Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer
- Stephanie Weibel†1,
- Elisabeth Hofmann†1,
- Thomas Christian Basse-Luesebrink1, 2, 3,
- Ulrike Donat1,
- Carolin Seubert1,
- Marion Adelfinger1,
- Prisca Gnamlin1,
- Christina Kober1,
- Alexa Frentzen7,
- Ivaylo Gentschev1, 7,
- Peter Michael Jakob2, 3 and
- Aladar A Szalay1, 4, 5, 6, 7Email author
© Weibel et al.; licensee BioMed Central Ltd. 2013
Received: 17 August 2012
Accepted: 23 April 2013
Published: 1 May 2013
Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options.
In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma.
We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment.
Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.
KeywordsOncolytic virotherapy Malignant effusion Lung cancer VEGF
More than 226,000 new cases of lung cancer are projected to occur in 2012 in the United States, and its high mortality rate makes it the leading cause of cancer-related death . Malignant pleural effusion (MPE) is a common complication of patients with highly symptomatic and advanced-stages of lung cancer. In the clinic, these patients are difficult to manage and have, in general, a short life expectancy of 4–9 month after diagnosis . In addition to lung cancer, MPE is a serious complication associated with different other tumor types including breast cancer and lymphomas affecting altogether 175,000 patients in the United States each year . Today, only palliative therapies such as thoracentesis and chemical pleurodesis are available in the clinic . Therefore, novel approaches for the therapy of MPE are needed.
Despite being a frequent and serious complication in cancer patients, the underlying mechanism of MPE formation is not fully understood. In all cases, fluid accumulates in the pleura as a result of increased pleural fluid formation and/or reduced drainage. However, increased local fluid formation is the principal underlying abnormality in the genesis of most exudative effusions, even if obstruction of fluid drainage co-exists . MPE formation is considered to develop on the basis of a locally disturbed tumor-microenvironment, with leakiness of local endothelial cell layers and pleural invasion by tumor cells [6, 7]. In various studies, vascular endothelial growth factor (VEGF) has been identified as a key mediator contributing to the formation of malignant effusions (ME) in solid tumors by modulation of the tumor vasculature [6, 8]. Strategies to antagonize the VEGF activity at various target points of the corresponding signaling pathway have shown success in vitro and in animal models of ME and ascites [5, 9–13].
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy based on the lytic destruction of solid tumors mediated by infection of the malignant tissue by tumor-specific viruses [14–17]. A multitude of different virus strains with oncolytic potential have been described and promising pre-clinical data as well as clinical trial reports from oncolytic virotherapy are available [18–20]. Recently, Zhang et al. [21, 22] have introduced the attenuated recombinant Vaccinia virus (rVACV) GLV-1h68 which was used as an oncolytic agent in several pre-clinical tumor models [23–27] and is currently applied in clinical trials (http://www.clinicaltrials.gov; references NCT00794131 and NCT01443260).
In the present study, we showed that the oncolytic VACV GLV-1h68 and its derivative GLV-1h108 , which encodes for a single chain antibody (scAb) against VEGF revealed significant tumor growth control and prevented formation of ME in a subcutaneous advanced-stage lung adenocarcinoma model. In addition, we showed for the first time that oncolytic virotherapy led to a reduction of tumor cell-derived VEGF-levels, to decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of blood vessel-invading tumor cells, thereby preventing formation of ME. These data support the use of oncolytic virotherapy against both the primary tumor as well as tumor-associated effusions.
PC14PE6-RFP human lung adenocarcinoma cells were stably transduced with the full-length dsRed2 cDNA as described by Kienast el al.  and kindly provided to us by F. Winkler (University of Heidelberg, Neurooncology, Heidelberg, Germany) in 2008. The PC14PE6-RFP cells used in this study were authenticated by the Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) by STR profiling to be identical with the parental cell line PC14 (Riken, Japan) in 2012. PC14PE6-RFP cells were cultured in DMEM supplemented with 1x MEM non-essential amino acids, 2 mM GlutaMAX, 10% FBS, 100 Units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C and 5% CO2.
Construction of the attenuated Vaccinia virus strains GLV-1h68 and GLV-1h108 was described previously by Zhang et al.  and Frentzen et al. , respectively. Briefly, three expression cassettes (encoding for Renilla luciferase-GFP fusion protein, β-galactosidase and β-glucuronidase) were recombined into the F14.5L, J2R and A56R loci, respectively, of the LIVP strain virus genome. In case of GLV-1h108 the glaf-1 coding sequence was recombined into the J2R locus of the parental GLV-1h68 virus strain. Viruses were propagated in CV-1 cells and purified through sucrose gradients.
