Research ArticleExperimental Studies

Development of New Oncolytic Virotherapy Targeting Breast Cancer Using Coxsackievirus B3

MIYAKO SAGARA, SHOHEI MIYAMOTO, SHUN ITOH, YASUSHI SODA and KENZABURO TANI
Anticancer Research January 2021, 41 (1) 81-89; DOI: https://doi.org/10.21873/anticanres.14753

Abstract

Background/Aim: Breast cancer is the most common cancer in women worldwide, and triple-negative breast cancer (TNBC) is highly refractory to current standard therapies. Oncolytic virotherapy has recently gathered attention as a new treatment candidate for refractory cancers. Materials and Methods: We previously developed a new Coxsackievirus B3 (CVB3) virotherapy targeting lung cancers, and demonstrated that miRNA target sequence insertion into CVB3 reduced its pathogenicity, retaining its original oncolytic activity. In this study, we examined the oncolytic effects of CVB3 against breast cancer cells including TNBC cells. Results: CVB3 infection killed breast cancer cells in a time- and titer-dependent manner, and induced apoptosis. Nude mice transplanted with human TNBC cells were successfully treated with both CVB3-WT and CVB3-HP. Importantly, mice treated with CVB3-HP showed very few adverse events. Conclusion: CVB3-HP is a strong oncolytic virus candidate for breast cancer, including TNBC, due to its remarkable oncolytic efficacy and improved safety profile.

Key Words:

Although recent advances in surgery, radiotherapy and chemotherapy have remarkably improved cure and survival rates of early-stage cancers, new therapeutic methods are still needed, particularly for refractory metastatic and recurrent cancers. In Japan, one in two people develops cancer during their lifetime and one in three dies of cancer (1). Breast cancer is the most common cancer in women worldwide, and both its morbidity and mortality rates have been increasing in Japan (2). In particular, triple-negative breast cancer (TNBC) is highly refractory to current standard therapies, including cytotoxic chemotherapies, molecular targeted drugs and hormonal therapies, because it lacks estrogen and progesterone receptors as well as human epidermal growth factor receptor 2 (ERBB2; formerly HER2) (3). New therapeutic modalities targeting breast cancer, including TNBC, are needed in clinical settings.

Oncolytic virotherapy is an emerging form of anticancer treatment that involves viruses with a natural ability to lyse tumor cells (4, 5). Clinical trials are investigating DNA viruses, such as adenovirus, herpes simplex virus, poxvirus and vaccinia virus, as well as RNA viruses, including Coxsackievirus A21, measles virus, Newcastle virus, reovirus and Seneca valley virus. There has been particularly extensive investigation of oncolytic adenovirus and herpes simplex viruses, and both have demonstrated clinical benefits and a good safety profile (6-8). Two genetically modified oncolytic viruses are already used commercially as cancer therapies: H101, an adenovirus, was approved in China in 2005 (9), and talimogene laherparepvec, a genetically modified oncolytic herpes simplex virus, was approved in the United States and Europe in 2015. Additional viruses are expected to be useful as new anti-cancer treatments (10, 11).

Picornaviruses have recently gained much attention as potential oncolytic viruses, for a number of reasons. First, since picornaviruses are RNA viruses, they are not transferred to the nucleus and therefore there is no risk of insertional mutagenesis due to external gene integration into the host cellular genome DNA. Second, RNA viruses with icosahedral capsids but no envelope have a diameter of almost 25 nm, and they replicate rapidly in the host cytoplasm (12). Based on these benefits, clinical trials have examined oncolytic virotherapy using enterovirus. Recent reports showed the clinical benefits of Coxsackievirus A21 (CVA21) for human malignant melanoma and breast cancer, and echovirus 1 for stomach cancer and ovarian cancer (12-15). However, CVA21 was reported to cause fatal myositis shortly after its administration in mice, and its safety is therefore being carefully monitored (16). Based on in vitro screening, we recently reported that Coxsackievirus B3 (CVB3) is a promising oncolytic virus against various cancers (17).

In this study, we performed in vitro and in vivo experiments and demonstrated that the CVB3 is a strong candidate oncolytic virus for the treatment of breast cancer, including poor-prognosis TNBC.

Materials and Methods

Mice. Four-week-old nude mice were purchased from Charles River Laboratories Japan (Kanagawa, Japan) and used for the following experiments. All animal experiments were carried out under the Guidelines for Animal Experiments of Kyushu Universuty, The University of Tokyo and Law 105 Notification 6 of the Japanese Government.

Cells and culture methods. Human breast cancer cell lines MDA-MB-231, MDA-MB-468, ZR-75-1 and SK-BR-3, and human normal breast epithelial cell line MCF10A were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Human non-small cell lung cancer cell line NCI-H1299 was purchased from ATCC and used in viral titration assays. The human breast cancer cell lines MDA-MB-453 (RCB1192) and MCF7 (RCB1904) were purchased from RIKEN BioResource Research Center (Ibaraki, Japan). MDA-MB-231, MDA-MB-468 and MDA-MB-453 were TNBC cell lines, and ZR-75-1, SK-BR-3 and MCF-7 were non-TNBC cell lines. MDA-MB-231, MDA-MB-486 and MDA-MB-453 cells were cultured in Leibovitz’s L-15 Medium (GIBCO, Thermo Fisher Scientific, Waltham, MS, USA) containing 10% fetal bovine serum (FBS) (GIBCO). MCF7 cells were cultured in MEM (GIBCO) containing 10% FBS, 1% MEM Non-Essential Amino Acid (GIBCO) and 1% sodium pyruvate (GIBCO). Both ZR-75-1 and H1299 cells were cultured in RPMI1640 (Nacalai Tesque, Kyoto, Japan) containing 10% FBS. SK-BR-3 cells were cultured in McCoy’s 5A (Modified) Medium (GIBCO) containing 10% FBS. MCF10A cells were cultured in serum-free MEGM BulletKit (Lonza, Basel, Switzerland) containing ReagentPack™ (Lonza) and 100 ng/ml cholera toxin. All FBS was heat inactivated at 56°C for 30 min before use. All cell lines were cultured in 5% CO2 at 37°C.

Viruses. Coxsackievirus B3 (CVB3) was a kind gift of Dr. Hiroyuki Shimizu, National Institute of Infectious Diseases, Japan. CVB3 was diluted with Opti-MEMI (GIBCO) and the diluted virus solution was added to H1299 cells and incubated for 1 h in 5% CO2 at 37°C. The virus solution was replaced with RPMI1640 containing 10% FBS and incubated in 5% CO2 at 37°C until cytopathic effects were detected. At that time, the culture solution was changed to Opti-MEMI and cells were detached with a cell scraper and collected. Cell suspensions were frozen and thawed three times using liquid nitrogen. Viral supernatant was collected after centrifugation at 1,860 × g for 15 min at 4°C. Collected virus solution was frozen at –80°C until use.

To increase the safety of CVB3 wild type (CVB3-WT), we constructed two new recombinant miRNAs targeting CVB3, specifically miR-1 and miR-217. Briefly, CVB3 cDNA was amplified by PCR using primers with internal restriction sites and cloned into pBluescript II KS+ previously linearized with Not I and Sal I. The miRNA sequences were cloned into the 3’UTR of pBluescript-CVB3 by overlap extension PCR. We constructed the novel recombinant CVB-HP by inserting two copies each of miR-1, which is specifically expressed in the heart, and miR-217, which is specifically expressed in pancreas, into the 3’ UTR of the CVB3 genome. We next recovered infectious CVB3-HP viruses by RNA transfection in NCI-H1299 cells and used the viruses in the following experiments.

Virus titrations. H1299 cells were plated at 5×103 cells/well in 96-well microplates and incubated for 7 h at 37°C. Serial 10-fold dilutions of each virus, each at a volume of 50 μl, were added to each of eight replicate wells. After 5 d of culture, cell lysis in each well was evaluated under a stereoscopic microscope, and the virus titer was calculated based on the median tissue culture infectious dose (TCID50) as previously described (18).

Crystal violet staining. Crystal violet staining was performed as previously described (17). Briefly, cells were infected with viruses at an appropriate multiplicity of infection (MOI) for 1 h and the viral supernatant was changed to fresh media. After 72 h, the cells were stained with crystal violet as previously described (17).

Flow cytometry. To quantitate the cell surface expression of two CVB3 receptors, namely Coxsackie and adenovirus receptor (CAR) and decay-accelerating factor (DAF), immunofluorescently labeled cells were analyzed using flow cytometry. Briefly, harvested cultured cells were suspended with PBS to 1×106 cells/ml and 100 μl of each cell was seeded in 96-well round plates. After centrifugation at 830 × g for 5 min at 4°C, cell pellets were suspended with 100 μl of 250-fold diluted anti-CAR antibody (Sigma-Aldrich, St. Louis, MO, USA) or 500-fold diluted phycoerythrin-labeled anti-DAF antibody (Becton Dickinson Pharmingen, San Jose, CA, USA) with PBS containing 2% bovine serum albumin (BSA) (Sigma-Aldrich) and incubated for 1 h on ice in the dark. The cells treated with anti-CAR antibody were washed with PBS containing 2% BSA and centrifuged as above, and the cell pellets were suspended with 100 μl of 2,000-fold diluted allophycocyanin (APC)-labeled anti-mouse IgG antibody (eBioscience, Thermo Fisher Scientific) and incubated for 1 h on ice in the dark. After labeling, cells were washed twice with PBS and then analyzed by FACS Verse (Becton Dickinson Pharmingen) and quantitated using FlowJo Software Ver 10.5.3 (TreeStar, Ashland, OR, USA).

Cell viability assay. We assessed cell viability using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay or a CellTiter-Glo (Promega, Madison, WI, USA) assay. The MTS assay was essentially performed as detailed in a previous report (17). Briefly, in vitro cell viability was measured using 1×104 cells infected with CVB3 and analyzed with a CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). The CellTiter-Glo 2.0 assay is a method of determining the number of living cells in culture by quantifying the amount of ATP present in metabolically active cells.

Western blot analysis. To demonstrate whether CVB3 infection induced apoptosis of the target tumor cells, western blot analysis was performed to detect the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) fragments in the virus-infected cells. Cancer cells infected with CVB3 for 1 h were incubated for 18 h, harvested using a cell scraper, and centrifuged at 400 × g for 10 min. The cell pellets were suspended with cell lysis buffer [25 mM Tris-HCl (pH=7.6) (Nacalai Tesque), 150 mM NaCl (Nacalai Tesque), 1% NP-40 (Sigma-Aldrich), 1% sodium deoxycholate (Nacalai Tesque), 0.1% sodium dodecyl sulfate (SDS) (WAKO, Osaka, Japan)], and the supernatant was obtained by centrifugation at 20,400 × g for 15 min at 4°C. Cell lysate samples were resolved by SDS-PAGE and immunoblotted as previously described (17). The primary antibodies used were monoclonal antibodies against PARP and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Annexin V staining and cell-cycle analysis. To investigate whether apoptosis is involved in the oncolytic effect of CVB3, and to quantify the proportion of apoptotic cells, Annexin V–positive cells were detected using the Annexin V-PE apoptosis detection kit (BD Bioscience, San Diego, CA, USA) and analyzed with FACS-Verse as previously described (17).

In vivo tumor retraction study using human breast cancer–bearing nude mice. A total of 1×107 MDA-MB-468 cells were subcutaneously (s.c.) injected to the right flank of nude mice to establish xenograft mouse models. When each tumor reached 0.5 cm in diameter, 5×106 TCID50 of CVB3-WT, CVB3-HP or vehicle (Opti-MEM) was injected intratumorally (i.t.). Viruses or vehicle were injected i.t. on days 4, 6, 8, 10 and 12. Tumor sizes and body weights were recorded every other day for a total of 55 days after tumor transplantation, and the tumor volumes were calculated as (length × width2)/2. Mice were euthanized when the tumor diameter exceeded 1.0 cm or signs of skin ulceration became evident.

Histopathological examination. The pancreas and heart of nude mice were removed and fixed with 10% HCHO. The organs were embedded in paraffin and processed for sectioning and H&E staining by the technical office of the Medical Institute of Bioregulation, Kyushu University (Fukuoka, Japan), to perform paraffin infiltration treatment and block preparation.

