Histone deacetylase inhibitor boosts anticancer potential of fusogenic oncolytic vaccinia virus by enhancing cell-cell fusion.

Oncolytic viruses have two anticancer functions: direct oncolysis and elicitation of antitumor immunity. We previously developed a novel fusogenic oncolytic vaccinia virus (FUVAC) from a non-fusogenic vaccinia virus (VV) and, by remodeling the tumor immune microenvironment, we demonstrated that FUVAC induced stronger oncolysis and antitumor immune responses compared with non-fusogenic VV. These functions depend strongly on cell-cell fusion induction. However, FUVAC tends to have decreased fusion activity in cells with low virus replication efficacy. Therefore, another combination strategy was required to increase cell-cell fusion in these cells. Histone deacetylase (HDAC) inhibitors suppress the host virus defense response and promote viral replication. Therefore, in this study, we selected an HDAC inhibitor, trichostatin A (TSA), as the combination agent for FUVAC to enhance its fusion-based antitumor potential. TSA was added prior to FUVAC treatment of murine tumor B16-F10 and CT26 cells. TSA increased the replication of both FUVAC and parental non-fusogenic VV. Moreover, TSA enhanced cell-cell fusion and FUVAC cytotoxicity in these tumor cells in a dose-dependent manner. Transcriptome analysis revealed that TSA-treated tumors showed altered expression of cellular component-related genes, which may affect fusion tolerance. In a bilateral tumor-bearing mouse model, combination treatment of TSA and FUVAC significantly prolonged mouse survival compared with either treatment alone or in combination with non-fusogenic VV. Our findings demonstrate that TSA is a potent enhancer of cell-cell fusion efficacy of FUVAC.

Fusogenic oncolytic vaccinia virus enhances systemic antitumor immune response by modulating the tumor microenvironment.

Oncolytic viruses induce antitumor immunity following direct viral oncolysis. However, their therapeutic effects are limited in distant untreated tumors because their antitumor function depends on indirect antitumor immunity. Here, we generated a novel fusogenic oncolytic vaccinia virus (FUVAC) and compared its antitumor activity with that of its parental non-fusogenic virus. Compared with the parent, FUVAC exerted the cytopathic effect and induced immunogenic cell death in human and murine cancer cells more efficiently. In a bilateral tumor-bearing syngeneic mouse model, FUVAC administration significantly inhibited tumor growth in both treated and untreated tumors. However, its antitumor effects were completely suppressed by CD8+ T cell depletion. Notably, FUVAC reduced the number of tumor-associated immune-suppressive cells in treated tumors, but not in untreated tumors. Mice treated with FUVAC before an immune checkpoint inhibitor (ICI) treatment achieved complete response (CR) in both treated and untreated tumors, whereas ICI alone did not show antitumor activity. Mice achieving CR rejected rechallenge with the same tumor cells, suggesting establishment of a long-term tumor-specific immune memory. Thus, FUVAC improves the tumor immune microenvironment and enhances systemic antitumor immunity, suggesting that, alone and in combination with ICI, it is a novel immune modulator for overcoming oncolytic virus-resistant tumors.

IL-7 coupled with IL-12 increases intratumoral T cell clonality, leading to complete regression of non-immunogenic tumors.

Immune checkpoint inhibitors against PD-1, PD-L1 and CTLA-4 have altered the treatment paradigm for various types of cancers in the past decade. However, they offer clinical benefits to only a subset of patients. Evaluation and identification of an appropriate therapeutic approach to improve intratumoral immune status are needed for better treatment outcomes. We previously demonstrated that intratumoral expression of IL-7 and IL-12 increased tumor-infiltrating lymphocytes in poorly immunogenic tumors, resulting in a higher tumor regression rate than IL-12 alone. However, the mechanism underlying the difference in efficacy with and without IL-7 remains unclear. Here, we identified a previously unknown effect of IL-7 on the T cell receptor (TCR) repertoire of intratumoral CD8+ T cells, which is induced in the presence of IL-12. While IL-7 alone increased the diversity of intratumoral CD8+ T cells, IL-7 with IL-12 increased a limited number of high-frequency clones, conversely augmenting IL-12 function to increase the clonality. The proportion of mice with multiple high-frequency clones in tumors correlated with that achieving complete tumor regression in efficacy studies. These findings provide a scientific rationale for combining IL-7 and IL-12 in anticancer immunotherapy and unveil a novel IL-7 function on intratumoral TCR repertoire.

Long noncoding RNA UCA1 enhances sensitivity to oncolytic vaccinia virus by sponging miR-18a/miR-182 and modulating the Cdc42/filopodia axis in colorectal cancer.

The promising anti-tumor effects of oncolytic vaccinia virus (OVV) have been demonstrated. Further, we previously showed that long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) enhances OVV cell-to-cell spread via the activation of Cdc42 in ovarian cancer. However, its role in other cancer types and the molecular mechanism underlying its effects remain to be explored. In this study, we first demonstrated that UCA1 upregulates OVV cell-to-cell spread but not its binding, entry, and replication in colorectal cancer cells. Functional analysis indicated that Cdc42 activation and filopodia formation play an important role in this process. Moreover, expression analysis of various miRNAs suggested that UCA1 inhibits both miR-18a and miR-182, thereby promoting Cdc42 activation, which in turn, regulates OVV cell-to-cell spread. Furthermore, UCA1 was found to modulate tumor malignancy, drug resistance, and sensitivity to OVV via different miRNAs in colorectal cancer. These findings indicate that a three-marker panel, which includes UCA1 expression, Cdc42 activation, and filopodia formation, could potentially be used to predict the therapeutic effect of OVV in colorectal cancer.

The Analysis of Cell Cycle-related Proteins in Ovarian Clear Cell Carcinoma Versus High-grade Serous Carcinoma.

In Japan, the frequency of ovarian clear cell carcinoma (CCC) is twice as high as that in the United States and Europe. Often, patient prognosis with CCC is poor because of chemoresistance. Here, we focus on the cell cycle, which is one of the mechanisms of chemoresistance. To detect the informative markers and improve the strategy of chemotherapy for CCC, we performed immunohistochemical staining of cell cycle-related proteins in ovarian malignant tumors. We detected that each of the 29 samples of CCC and high-grade serous carcinoma (HGSC) were necessary to reveal the significant differences in immunostaining and prognosis. We performed the immunostaining analysis using the antibodies of cell cycle-related proteins such as Ki-67, Cdt1, MCM7, and geminin. The positive rate of Cdt1 in the CCC group was significantly higher than that in the HGSC group (P<0.0001). However, the positive rate of geminin in the HGSC group was significantly higher than that in the CCC group (P<0.0001). The overall survival of CCC patients with high labeling index of Cdt1 was significantly worse than that of CCC patients with low labeling index of Cdt1 (P=0.004). The study results suggested that the cancer cells of CCC and HGSC exist in the G1 phase and S, G2, and M phases, respectively. The differences in cell cycle of CCC might be one of the reasons for chemotherapy resistance. Further investigations are necessary to reveal the usefulness of Cdt1 as a biomarker in CCC.

Does local infiltration anesthesia on laparoscopic surgical wounds reduce postoperative pain? Randomized control study.

PURPOSE: Recently, endoscopic surgeries are widely performed in the gynecological field. Several studies on the use of local anesthesia for pain control after laparoscopic surgery have been conducted; however, its effects remain controversial. Herein, a randomized control study on gynecological laparoscopic surgeries was conducted to analyze the effectiveness of local anesthesia on postoperative pain. METHODS: Patients who underwent laparoscopic surgeries due to gynecologic benign diseases or endometrial cancer in the early stage were enrolled, and randomly divided into intervention (injected with levobupivacaine), and control (injected with saline) groups. The primary outcome was the dosage of analgesic consumption within 12 hours postoperatively. RESULTS: A total of 147 patients were enrolled in the intervention group and 147 in the control group. The outcome of local anesthesia was not significantly different between the two groups during the whole analysis. We analyzed the effects of local anesthesia in the laparoscopic surgery subgroup. The dosage of analgesic consumption within 12 h after a laparoscopic hysterectomy (TLH) or TLH with pelvic lymph node dissection (TLH+PLD) in the intervention group was significantly smaller than that in the control group. CONCLUSION: Local infiltration anesthesia can effectively reduce postoperative pain in patients who underwent TLH or TLH +PLD.

[A Case Report of Surgery and Chemotherapy for a Patient with Rapidly Progressing Breast Cancer during Pregnancy].

A 33-year-old woman became aware of a right breast mass at her 28th week of pregnancy. From the biopsy results, we diagnosed her with right breast cancer. At her 33rd week of pregnancy, she underwent modified radical mastectomy (pT2N3aM0, Stage III C, ER-negative, PR-negative, HER2-positive), and she elected to receive adjuvant therapy after the surgery during her pregnancy. She received the first course of EC (epirubicin plus cyclophosphamide) therapy on the 13th postoperative day (35 weeks of gestation) and gave a natural, vaginal delivery at 36 weeks and 5 days of gestation. On the 4th day after birth, the patient noticed a contralateral left breast mass and was diagnosed with left breast cancer, after core needle biopsy. She received 4 courses of EC therapy and is currently undergoing PTX plus HER (paclitaxel plus trastuzumab) therapy. Regarding chemotherapy during pregnancy, we recommend that there is no need to perform artificial preterm birth, because chemotherapy has little influence on children after their second-trimester. After the second-trimester, chemotherapy can be safely performed.

Cortical control of object-specific grasp relies on adjustments of both activity and effective connectivity: a common marmoset study.

KEY POINTS: The cortical mechanisms of grasping have been extensively studied in macaques and humans; here, we investigated whether common marmosets could rely on similar mechanisms despite strong differences in hand morphology and grip diversity. We recorded electrocorticographic activity over the sensorimotor cortex of two common marmosets during the execution of different grip types, which allowed us to study cortical activity (power spectrum) and physiologically inferred connectivity (phase-slope index). Analyses were performed in beta (16-35 Hz) and gamma (75-100 Hz) frequency bands and our results showed that beta power varied depending on grip type, whereas gamma power displayed clear epoch-related modulation. Strength and direction of inter-area connectivity varied depending on grip type and epoch. These findings suggest that fundamental control mechanisms are conserved across primates and, in future research, marmosets could represent an adequate model to investigate primate brain mechanisms. ABSTRACT: The cortical mechanisms of grasping have been extensively studied in macaques and humans. Here, we investigated whether common marmosets could rely on similar mechanisms despite striking differences in manual dexterity. Two common marmosets were trained to grasp-and-pull three objects eliciting different hand configurations: whole-hand, finger and scissor grips. The animals were then chronically implanted with 64-channel electrocorticogram arrays positioned over the left premotor, primary motor and somatosensory cortex. Power spectra, reflecting predominantly cortical activity, and phase-slope index, reflecting the direction of information flux, were studied in beta (16-35 Hz) and gamma (75-100 Hz) bands. Differences related to grip type, epoch (reach, grasp) and cortical area were statistically assessed. Results showed that whole-hand and scissor grips triggered stronger beta desynchronization than finger grip. Task epochs clearly modulated gamma power, especially for finger and scissor grips. Considering effective connectivity, finger and scissor grips evoked stronger outflow from primary motor to premotor cortex, whereas whole-hand grip displayed the opposite pattern. These findings suggest that fundamental control mechanisms, relying on adjustments of cortical activity and connectivity, are conserved across primates. Consistently, marmosets could represent a good model to investigate primate brain mechanisms.

Stabilization of lead in an alkali-activated municipal solid waste incineration fly ash-Pyrophyllite-based system.

This work focuses on the stabilization and speciation of lead (Pb) in a composite solid produced from an alkali-activated municipal solid waste incineration fly ash (MSWIFA)-pyophyllite-based system. The solid product was synthesized after mixtures of raw materials (dehydrated pyrophyllite, MSWIFA, 14 mol/L aqueous sodium hydroxide, and sodium silicate solution) were cured at 105 °C for 24 h. The product could reduce the leaching of Pb and the Pb concentration in the leachate was 7.0 × 10-3 using the Japanese leaching test and 9.7 × 10-4 mg/L using toxicity characteristics leaching procedure method, which satisfied the respective test criteria and successfully stabilized Pb in this system. The solid product had a compressive strength of 2 MPa and consisted mainly of crystalline phases. Scanning electron microscopy with X-ray analysis and X-ray absorption fine structure suggested that Pb was present along with Al, Si, and O, and that the atomic environment around the Pb was similar to that of PbSiO3. These results suggest that the alkali-activated MSWIFA-pyrophyllite-based system could be used to stabilize Pb in MSWIFA.

Retargeting of microcell fusion towards recipient cell-oriented transfer of human artificial chromosome.

BACKGROUND: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction. Application of Measles Virus (MV) fusogenic proteins to MMCT instead of polyethylene glycol (PEG) has partly solved this drawback, whereas the tropism of MV fusogenic proteins is restricted to human CD46- or SLAM-positive cells. RESULTS: Here, we show that retargeting of microcell fusion by adding anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of the MV-H protein improves the efficiency of MV-MMCT to human fibroblasts which originally barely express both native MV receptors, and are therefore resistant to MV-MMCT. Efficacy of chimeric fusogenic proteins was evaluated by the evidence that the HAC, tagged with a drug-resistant gene and an EGFP gene, was transferred from CHO donor cells into human fibroblasts. Furthermore, it was demonstrated that no perturbation of either the HAC status or the functions of transgenes was observed on account of retargeted MV-MMCT when another HAC carrying four reprogramming factors (iHAC) was transferred into human fibroblasts. CONCLUSIONS: Retargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors. Therefore, this technology could facilitate the systematic cell engineering by HACs.

Angiopoietin-like protein 2 promotes inflammatory conditions in the ligamentum flavum in the pathogenesis of lumbar spinal canal stenosis by activating interleukin-6 expression.

PURPOSE: Chronic inflammation is thought to cause ligamentum flavum (LF) degeneration and hypertrophy in lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is highly expressed in hypertrophied LF. Because Angptl2 regulates interleukin-6 (IL-6) expression in various tissues, we investigated whether IL-6 is expressed in hypertrophied LF and, if so, does Angptl2 induce IL-6 expression in LF fibroblasts. METHODS: LF tissue was obtained from LSCS patients and non-LSCS patients. Polymerase chain reaction (PCR) for Angptl2 and IL-6 genes and immunohistochemistry for IL-6 protein were performed in LF tissue. Fibroblasts from LF tissue were used for in vitro experiments. Expression of integrin α5ß1 (an Angptl2 receptor) and Angptl2 binding to receptors on LF fibroblasts were examined by fluorescence-activated cell sorter analysis and cell adhesion assays. After Angptl2 recombinant protein treatment, NF-κB activation and IL-6 expression in LF fibroblasts were investigated by immunocytochemistry, PCR, and enzyme-linked immunosorbent assay. RESULTS: IL-6 mRNA expression was increased in hypertrophied LF tissue from LSCS patients and positively correlated with LF thickness and Angptl2 mRNA expression. IL-6 protein was highly expressed in LF fibroblasts in hypertrophied LF tissue. In vitro experiments demonstrated integrin α5ß1 expression on LF fibroblasts and Angptl2 binding to cells via receptors. Angptl2 stimulation promoted NF-κB nuclear translocation and induced IL-6 expression and secretion in LF fibroblasts. CONCLUSIONS: Angptl2 promotes inflammation in LF tissue by activating IL-6 expression, leading to LF degeneration and hypertrophy.

Massive cystic degeneration of a uterine leiomyoma in a patient with autosomal dominant polycystic kidney disease.

BACKGROUND: A uterine cyst occurring as an extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) is extremely rare. CASE: A 46-year-old Japanese woman was referred with a large abdominal mass causing severe abdominal distension. A large uterine cyst as an extrarenal manifestation of ADPKD was strongly suspected. First, we managed this patient by aspirating the cyst fluid through a small laparotomy. A year later, the cyst recurred and the patient underwent hysterectomy. Massive cystic degeneration of a uterine leiomyoma was diagnosed histologically. CONCLUSION: We described the rare case of massive cystic degeneration of a uterine leiomyoma in a patient with ADPKD, in which a causal relationship was suspected.

MicroRNA regulation of glycoprotein B5R in oncolytic vaccinia virus reduces viral pathogenicity without impairing its antitumor efficacy.

Vaccinia virus, once widely used for smallpox vaccine, has recently been engineered and used as an oncolytic virus for cancer virotherapy. Their replication has been restricted to tumors by disrupting viral genes and complementing them with products that are found specifically in tumor cells. Here, we show that microRNA (miRNA) regulation also enables tumor-specific viral replication by altering the expression of a targeted viral gene. Since the deletion of viral glycoprotein B5R not only decreases viral pathogenicity but also impairs the oncolytic activity of vaccinia virus, we used miRNA-based gene regulation to suppress B5R expression through let-7a, a miRNA that is downregulated in many tumors. The expression of B5R and the replication of miRNA-regulated vaccinia virus (MRVV) with target sequences complementary to let-7a in the 3'-untranslated region (UTR) of the B5R gene depended on the endogenous expression level of let-7a in the infected cells. Intratumoral administration of MRVV in mice with human cancer xenografts that expressed low levels of let-7a resulted in tumor-specific viral replication and significant tumor regression without side effects, which were observed in the control virus. These results demonstrate that miRNA-based gene regulation is a potentially novel and versatile platform for engineering vaccinia viruses for cancer virotherapy.

A case of serous endometrial intraepithelial carcinoma with p53 positivity for six years.

A 69-year-old postmenopausal woman was referred because she had been taking tamoxifen for four years. Tissues obtained by endometrial curettage were immunopositive for p53, but there was no definite malignancy. At age 73, cytology again showed abnormalities, so we repeated complete endometrial curettage. Again, there was no malignancy, but p53 immunostaining was widely positive. At age 75, hysterectomy was performed because cytological examination showed increasingly abnormal findings and the patient opted for surgery. In the resected uterus, endometrial glands were replaced by malignant cells resembling papillary serous carcinoma cells with high-grade nuclei, but there was no stromal or myometrial invasion. The pathological diagnosis was intraepithelial serous endometrial carcinoma. This is a rare case because we could follow the patient for 6 years by endometrial cytology or endometrial curettage and we observed gradual transformation into endometrial intraepithelial carcinoma.

An artificial intelligence-assisted diagnostic system improves the accuracy of image diagnosis of uterine cervical lesions.