Tumor inoculation and administration of the virus
All animal experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Explora Biolabs (San Diego, USA, protocol number EB11-025) or the government of Unterfranken (Würzburg, Germany, protocol number AZ 55.2-2531.01-17/08).
Six-week-old female athymic nude Foxn1 nu mice were obtained from Harlan Laboratories (Netherlands and Indianapolis). PC14PE6-RFP tumor cells (4 × 105/100 μl PBS) were subcutaneously (sc) injected into the abdominal right flank. Tumor volume was calculated as length × width2 × 0.52. For all experiments, tumors were grown up to 100–200 mm3 in size (13–14 days) before viral administration. A single viral dose of 1 × 107 plaque forming units (pfu) in 100 μl PBS was injected intravenously (iv) via the tail vein.
Fluorescence live-animal imaging
Tumor cell growth and viral infection were monitored directly by the RFP expression of tumor cells and GFP expression of Vaccinia virus-infected cells and quantified with the Maestro EX imaging system (CRI, Woburn, MA) using appropriate filters for RFP (tumor; excitation: 503–555 nm, emission: 580 nm cut-in) and GFP (virus; excitation: 445–490 nm, emission: 515 nm cut-in). Images were evaluated and quantified using the Maestro Version 2.10.0 software.
For flow cytometric analysis of tumors and exudates, four end-stage PC14PE6 tumor-bearing mice (28 dpim) were sacrificed by CO2 inhalation. Effusions were punctured and 400 μl of the exudates were collected. Preparation of tumors were performed as previously described . In both effusion and tumor preparations lysis of erythrocytes using an isotonic ammonium cloride lysis buffer was performed and DNA was digested using 5 MU/ml DNase I.
Blocking of unspecific binding-sites and antibody-labeling using anti-mouse CD45-PECy7 (eBioscience, San Diego, CA, USA) was performed as decribed elsewhere . Immediately before use, dead cells were labeled with propidium iodide (PI) solution.
Labeled cells were subsequently analyzed, using the Accuri C6 Cytometer and FACS analysis software CFlow Version 126.96.36.199 (Accuri Cytometers, Inc. Ann Arbor, MI USA).
In vivo MRI
In vivo MRI measurements of tumor-bearing mice were performed at room temperature on a 7 Tesla Bruker Biospec System (Bruker BioSpin GmbH, Reinstetten, Germany) using a 35 mm diameter home-built quadrature birdcage coil. For in vivo imaging the animals received inhalation anesthesia (1-2% isoflurane) during the measurement and were placed in a home-built measurement container according to safety regulations.
T1 weighted (T1w) spin echo experiments were performed at different time points following an injection (iv) of 0.1 mmol/kg body weight Gadopentetate-Dimeglumine (Gd-DTPA, Magnevist, Bayer Schering Pharma AG, Berlin, Germany) of exemplary mock infected animals. Furthermore, for the shown data, a T2 weighted (T2w) spin echo experiment was performed for anatomical correlation approximately half an hour after injection of the contrast agent.
For n = 4 additional animals (n = 2 mock/ GLV-1h68) multi spin echo (MSE) experiments were performed for the evaluation of tissue T2 times (without contrast agent). Data processing of the MRI data was performed in MATLAB (The MathWorks Inc., Natick, USA) using home-written software routines. The chosen MRI sequence parameters are provided in the Additional file 1.
Protein isolation and hVEGF/mVEGF ELISA of tumor samples
Quantitative evaluation of VEGF concentrations in tumor tissues was performed using a human (Thermo Scientific, EHVEGF) and mouse specific VEGF ELISA kit (abcam, ab100751) according to the manufacturer’s protocol. Tumors were isolated 7 dpi, lysates were prepared as described previously . Absorbance was measured using a Tecan sunrise absorbance reader (Tecan, Crailsheim, Germany). Protein concentrations for each sample were interpolated from a VEGF-specific standard curve.
RT-PCR of ß-actin
Tissue samples were analyzed for the presence of PC14PE6-RFP cells by RT-PCR. Brains, lungs, and livers of PC14PE6-bearing mice (28 dpim) were homogenized in TRIzol Reagent (Invitrogen) to isolate total RNA. Samples were further treated as described elsewhere . Primer sequence for human ß-actin: (forward: 5′-CCT CTC CCA AGT CCA CAC AG-3′ and reverse: 5′- CTG CCT CCA CCC ACT C-3′) and for murine ß-actin: (forward: 5′-CGT CCA TGC CCT GAG TC- 3′ and reverse: 5′-GCT GCC TCA ACA CCT CAA C-3′). PC14PE6-RFP cell lysates were used as positive control for human ß-actin. The PCR reaction was performed in a T-Gradient Thermoblock PCR machine (Biometra, Göttingen, Germany).
For histological studies, tumors were excised and snap-frozen in liquid N2, followed by fixation in 4% paraformaldehyde/PBS pH 7.4 for 16 h at 4°C. Fixed tumors were rinsed in PBS followed by dehydration in 10% and 30% sucrose/PBS (Carl Roth, Karlsruhe, Germany) and finally embedded in Tissue-Tek® O.C.T. (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands). Tumor samples were sectioned (15 μm) with a cryostat 2800 Frigocut (Leica Microsystems GmbH, Wetzlar, Germany) and stored at −80°C. Antibody-labeling was performed following fixation in ice-cold aceton. The primary antibody was incubated for 1 h. After washing with PBS, sections were labeled for 30 min with the secondary antibody and finally mounted in Mowiol 4–88.
Whole animal sectioning and H&E staining of tissue sections
Mice were euthanized, perfused with 4% paraformaldehyde, and whole mice were fixed in 4% formalin for 2 days followed by decalcification in 10% formic acid/4% formalin for up to 5 days. Decalcified specimens were dehydrated and embedded in paraffin. Tissue sections (5 μm) were stained by H&E staining.
The fluorescence-labeled preparations were examined using a MZ16 FA Stereo-Fluorescence microscope (Leica) equipped with a digital DC500 CCD camera and the Leica IM1000 4.0 software (1300 × 1030 pixel RGB-color images). Furthermore, a Leica TCS SP2 AOBS confocal laser microscope equipped with argon, helium-neon and UV lasers and the LCS 2.16 software (1024 × 1024 pixel RGB-color images) was used. Digital images were processed with Photoshop 7.0 (Adobe Systems, Mountain View, CA) and merged to generate overlay images.
Fluorescence intensity measurements and microvessel density
Fluorescence intensity of the CD31-, Ly-6G-, MHCII-labeling as well as the vascular density was measured in 15-μm-thick cryostat sections of control tumors and infected areas of GLV-1h68-colonized tumors on digital images as described earlier . For all experiments the mean value was calculated for nine images (three images each of three different control and GLV-1h68-infected tumors) and presented with standard deviation.
Antibodies, reagents and treatment of animals
Endothelial cells of blood vessels were labeled with hamster anti-mouse CD31 antibody (Chemicon, International, Temecula, CA). Platelets were labeled with rat anti-mouse CD41 antibody (GeneTex Inc., Irvine, CA). Immune cells were labeled using rat anti-mouse MHCII antibody (B, dendritic cells, monocytes, macrophages) and rat-anti Ly-6G antibody (neutrophils) (eBioscience, San Diego, CA). Vaccinia virus was labeled with rabbit anti-Vaccinia virus (abcam, Cambridge, UK).
The DyLight649-conjugated secondary antibodies (donkey) were obtained from Jackson ImmunoResearch (West Grove, PA).
For the labeling of functional blood vessels in tumors, mice were intravenously injected with 100 μg of biotinylated-Lycopersicum esculentum Lectin (Vector Laboratories, Burlingame, CA). Three minutes later, tumors were removed and prepared for histology. Tumor cross-sections (15 μm) were labeled with DyLight649-conjugated streptavidin (Sigma Aldrich, Taufkirchen, Germany) to visualize the Lectin-labeled functional tumor vasculature.
A two-tailed Student’s t test was used for statistical analysis. P values of < 0.05 were considered statistically significant (*p < 0.05, ** p < 0.01, *** p < 0.001).
Subcutaneously implanted PC14PE6-RFP lung adenocarcinomas developed malignant effusions in athymic nude mice
Exemplarily, the injection of the contrast-agent Gd-DTPA into a tumor-bearing mouse with ME followed by a time course analysis of the contrast agent distribution using T1w MRI revealed a fast and strong accumulation of the contrast agent in well-vascularized tumor areas (blue, Figure 1C, D) as well as in the effusion (green, Figure 1C, D). During the time course the signal to noise ratio (SNR) within the vascularized tumor areas (blue) started to decline 5 min after injection while it further accumulated in the effusion (blue, light blue) reaching a plateau after approximately 15 min.
