Oncolytic activity of a coxsackievirus B3 strain in human endometrial cancer cell lines
Endometrial cancer (EC) is one of the most common gynecological malignancies globally. Although progress has been made in surgical and other adjuvant therapies, there is still a great need to develop new approaches to further reduce the incidence and mortality of EC. Oncolytic virotherapy offers a novel promising option of cancer treatment and has demonstrated good efficacy in preclinical models and clinical trials. However, only few oncolytic viruses have been tested for EC treatment. In this study, the potential of an oncolytic coxsackievirus B3 (CV-B3) strain 2035A (CV-B3/2035A) was investigated as a novel biotherapeutic agent against EC.
Human EC cell lines (Ishikawa, HEC-1-A and HEC-1-B) were infected with CV-B3/2035A, and viral replication and cytotoxic effects were evaluated in vitro. CV-B3/2035A-induced oncolysis was also investigated in nude mice bearing EC xenografts in vivo and in patient-derived EC samples ex vivo.
Human EC cell lines expressing different levels of CAR and DAF were all susceptible to infection by CV-B3/2035A and supported efficient viral replication in vitro. In the EC xenograft/nude mouse model, both intratumoral and intravenous administrations of CV-B3-2035A exerted significant therapeutic effects against pre-established EC tumors without causing significant treatment-related toxicity and mortality in nude mice. Moreover, CV-B3/2035A treatment resulted in decreased viability of patient-derived EC samples ex vivo.
CV-B3/2035A showed oncolytic activity in human EC cell lines both in vitro and in vivo as well as in patient-derived EC samples ex vivo and thus could be used as an alternative virotherapy agent for the treatment of EC.
KeywordsCoxsackievirus B3 CV-B3 Oncolytic virus Endometrial cancer Virotherapy
Coxsackievirus and adenovirus receptor
Decay accelerating factor
Histoculture drug response assay
Hand, foot and mouse disease
Non–small cell lung cancer
Endometrial cancer (EC) is one of the most common gynaecological cancers in the world . There are two histological types of EC. The most common type is endometrioid adenocarcinoma, or type I EC, and type II ECs are predominantly aggressive carcinomas . Today, the prevalence of EC is increasing due to the increasing number of elderly women and increasing rates of obesity and metabolic syndromes, and thus EC will continue to be a major problem for health-care systems worldwide [1, 3, 4]. Presently for women with early-stage EC, standard management with hysterectomy, bilateral salpingoophorectomy with or without pelvic/para-aortic lymph node dissection confers an excellent 5-year overall survival rate (> 90%) [5, 6, 7, 8]. In more advanced cases, surgery can be followed by adjuvant therapies including radiation therapy, chemotherapy or hormone therapy [9, 10, 11, 12, 13, 14]. However, current treatments result in low response rates and survival benefit for patients with high-graded, metastatic or recurrent EC [14, 15, 16], which calls for the necessity of new innovative therapeutic approaches.
Oncolytic virotherapy has gained increasing interest as a novel immunotherapeutic approach to treat cancers in recent years (reviewed in [17, 18]). Although not fully understood, oncolytic viruses are thought to exert anti-tumor effects by inducing selective tumor cell death, which not only directly eliminates viable tumor cells but also sets the stage for initiation of systemic anti-tumor immune responses. Targeting of these viruses to cancer cells is mainly based on the overexpression of viral entry receptors on tumor cells and aberrant signaling pathways within tumor cells. To date, a number of oncolytic viruses, including adenoviruses, herpesviruses, reoviruses, poxviruses and picornaviruses, have demonstrated appreciable efficacy in preclinical models and clinical trials (reviewed in [17, 18]). A modified herpes simplex virus type 1 (HSV-1), T-VEC from Amgen, is the first and so far only oncolytic virotherapy to be approved for cancer treatment by the US Food and Drug Administration (FDA) [19, 20]. However, only few oncolytic viruses have been tested for EC treatment.
