.167) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions