Fig 1: Serum programmed cell death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) levels are strongly correlated in cats with HER2-positive and TN normal-like mammary carcinoma. (A) Correlation between sPD-1 and sPD-L1 levels in cats with mammary carcinoma (B) and in cats with HER2-positive and (C) triple negative (TN) normal-like carcinoma subtypes.
Fig 2: Programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) staining analysis of tumor-infiltrating lymphocytes (TILs) and cancer cells in HER2-positive and triple negative (TN) normal-like tumor samples (range values). (A) The score of PD-L1-positive TILs (B) and cancer cells in HER2-positive mammary carcinomas in comparison to TN normal-like tumors. (C) The score of PD-1-positive TILs between HER2-positive tumors and TN normal-like mammary carcinomas and (D) cancer cells.
Fig 3: vPD1/IL-12 displays distinct response patterns across a variety of tumor models. (A) Schematic representation of experimental design. Syngeneic C57/Bl6 mice were injected SQ on both the left and right flanks with LLC cells. Tumors were allowed to establish and then either mock treated or treated with three IT injections of vPD1, vIL-12, or vPD1/IL-12 (n=5–6 for all groups). (B) Progression of individual tumors after treatment. Data are displayed as the fold increase or decrease of tumor area compared with area at the initiation of treatment. (C) Overall survival of animals treated as indicated. Significance was determined using log-rank analysis (***p<0.001). Data in A–C are from a single experiment, which is representative of two independent experiments. (D) Schematic representation of experimental design. Syngeneic C57/Bl6 mice were injected with B16/F10 cells on both flanks. Tumors were allowed to establish and then either mock treated or treated with three IT injections of vPD1, vIL-12, or vPD1/IL-12 (n=5 for all groups). (E) Progression of individual tumors after treatment. Data are displayed as the fold increase or decrease of tumor area compared with area at the initiation of treatment. (F) Overall survival of animals treated as indicated. Significance was determined using log-rank analysis (**p<0.01). Data in D–F are from a single experiment, which is representative of two independent experiments. (G) Schematic representation of experimental design. Syngeneic FVB mice were injected with BR5-luc cells into the peritoneal cavity. Mice were sorted into their appropriate groups with equally representative tumor burden. Groups were then mock treated or treated with vPD1, vIL-12 or vPD1/IL-12 into the peritoneal cavity in 50 µL sterile PBS (n=5 for each group). (H) Bioluminescent signal indicative of tumor burden per group at 10 and 25 days after initial injection. (I) Overall survival of animals treated as indicated. Significance was determined using log-rank analysis (**p<0.01). Data in G–I are from a single experiment, which is representative of three independent experiments. (J) Schematic representation of experimental design. Syngeneic C57/Bl6 mice were injected with MC38 cells on both flanks. Tumors were allowed to establish and then either mock treated or treated with three IT injections of vPD1, vIL-12, or vPD1/IL-12 (n=5 for all groups). (K) Progression of individual tumors after treatment. Data are displayed as the fold increase or decrease of tumor area compared with area at the initiation of treatment. (L) Overall survival of animals treated as indicated. Significance was determined using log-rank analysis (***p<0.001). Data in J–L are from a single experiment, which is representative of three independent experiments. IL-12, interleukin 12; IT, intratumoral; LLC, Lewis lung carcinoma; MYXV, myxoma virus; PBS, phosphate-buffered saline; PD1, programmed cell death protein 1; SQ, subcutaneously; vPD1, virus expressing PD1; vPD1/IL-12, virus expressing PD1 and IL-12.
Fig 4: vPD1/IL-12 displays normal oncolytic potential in vitro. (A) The indicated cell types were infected with vGFP, vPD1, vIL-12, or vPD1/IL-12 at an MOI of 10. At 72 hours postinfection, cells were harvested and the number of infectious progeny virus was determined using standard foci forming assays. Data are representative of three independent experiments. (B) The indicated cell types were mock infected or infected with vGFP, vPD1, vIL-12, or vPD1/IL-12 at MOIs of 0.005, 0.01, 0.05, 0.1, 0.5, and 1.0. At 24 hours postinfection, cellular viability was determined using MTT assay. Data are shown as per cent viability compared with mock-infected control and represent the summation of two independent experiments. (C and D) BSC40 cells were infected with the indicated viruses at an MOI of 10. 72 hours postinfection, supernatant was harvested and analyzed for the presence of both soluble PD1 (C) and IL-12 (D) using ELISA. Data are representative of two independent experiments. Significance was determined using Student’s t-test (**p<0.01). FFU, foci forming units; LLC, Lewis lung carcinomav; vGFP, virus expressing green fluorescent protein; vIL-12, virus expressing interleukin 12; vPD1, virus expressing programmed cell death protein 1; vPD1/IL-12, virus expressing PD1 and IL-12.
Fig 5: Localized vPD1/IL-12 treatment induces high levels of TNF. Syngeneic C57/Bl6 mice were injected SQ on both the left and right flanks with B16/F10 cells. Tumors were allowed to establish and then either mock treated or treated with three IT injections of vPD1, vIL-12, or vPD1/IL-12 (n=3 for all groups). Six days after the initiation of treatment, total RNA was extracted from each tumor and analyzed using RNA-seq. (A) Unsupervised principle component analysis of gene expression from the indicated cohorts. (B) Gene Ontology pathway analysis of all genes which were differentially expressed between vPD1/IL-12 treated mice and all other cohorts. (C) Volcano plot showing the expression of all cytokines and interleukins. Data shown compare tumors treated with vPD1/IL-12 to tumors treated with control virus. (D) ELISA data showing expression of IFN and TNF protein in the supernatants of tumors 8 days after being treated as indicated. (E) Analysis of RNA-seq data (from A) demonstrating the presence of previously identified gene response signatures to IFN and TNF. FC, fold change; IFN, interferon; IL-12, interleukin 12; IT, intratumoral; MYXV, myxoma virus; PD1, programmed cell death protein 1; SQ, subcutaneously; TNF, tumor necrosis factor; vGFP, virus expressing green fluorescent protein; vPD1, virus expressing PD1; vPD1/IL-12, virus expressing PD1 and IL-12.
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