Coxsackievirus B3 (CV-B3) is a small, nonenveloped enterovirus with a single-stranded, positive-stranded RNA genome in the Picornaviridae family. Previously, CV-B3 prototype Nancy strain (CV-B3/Nancy) has demonstrated oncolytic activity against various human tumor cell lines including colon carcinoma, pancreatic carcinoma, breast carcinoma, cervical carcinoma, human non–small cell lung cancer (NSCLC), etc.  These data indicate that CV-B3 may hold promise as a broad-spectrum oncolytic agent that can infect and kill a wide variety of human tumor cell types, possibly including human EC cells. In the present study, the oncolytic activity of a CV-B3 2035A strain (CV-B3/2035A) was investigated in different human EC cell lines both in vitro and in vivo. Additionally, CV-B3/2035A-induced oncolysis was assessed in patient-derived EC samples ex vivo. The results of this study indicate that CV-B3/2035A could be used as an effective oncolytic and therapeutic agent against human EC.
Cells and virus
Two human EC cell lines (HEC-1-A and Ishikawa) and a human rhabdomyosarcoma cell line (RD) were purchased from the American Type Culture Collection (Manassas, USA). A human EC cell line (HEC-1-B) was purchased from the China Infrastructure of Cell Line Resources (Beijing, China). All cells were maintained in 5% CO2 at 37 °C in their respective media as follows: HEC-1-A in McCoy’s 5a medium, HEC-1-B and Ishikawa in Minimum Essential Medium (MEM) and RD in Dulbecco’s modified Eagle’s medium (DMEM) (all from Invitrogen). The culture media used were all supplemented with 10% (v/v) fetal bovine serum (FBS), 50 U/ml penicillin, and 50 μg/ml streptomycin (Invitrogen).
CV-B3 2035A strain (GenBank Accession No. KY286529.1) was isolated from a throat swab of a patient with mild hand, foot and mouse disease (HFMD) from the Centers for Disease Control and Prevention (CDC) of Xiamen, China in 2008, and was propagated in RD cells. Virus titers were determined by TCID50 assay on RD cells according to the Reed-Muench method , and were used for all multiplicity of infection (MOI) determinations described.
Flow cytometry analysis
Dispersed EC cells (1 × 106) were incubated with either a mouse monoclonal antibody (mAb) against coxsackievirus and adenovirus receptor (CAR; clone RmCB; Millipore) followed by incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (Sigma) or a FITC-conjugated mouse mAb against decay-accelerating factor (DAF; BD Biosciences) for 30 min (or mock treated) at room temperature. Cells were then washed, pelleted, resuspended in phosphate buffered saline (PBS), and analyzed for receptor expression using a flow cytometer (BD FACSAria™ III, BD Bioscience), with data analyzed using FlowJo software (TreeStar, USA).
In vitro viral infectivity assay
EC cells were seeded in 96-well plates in the appropriate medium without FBS and then infected with ten-fold serial dilutions of CV-B3/2035A (100 μl/well in quadruplicate, from MOI = 10 to MOI = 10− 3). After incubation in 5% CO2 at 37 °C for 72 h, cytotoxicity was assessed using a Cell Counting Kit-8 (CCK-8) assay according to the manufacture’s instructions (Beyotime Institute of Biotechnology, China).
In vitro viral growth kinetics in infected tumor cells
EC cells were seeded in 24-well plates and then infected with CV-B3/2035A at an MOI of 0.1 in triplicate and allowed to adsorb for 1 h at 37 °C. Then cells were washed three times with media to remove unbound virus and fresh media were added. Cells were incubated in 5% CO2 at 37 °C and harvested at 2, 24 or 48 h post-infection (hpi). The harvested cells and supernatants were freeze-thawed twice and the lysates were centrifuged at 5000 g for 10 min at 4 °C. Virus yield in the obtained supernatants were titrated on RD cells as described above.
Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from tissues using a GenMagSpin Viral DNA/RNA Kit (GenMag Bio, China). The qRT-PCR was performed with the One-Step RT-PCR Kit (GenMag Bio, China) following the manufacturer’s instructions in the LightCycler® 96 system (Roche, Switzerland). Primer and probe sequences are shown in Additional file 1: Table S1. The thermal conditions were set to 50 °C for 10 min, 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 55 °C for 50 s. Relative cDNA level was calculated using a standard curve of Ct (cycle threshold) values generated from the dilution series of pMD18-CV-B3/2035A plasmid containing the target gene.
In vivo anti-tumor studies using subcutaneous xenografts in nude mice
All animal procedures were carried out under specific-pathogen-free (SPF) conditions and in accordance with the approved animal use protocols of Xiamen University Laboratory Animal Center. EC cells (5 × 106 cells) were injected subcutaneously into the right or bilateral flanks of 6–8 week old female nude mice (BALB/c-nu/nu mice; Beijing Vital River Laboratory Animal Technology Co., Ltd., China). Treatment started when tumors reached diameters of ~ 0.5 cm. For evaluating intratumoral use of CV-B3/2035A, the right flank tumors were injected with five consecutive doses (5 × 106 TCID50 per dose) of CV-B3/2035A at 2-day intervals, or with PBS as a negative control. For evaluating intravenous use of CV-B3, nude mice with bilateral EC xenografts were administered with a single dose of 1 × 106 TCID50 of CV-B3/2035A or PBS via lateral tail veins. Tumor growth was monitored and measured with calipers every other day for 20 days. The tumor volume was calculated as length × width × width/2 and expressed as means ± SD. For the biodistribution analysis of CV-B3/2035A, presence of virus in nude mice 2 and 6 days after a single intravenous injection was quantified by qRT-PCR from tissue samples, including tumor, brain, heart, liver, spleen, lung and kidney. For the preliminary toxicity evaluation of CV-B3/2035A, body weight of all groups of nude mice was monitored regularly, and vital tissues of nude mice given a single intravenous injection were histologically analyzed after H&E staining on day 20.
Histology and immunohistochemistry
Paraffin sections (5 μm thick) of tissues (including tumor, brain, heart, liver, spleen, lung and kidney) of nude mice were deparaffinized in xylene and rehydrated in graded alcohols. For histological analysis, sections of organs of nude mice were stained with hematoxylin and eosin (H&E). For analysis of CV-B3/2035A replication in tumor xenografts, immunohistochemistry (IHC) was performed for viral antigen expression as previously described . Briefly, sections were subjected to heat-induced antigen retrieval using citrate buffer, followed by endogenous peroxidase quenching using hydrogen peroxide. Then, sections were blocked with normal goat serum and incubated with a mouse mAb against CV-B3 (clone L8F12, unpublished). Next, IHC staining was performed using an Ultrasensitive TMS-P kit (Fuzhou Maixin Biotechnology Development Co., Ltd., China) and a DAB detection kit (streptavidin-biotin; Fuzhou Maixin Biotechnology Development Co., Ltd., China) according to the manufacturer’s instructions. Finally, sections were counterstained with hematoxylin, dehydrated and cover-slipped.
Histoculture drug response assay
Nucleotide and amino acid identities (%) between CV-B3/2035A and CV-B3/Nancy
5′ UTR nucleotide
P1 amino acid
P2 amino acid
P3 amino acid
3′ UTR nucleotide
The histoculture drug response assay (HDRA) was used for assessment of the oncolytic ability of CV-B3/2035A in patient-derived tumor samples ex vivo as described elsewhere , with slight modifications. Briefly, EC tissues were cut into pieces (~ 1 mm3) under sterile condition and placed on 0.5 cm2 filter papers, which were not completely submerged in DMEM containing 15% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, in equal quantities in each well of 24-well plates. After overnight culture at 37 °C, EC tissues were exposed to 106 TCID50 of CV-B3/2035A per well or left untreated. Following incubation for 72 h, 100 μl of 0.06% collagenase (type IV; Sigma) in DMEM was added to each well and the plates were incubated at 37 °C for another 4 h. Next, tissues and supernatants from each well were harvested and centrifuged at 5000 g for 10 min at 4 °C. The resultant pellets from each well were resuspended in 100 μl media and cell viability was assessed by CCK-8 assay in 96-well plates. The inhibition rate was calculated as: Inhibition rate (%) = (1 – A/B) × 100, where A is the mean absorbance of the treated wells per 1 g of tumor and B is the mean absorbance of the control wells per 1 g of tumor.
All statistical analysis were done using GraphPad Prism 7 (GraphPad Software Inc., USA). Comparisons of viral titers were performed using unpaired Student t-test. Comparisons of tumor growth curves and body weight curves between the CV-B3-treatment groups and control groups were performed using two-way repeated measures ANOVA.
Oncolytic activity of CV-B3/2035A in human EC cell lines in vitro
Growth kinetics of CV-B3/2035A in human EC cells in vitro
Since replication of oncolytic virus in cancer cells typically results in a substantial amplification of the input viral load, which may facilitate oncolysis of cancer cells nearby or at distant sites of metastasis, the production of CV-B3/2035A progeny virus in the EC cell lines was further assessed by evaluating viral growth kinetics. EC cell monolayers were challenged with CV-B3/2035A at an MOI of 0.1, and viral replication was monitored for 48 h. As shown in Fig. 3c, similar viral growth kinetics were obtained in HEC-1-B and Ishikawa cells, with the exception of HEC-1-A cells, where viral growth kinetics was slower. All EC cell lines produced high virus yields (106–108 TCID50/well) at 48 h post-infection (hpi). Due to a slower growth kinetics, HEC-1-A cells had approximately 5-fold-less virus yields than HEC-1-B and Ishikawa cells. These data indicated that CV-B3/2035A initiated a robust productive infection in all EC cell lines in vitro.
Oncolytic activity of CV-B3/2035A in human EC xenografts in nude mice in vivo
Oncolytic activity of CV-B3/2035A in patient-derived EC samples ex vivo
Patient-derived endometrial cancer samples used in this study
Stage IA, grade 2
Stage II, grade 2
Stage IIIA, grade 1
Stage IIIC, grade 1
Stage IIIC, grade 1
Malignancies resistant to current treatments require novel therapeutic approaches. A promising option is oncolytic virotherapy with picornaviruses, which have shown encouraging results in recent clinical trials, including coxsackievirus A21 (CV-A21; CAVATAK), poliovirus (PVS-RIPO) and Seneca Valley virus (NTX-010) (reviewed in ). Recently, CV-B3 (prototype Nancy strain) has been found to have natural oncolytic activity against a variety of human tumor cell types, especially in NSCLC cell lines . These observations indicated that CV-B3 strains may have broad-spectrum antitumor effects and thus warrant further investigation.
EC is one of the most common gynecological malignancies and its incidence is rising among pre- and postmenopausal women globally. Although the outcome of standard treatment of early-stage EC by surgery is generally favorable, available treatment options for advanced, recurrent and metastatic ECs remain very limited . Oncolytic virotherapy as an emerging immunotherapeutic modality for cancer therapy may improve outcomes in EC, but there have been only few reports about oncolytic viruses for EC treatment in recent years [34, 35]. In this context, the aim of the present study was to investigate the potential of a CV-B3 strain 2035A as a novel viral therapeutic agent against EC.
CV-B3/2035A was isolated in China and possessed oncolytic ability in a wide range of human tumor cells (Additional file 1: Table S2). In vitro assays demonstrated that all examined EC cell lines expressed CV-B3 receptors (DAF and CAR) (Fig. 2) and were markedly susceptible to CV-B3/2035A-induced cytotoxicity (Fig. 3). Previous studies have shown that CAR and DAF are expressed on EC cells of different histological types, grades and stages [32, 33]. CAR expression was reported to be increased with increasing grade of tumor in EC patients , while DAF expression is upregulated mainly on well-differentiated early-stage EC cells . These findings suggest that EC may be a good target for CV-B3/2035A.