Statistical analysis. All statistical analyses and graphical representations were performed as previously described using GraphPad Prism software, version 7.0d (GraphPad Prism, La Jolla, CA, USA) (17). Statistical analysis was performed using a two-tailed unpaired Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test. Survival curves were plotted according to the Kaplan–Meier method, and statistical differences were evaluated by log-rank test. p-Values less than 0.05 were considered statistically significant (17).

Results

Anti–breast cancer effects of CVB3-WT in vitro. To visualize viral cytopathic effects, crystal violet staining was performed. CVB3-WT at MOIs of 10–3, 10–2, 10–1, 100 and 10 was used to infect the following: human TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453, human non-TNBC cell lines ZR-75-1, SK-BR-3 and MCF7, and human normal mammary epithelial cell line of MCF10A. Seventy-two hours after infection, crystal violet staining indicated that CVB3-WT showed strong cytopathic effects at MOI 0.1 against human TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453, and against non-TNBC breast cell lines ZR-75-1, SK-BR-3 and MCF7. On the other hand, CVB3-WT showed no cytopathic effects against the human normal mammary epithelial cell line MCF10A, even at an MOI of 1 (Figure 1A).

Figure 1.
Figure 1.

Cytotoxic effects of CVB3 infection for human breast cancer cells in vitro. A. Human TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453, human non-TNBC cell lines MCF-7, ZR-75-1 and SK-BR-3, and human normal mammary epithelial cell line MCF10A were infected with CVB3-WT at MOIs of 10–2, 10–1, 100 and 10. Oncolytic activities were evaluated by crystal violet staining after 72 h. B. Surface expression of the CVB3 receptors CAR and DAF on the normal breast epithelial cell line MCF10A and the breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF7 and ZR-75-1 were quantified by flow cytometry. Histograms represent the measured fluorescence of cells incubated with an isotype control antibody (unshaded) and anti-CAR or anti-DAF antibody (shaded). C. Two TNBC cell lines, MDA-MB-231 and MDA-MB-468, were infected in vitro with CVB3 at MOIs of 0.1, 1 and 10, and analyzed every 12 h for cell viability by MTS assay. Each value was normalized to Opti-MEM–treated cells (Mock) and represents the mean±SD.

Since CVB3-WT was reported to lyse cells expressing high levels of CAR and DAF (17), we performed flow cytometric analysis to detect the expression of both proteins. All breast cancer cell lines expressed CAR and DAF, whereas MCF10A expressed low levels of both (Figure 1B). Furthermore, MTS assays showed that CVB3-WT killed both MDA-MB-231 and MDA-MB-468 in a time- and dose-dependent manner (Figure 1C).

Apoptosis induction by CVB3 infection. CVB3-infection has been reported to induce apoptosis in various cells (17, 19). To demonstrate whether CVB3 infection induced cellular apoptosis in target tumor cells in this study, western blotting analysis was performed to detect the proteolytic cleavage of PARP fragments that are produced following caspase activation during the apoptotic process in infected cells. An 89-kDa PARP fragment was detected in MDA-MB-468 cells, suggesting the induction of apoptosis. On the other hand, no PARP fragments were detected in MDA-MB-231 cells (Figure 2A).

Figure 2.
Figure 2.

Correlation between caspase-dependent apoptosis and CVB3-mediated cytotoxicity in breast cancer cells. Breast cancer cell lines were infected with CVB3 (MOI=0.01 or 0.1) and analyzed 18 h later. A. Each cellular lysate was subjected to immunoblot analysis. Full-length PARP (116 kDa) and cleaved PARP (89 kDa) are shown. B. The early apoptotic population consists of Annexin V-PE+/7-AAD– cells.

Furthermore, to confirm the induction of apoptosis in CVB3-infected TNBC cells, we assayed for Annexin V–positive cells, which usually appear in the early phase of apoptosis. Twenty-four hours after CVB3-WT infection, the population of Annexin V–positive cells (Annexin V-APC+/7-AAD−) was increased compared with mock infected cells (Mock) (Figure 2B). These results suggested that both direct cell destruction and apoptosis contributed to the cytopathic effects of CVB3 infection in breast cancer cells.