The present study created an artificial intelligence (AI)-automated diagnostics system for uterine cervical lesions and assessed the performance of these images for AI diagnostic imaging of pathological cervical lesions. A total of 463 colposcopic images were analyzed. The traditional colposcopy diagnoses were compared to those obtained by AI image diagnosis. Next, 100 images were presented to a panel of 32 gynecologists who independently examined each image in a blinded fashion and diagnosed them for four categories of tumors. Then, the 32 gynecologists revisited their diagnosis for each image after being informed of the AI diagnosis. The present study assessed any changes in physician diagnosis and the accuracy of AI-image-assisted diagnosis (AISD). The accuracy of AI was 57.8% for normal, 35.4% for cervical intraepithelial neoplasia (CIN)1, 40.5% for CIN2-3 and 44.2% for invasive cancer. The accuracy of gynecologist diagnoses from cervical pathological images, before knowing the AI image diagnosis, was 54.4% for CIN2-3 and 38.9% for invasive cancer. After learning of the AISD, their accuracy improved to 58.0% for CIN2-3 and 48.5% for invasive cancer. AI-assisted image diagnosis was able to improve gynecologist diagnosis accuracy significantly (P<0.01) for invasive cancer and tended to improve their accuracy for CIN2-3 (P=0.14).

Enhanced antitumor effects of an engineered measles virus Edmonston strain expressing the wild-type N, P, L genes on human renal cell carcinoma.

Measles virus Edmonston strain (MV-Edm) is thought to have remarkable oncolytic activity that selectively destroys human tumor cells. The P/V/C protein of wild-type MV was shown to resist the antiviral effects of interferon (IFN)-alpha. Here, we engineered new MVs by arming MV-Edm tag strain (a V-defective vaccine-lineage strain, MV-Etag) with the P or N, P, and L genes of wild-type MV (MV-P and MV-NPL, respectively). The oncolytic activities of the MVs were determined in human renal cell carcinoma (RCC) cell lines and primary human RCC cells by the MTT assay. The antitumor efficacy of the MVs was evaluated in A-498 xenografts in nude mice. IFN-alpha effectively inhibited the replication of MV-Etag and MV-P, but not MV-NPL. MV-NPL more efficiently induced cytopathic effects (CPEs) in OS-RC-2 cells, even in the presence of human IFN-alpha. MV-NPL replicated more rapidly than MV-P and MV-Etag in A-498 cells. Apoptosis was induced earlier in A-498 cells by MV-NPL than MV-Etag and MV-P. MV-NPL showed more significant antitumoral effects and had prolonged replication compared to MV-Etag and MV-P. In this study, we demonstrated that the newly engineered MV-NPL has more effective oncolytic activity and may help establish an innovative cancer therapy.

Effects of 4-hydroxy-2-nonenal, a major lipid peroxidation-derived aldehyde, and N-acetylcysteine on the cyclooxygenase-2 expression in human uterine myometrium.

BACKGROUND: Chorioamnionitis is one of the important causes of preterm labor. Preterm labor with chorioamnionitis is associated with oxidative stress. We reported that 4-hydroxy-2-nonenal (4-HNE), a major end product of oxidative fatty acid metabolism, is accumulated in the placenta with chorioamnionitis. The aim of this study was to confirm the effect of 4-HNE on cyclooxygenase-2 (COX-2) and prostaglandin (PG) induction in the uterine myometrial tissues. We also examined the effect of N-acetylcysteine (NAC) on 4-HNE-induced COX-2 expression. METHODS: Uterine myometrial tissues were obtained from 5 patients. One of them underwent elective cesarean section without labor, and 4 of them underwent hysterectomy because of placental previa or atonic bleeding. We stimulated the uterine myometrial tissues with 4-HNE. In addition, the tissues were pretreated with NAC before 4-HNE treatment. The expression of COX-2 mRNA was observed by real-time PCR. PGE2 and prostacyclin release into the supernatants of the tissue cultures was measured by ELISA. RESULTS: 4-HNE induced the COX-2 mRNA expression and PGE2 production in the uterine myometrial tissue culture in a dose-dependent and time-dependent manner. NAC inhibited 4-HNE-induced COX-2 expression. CONCLUSION: 4-HNE may play an important role in preterm labor. NAC might be protective against preterm labor under oxidative stress.

Change of salivary stress marker concentrations during pregnancy: maternal depressive status suppress changes of those levels.