EC relapses both locally and at distant sites. Therefore, in nude mice models bearing subcutaneous EC xenografts, the in vivo therapeutic efficacy for both intratumoral and systemic administrations of CV-B3/2035A was evaluated. Firstly, the results demonstrated that the ability of CV-B3/2035A to inhibit tumor growth varied in different EC cell lines (Fig. 4). The growth inhibition by CV-B3/2035A on HEC-1-B and Ishikawa xenografts was marked and sustained until day 42 (the actual time of study termination; data not shown). In contrast, HEC-1-A xenografts were much less sensitive to growth inhibition by CV-B3/2035A, which may be explained by differences in the level of surface CAR expression (Fig. 2) or by other factors that help to define the viral tropism. Additionally, viremia was observed following intratumoral administration of CV-B3/2035A into the EC xenografts, which was probably the cause of viral accumulation and replication in the distant untreated tumors (Fig. 5b). Furthermore, IHC analysis demonstrated that CV-B3/2035A spread throughout the contralateral tumors by disrupting the tumor architecture, which could lead to their growth inhibition (Fig. 5c). As expected, a single intravenous injection of CV-B3/2035A that mimicked the viremia exerted significant therapeutic effects on the EC xenografts, thus supporting its systemic use (Fig. 6a). Besides the EC xenograft/nude mice model, CV-B3/2035A also exhibited therapeutic efficacy in patient-derived EC samples ex vivo (Fig. 7). Altogether, these data indicated that CV-B3/2035A could be effective for both local and systemic treatment of EC.
A preliminary toxicity assessment was conducted in nude mice bearing EC xenografts following a single intravenous injection of CV-B3/2035A. Neither death nor significant body weight loss was observed in the tumor-bearing mice throughout the study. Biodistribution analysis showed that CV-B3/2035A had high tumor selectivity in the EC xenograft/nude mice model (Fig. 6b). Except for the EC xenografts, viral RNA was mainly detected in the heart of nude mice. However, no cardiac pathology was found in any examined mouse (n = 5) by histological analysis on day 20 (Additional file 1: Figure S1). To date, a number of CV-B3 strains, including CV-B3/Nancy, CV-B3/MKP, CV-B3/20 and CV-B3/28, are known to induce viral myocarditis in nude mice [36, 37, 38]. On the other hand, some CV-B3 strains, including CV-B3/0, CV-B3/GA and CV-B3/CO, are non-cardiovirulent [38, 39]. These data indicated that CV-B3/2035A, isolated from a case of mild HFMD, may be a benign non-cardiovirulent strain; nonetheless, further studies are warranted to test this hypothesis.
Even if CV-B3/2035A does not cause systemic disease in mice, its direct use may still cause serious diseases, such as myocarditis, aseptic meningitis and encephalitis, in humans. Therefore, there is a need for genetic modification of CV-B3/2035A to reduce its cytotoxicity to normal tissues while retaining oncolytic properties. Recently, microRNAs (miRNA) have been exploited to regulate tropisms of replication-competent oncolytic viruses by insertion of miRNA target (miRT) sequences into viral genomes (reviewed in ). Such miRNA-based genetic modulation has proven an effective method to attenuate the cytotoxic effect of CV-B3 without affecting its antitumor activity in NSCLC cells and thus could overcome a major hurdle to clinical development of this virotherapeutics candidate [41, 42]. Therefore, by combining with miRNA regulation, CV-B3/2035A could be further improved in safety. Furthermore, since CV-B3 has been extensively studied as a recombinant viral vector [43, 44, 45], CV-B3/2035A could be further improved in oncolytic potency by inserting anti-tumor genes into the viral genome. In addition, future research may be needed to explore the synergies between virotherapy with CV-B3/2035A and small molecular anticancer compounds or immune checkpoint inhibitors in EC treatment.