Cytotoxic effects of CVB3-HP against human TNBC cells in vitro. We previously reported that mice treated with CVB3-WT showed moderate liver dysfunction and mild myocarditis (17). To increase the safety of CVB3 virotherapy, we constructed several new recombinant CVB3s using microRNA technology and obtained CVB3-HP (Figure 3A). Three human TNBC cell lines were infected at MOIs of 10–3, 10–2, 10–1, 100 and 10 with CVB3-HP or CVB3-WT. Seventy-two hours after infection, viral cytotoxicity was evaluated using crystal violet staining. CVB3-HP showed almost the same cytotoxic effects as CVB3-WT (Figure 3B). Additionally, CellTiter-Glo assays of cell viability showed that both viruses exerted similar cytotoxic effects against TNBC cells, regardless of virus titer and time since infection (Figure 3C).

Figure 3.
Figure 3.

Evaluation of cytotoxic effects of CVB3-HP infection. A. Schematic diagram of CVB3-HP, showing insertion sites of miR-1 and miR-217 target constructs in the 3’ UTR of the CVB3 genome. B. Human TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453, and a human non-TNBC cell line MCF-7 were infected with CVB3-WT and CVB3-HP at MOIs of 10–2, 10–1, 100 and 10. Oncolytic activities were evaluated by crystal violet staining after 72 h. C. Two TNBC cell lines, MDA-MB-231 and MDA-MB-468, were infected in vitro with CVB3 at MOIs of 1 and 10, and analyzed every 24 h for cell viability by CellTiter-Glo assay. Each value was normalized to Opti-MEM–treated cells (Mock) and represents the mean±SD.

Antitumor activity of CVB3s in mouse tumor models. The antitumor activity of CVB3s in vivo was investigated by injecting CVB3-HP or CVB3-WT into MDA-MB-468 cell-transplanted tumors in BALB/c nude mice, established as described above. On day 4, subcutaneously transplanted tumors in the right flank region reached 0.5 cm in diameter and we injected 5×106 TCID50 of CVB3-HP or CVB3-WT on days 4, 6, 8, 10 and 12. Control mice showed continuous tumor growth, whereas all virus-treated mice showed complete tumor regression at 2 days after i.t. injection (p<0.05) (Figure 4A). There were no deaths during the observation period, indicating that the antitumor activity of CVB3-HP remained as effective as that of the original CVB3-WT. Importantly, although CVB3-WT-treated mice showed remarkable weight loss, CVB3-HP–treated mice showed none (p<0.05). Mice treated with CVB3-WT and CVB3-HP exhibited the same survival rates (Figures 4B and C).

Figure 4.
Figure 4.

In vivo oncolytic effects of intratumoral CVB3 administration to mice bearing human breast cancer cell xenografts. Each nude mouse received five i.t. doses of CVB3-WT, CVB3-HP or Opti-MEM into established tumors every other day. For in vivo studies, BALB/c nude mice underwent s.c. transplantation of 1×107 MDA-MB-468 cells. Arrows indicate the timing of five i.t. injections (5×106 TCID50) of the indicated viruses or vehicle control. Tumor volume (A) and body weight (B) were monitored every 2 d. C. Kaplan–Meier survival curves of mice treated with the indicated viruses. Differences between the control group and each virus-treated group were statistically evaluated by the log-rank test (p=0.0169). Data represent means±SEM. D. Representative pathological images of mouse pancreas and heart treated with Opti-MEM (a, d), CVB3-WT (b, e) or CVB3-HP (c, f). Scale bars, 100 μM.

Pathological findings showed destruction of exocrine glands in the pancreas and myocardial fibrosis in the heart of mice infected with CVB3-WT. In contrast, there were no such pathological abnormalities in either controls or CVB3-HP–infected mice (Figure 4D).