AIM: The aim of the present study was to show changes in salivary cortisol and chromogranin A/protein concentrations as stress markers during pregnancy and to clarify the effect of chronic stress on stress markers. MATERIAL AND METHODS: Salivary samples were collected from 69 pregnant women during pregnancy. Salivary cortisol levels and chromogranin A/protein titers were determined. We surveyed the women's chronic stress using the Zung self-rating depression scale and General Health Questionnaire-28. RESULTS: Cortisol levels in the saliva of pregnant women showed biphasic change during pregnancy. Chromogranin A/protein levels in the saliva of pregnant women increased in the second and the early third trimesters and decreased to the puerperal period. Salivary cortisol concentrations of the chronic high stress group were significantly lower compared with those of the normal group. Salivary chromogranin A/protein concentrations of the chronic high stress group were also significantly lower than those of the normal group. CONCLUSION: The titration of salivary cortisol concentrations and chromogranin A/protein levels is a useful tool to determine maternal stress levels. The elevation of cortisol and chromogranin A/protein in the saliva was suppressed in the chronic high stress group during pregnancy.

Oncolytic vaccinia virus induces a novel phenotype of CD8+ effector T cells characterized by high ICOS expression.

Characterization of the intratumoral immune status is important for developing immunotherapies and evaluating their antitumor effectiveness. CD8+ T cells are one of the most important cell types that directly and indirectly contribute to antitumor efficacy by releasing cytolytic molecules and inflammatory cytokines in the tumor microenvironment. Previously, we engineered a tumor-selective oncolytic vaccinia virus that encodes interleukin-7 (IL-7) and IL-12 and demonstrated its usefulness as an agent for in situ vaccination against tumors, with data showing that antitumor efficacy was reliant upon CD8+ T cells recruited by viral treatment. Here, we investigated the phenotypic changes in intratumoral CD8+ T cells caused by this oncolytic virus and found increased expression of inducible co-stimulator (ICOS) in PD-1-CD8+ T cells. Unlike previously reported ICOS+CD8+ T cells, a subset of ICOS+PD-1-CD8+ T cells showed effector function characterized by granzyme B expression. ICOS expression was induced by the backbone virus, which did not encode any immune transgenes and was independent of upregulation of the type I interferon pathway. Not only did we identify a novel effector cell subset characterized by ICOS expression, but our findings also shed light on a potential unknown aspect of the mechanism of oncolytic vaccinia virotherapy.

Anti-Tumor Effects of MAPK-Dependent Tumor-Selective Oncolytic Vaccinia Virus Armed with CD/UPRT against Pancreatic Ductal Adenocarcinoma in Mice.

Engineered vaccinia virus serves as an oncolytic virus for cancer virotherapy. We evaluated the oncolytic characteristics of VGF- and O1-deleted recombinant mitogen-activated protein kinase (MAPK)-dependent vaccinia virus (MDRVV). We found that compared with viruses with the deletion of either gene alone, MDRVV is more attenuated in normal cells and can replicate in cancer cells that exhibit constitutive ERK1/2 activation in the MAPK pathway. We armed MDRVV with a bifunctional fusion gene encoding cytosine deaminase and uracil phosphoribosyltransferase (CD/UPRT), which converts 5-fluorocytosine (5-FC) into chemotherapeutic agents, and evaluated its oncolytic activity alone or in combination with 5-FC in human pancreatic cancer cell lines, tumor mouse models of peritoneal dissemination and liver metastasis, and ex vivo-infected live pancreatic cancer patient-derived tissues. CD/UPRT-armed MDRVV alone could efficiently eliminate pancreatic cancers, and its antitumor effects were partially enhanced in combination with 5-FC in vitro and in vivo. Moreover, the replication of MDRVV was detected in tumor cells of patient-derived, surgically resected tissues, which showed enlarged nuclei and high expression of pERK1/2 and Ki-67, and not in stromal cells. Our findings suggest that systemic injections of CD/UPRT-armed MDRVV alone or in combination with 5-FC are promising therapeutic strategies for pancreatic ductal adenocarcinoma.