In summary, CV-B3 2035A strain exhibited effective oncolytic activities in human EC cell lines both in vitro and in vivo as well as in patient-derived EC samples ex vivo. Future studies will be necessary to determine the mechanisms underlying the oncolytic effects of CV-B3/2035A in EC cells and to improve the safety and potency of CV-B3/2035A as a potential virotherapy agent for EC treatment.
This work was supported by the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003-005-003), National Natural Science Foundation of China (No. 31670933 and No. 81773291).
Availability of data and materials
YZL, WW, and YFW designed the experiments. YZL, JKW, YY, WKF, DQP and LLC carried out the experiments. YZL, WW, TC and XMH analyzed the data. YZL, WW, TC and YFW wrote and edited the paper. All authors read and approved the final manuscript.
Human EC samples and normal endometrial tissues were obtained from patient biopsy collected during surgery at Zhongshan Hospital of Xiamen University. Informed consent for this study was obtained from each patient. This study was approved by the Institutional Review Board of the hospital and the Research Ethics Committee of Xiamen University. Experiments were carried out in strict accordance with the guidelines and regulations of Xiamen University.
The mice were treated humanely and all animal procedures were carried out in accordance with the approved animal use protocols of Xiamen University Laboratory Animal Center.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 6.Janda M, Gebski V, Brand A, Hogg R, Jobling TW, Land R, Manolitsas T, McCartney A, Nascimento M, Neesham D, et al. Quality of life after total laparoscopic hysterectomy versus total abdominal hysterectomy for stage I endometrial cancer (LACE): a randomised trial. Lancet Oncol. 2010;11:772–80.CrossRefPubMedGoogle Scholar
- 10.Homesley HD, Filiaci V, Gibbons SK, Long HJ, Cella D, Spirtos NM, Morris RT, DeGeest K, Lee R, Montag A. A randomized phase III trial in advanced endometrial carcinoma of surgery and volume directed radiation followed by cisplatin and doxorubicin with or without paclitaxel: a gynecologic oncology group study. Gynecol Oncol. 2009;112:543–52.CrossRefPubMedGoogle Scholar
- 11.Randall ME, Filiaci VL, Muss H, Spirtos NM, Mannel RS, Fowler J, Thigpen JT, Benda JA. Gynecologic Oncology Group S: randomized phase III trial of whole-abdominal irradiation versus doxorubicin and cisplatin chemotherapy in advanced endometrial carcinoma: a Gynecologic Oncology Group Study. J Clin Oncol. 2006;24:36–44.CrossRefPubMedGoogle Scholar
- 12.Kokka F, Brockbank E, Oram D, Gallagher C, Bryant A. Hormonal therapy in advanced or recurrent endometrial cancer. Cochrane Database Syst Rev. 2010:CD007926.Google Scholar
- 22.Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Hyg. 1938;27:493–7.Google Scholar
- 29.Pan J, Zhang L, Organtini LJ, Hafenstein S, Bergelson JM. Specificity of coxsackievirus B3 interaction with human, but not murine, decay-accelerating factor: replacement of a single residue within short consensus repeat 2 prevents virus attachment. J Virol. 2015;89:1324–8.CrossRefPubMedGoogle Scholar
- 38.Tracy S, Drescher KM, Chapman NM, Kim KS, Carson SD, Pirruccello S, Lane PH, Romero JR, Leser JS. Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence. J Virol. 2002;76:12097–111.CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Miyamoto S, Inoue H, Sagara M, Wang BB, Takayama K, Shimizu H, Nakanishi Y, Tani K. Dual microRNA-regulated oncolytic Coxsackievirus B3 infection displays antitumor activity with attenuated pathogenicity in mice. Mol Ther. 2013;21:S28–9.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.