Discussion

Breast cancer is categorized into three major subtypes based on the presence or absence of estrogen and progesterone receptors and ERBB2: hormone receptor positive/ERBB2 negative (70% of patients), ERBB2 positive (15%-20%), and triple-negative (tumors lacking all three standard molecular markers; 15%). TNBC is more likely to recur than the other two subtypes; the 5-year breast cancer–specific survival for stage I triple-negative tumors is 85%, compared to 94% and 99% for hormone receptor–positive tumors and ERBB2-positive tumors, respectively (20). Further, the approximate median overall survival is 1 year for metastatic TNBC compared to 5 years for the other two subtypes. TNBC cells are resistant to hormone or molecular therapies, and the mortality rate is increasing in Japan. Since newly introduced therapies, including PARP inhibitors and checkpoint inhibitor immunotherapy, are effective in a very limited patient population, new therapeutic modalities are needed, and oncolytic virotherapy is one of the promising candidates (3). We previously reported that CVB3 exerted a strong antitumor effect in various tumors because of the broad expression of its receptors and its rapid replication in tumor cells (17). However, CVB3-WT caused non-negligible toxicities, including an increase of pancreatic amylase. We decided to develop a safer form of CVB3 oncolytic virotherapy and therefore, recently constructed CVB3-HP. In this report, we showed the effectiveness of CVB3-HP against breast cancer in vitro and in vivo using nude mice subcutaneously transplanted with human TNBC cells. Our results strongly suggest that this new CVB3 virus will be clinically useful for treating breast cancer, including TNBC, with higher safety.

The CVB3 CAR receptor was first identified as a molecule involved in CVB3 attachment and infection, and was subsequently reported to be a receptor for adenovirus (21-24). In contrast, DAF was shown to be a supportive protein that helps CVB3 enter into the cytoplasm (25-27). More specifically, it was suggested that DAF potentiates viral binding to CAR, similar to its role in CVA21 (28), because a previous study showed that rhabdomyosarcoma cells exhibited high sensitivity to CVB3 infection even though they expressed high levels of DAF but low levels of CAR (25).

We also demonstrated that the cytopathic effects of CVB3 against lung cancer cells strongly correlated with the combined cellular expression levels of CAR and DAF (17). Our flow cytometry study demonstrated that all four breast cancer cell lines expressed high levels of CAR or DAF. In particular, three breast cancer cell lines that expressed high levels of CAR showed high sensitivity to CVB3 infection (Figure 1B).

We also found that apoptosis played a role in the cytopathic effects of CVB3 (Figure 2). Since many cancers exhibit resistance to chemotherapy-induced apoptosis (29), the ability of CVB3 to induce apoptosis in TNBC cells is considered one of the major advantages of using CVB3 as a new anticancer treatment (30).

Lastly, we demonstrated that CVB3-HP was safer than CVB3-WT in human breast cancer–bearing immunodeficient nude mice, even though the two viruses were equivalent in terms of antitumor efficacy (Figure 4A), prevention of body weight loss (Figure 4B) and frequency of adverse reactions (Figure 4D). In future research we plan to examine the effects of purified CVB3-HP on various laboratory tests, including those involving serum, liver, pancreas and muscle, as these are required before translating CVB3-HP to clinical use.

Acknowledgements

The Authors thank Michiko Ushijima for administrative assistance. They have received technical support from the Research Support Center, Graduate School of Medical Sciences, Kyushu University. This work was mainly supported by Grant-in-Aid for Scientific Research A from the Ministry of Education, Culture, Sports, Science and Technology (23240133), Japan.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Authors’ Contributions

    M.S. conducted the experiments, prepared figures, and participated in study design and writing manuscript. S.M. designed the study and provided expertise and advice. S.I supported the experiments. Y.S. provided advice. K.T. contributed to the designs of the study and writing the manuscript, and provided advice.

  • Conflicts of Interest

    K.T. reports receiving research grants from Neopharma Japan, Co., Ltd., Neoprecision therapeutics Co., Ltd. and Shinnihonseiyaku Co., Ltd.; S.M., Y.S. and K.T. has stock in Neoprecision therapeutics Co., Ltd. And K.T. has stock in Shinnihonseiyaku Cο., Ltd.; No potential conflicts of interest were disclosed by the other authors.

  • Received November 21, 2020.
  • Revision received December 6, 2020.
  • Accepted December 7, 2020.

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Development of New Oncolytic Virotherapy Targeting Breast Cancer Using Coxsackievirus B3
MIYAKO SAGARA, SHOHEI MIYAMOTO, SHUN ITOH, YASUSHI SODA, KENZABURO TANI
Anticancer Research Jan 2021, 41 (1) 81-89; DOI: 10.21873/anticanres.14753
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