The Fluorescence-microscopic analysis of the effusion content revealed a large number of erythrocytes, red-fluorescent PC14PE6-RFP tumor cells, as well as non-fluorescent cells, confirming that these effusions are indeed of the malignant type (Figure 1E). Furthermore, FACS analysis of the effusion content showed that most of the cells within the ME were already dead red-fluorescent PC14PE6-RFP tumor cells (57.46%), however, 6.32% of the tumor cells were still alive (Figure 1F). When compared to tumors with only 28.96% dead and 34.32% live tumor cells, the environment of the effusions seems to be cytotoxic. Interestingly, CD45-positive immune cells, which were the other nucleated cell component of effusions, were enriched in ME (20.26%) when compared to tumors (11.63%) indicating an inflammatory environment of ME.
Taken together, the results showed that PC14PE6 tumor-associated ME can be established in the flank area of mice indicating that the local microenvironment of the lung is not essential for ME formation.
PC14PE6-RFP tumors exhibited enlarged CD31-positive blood vessels containing clusters of tumor cells
Since PC14PE6-RFP tumors were described to be highly metastatic and easily form brain metastases , the intra-vascular PC14PE6-RFP cells may be actively migrating metastatic tumor cells. Therefore, we analyzed different organs of late-stage PC14PE6-RFP tumor-bearing mice (28 dpim) for local metastases using human-specific ß-actin primers as target for RT-PCR analysis. Interestingly, neither brains nor lungs or livers of the analyzed mice were positively tested for human PC14PE6-RFP tumor cells (Figure 2G). Thus, we assume that TCCBVs are statically associated with the tumor vasculature of PC14PE6-RFP lung adenocarcinomas rather than are being snapshots of metastatic seeding.
rVACV colonization of PC14PE6-RFP tumors retained both tumor growth and ME formation
To determine the anatomical context of ME, we scanned untreated and virus-treated animals first by in vivo MRI (Figure 3C) followed by whole mouse sectioning (Figure 3D-F). In untreated PC14PE6-RFP tumor-bearing mice ME was visualized already 7 dpi by MRI (Figure 3C). These animals showed highly necrotic tumors surrounded by enlarged effusions. In contrast, rVACV-treated tumors showed highly attenuated ME. Histological analysis of PC14PE6-RFP tumor-associated ME revealed erythrocyte-containing blood lakes with highest frequency at the tumor rim in untreated tumors (Figure 3E, F). Moreover, these tumors did not show defined tumor borders and blood leaks into the surrounding tissue/cavity. In GLV-1h68-treated tumors defined tumor border regions and extended necrotic tumor areas were observed.
In summary, oncolytic therapy using rVACV proved to be an effective treatment modality for both growth control of lung adenocarcinomas and therapy of ME.
rVACV colonization of PC14PE6-RFP tumors activates the tumor endothelium
Altogether, rVACV treatment of PC14PE6-RFP tumors led to inflammation-mediated activation of the endothelium and did not lead to a direct destruction of the tumor vasculature.
rVACV colonization of PC14PE6-RFP tumors led to a decrease in the intratumoral human VEGF (hVEGF) content and to infected TCCBVs
Investigation of rVACV-treated tumors (7dpi) revealed that tumor cells located in CD31-positive TCCBVs did express GFP, a direct visual marker for viral infection (Figure 5C). Interestingly, different morphologies of TCCBVs such as glomeruloid bodies and vessels with intussusceptive vascular growth were infected by rVACV (Figure 5D-F). Moreover, specific anti-VACV antibody labeling of tumor sections confirmed viral infection of TCCBVs by positively labeling of GFP-positive tumor areas (Figure 5G). In addition to the VEGF over-expression the invasive potential of the tumor cells seems to be critical for ME formation . The investigation of the tumor border region in rVACV-treated tumors revealed GLV-1h68-infected PC14PE6-RFP tumor cells, thereby directly preventing tumor cell invasion of the peritumoral tissue (Figure 5H).
In summary, viral colonization of PC14PE6-RFP tumors decreased tumor cell-derived hVEGF levels, directly infected TCCBV’s, and inhibited tumor cell invasion of the peritumoral tissue, thereby rVACV potentially prevented the formation of ME.
rVACV encoding for GLAF-1 significantly enhanced both therapy of lung adenocarcinomas and ME
Taken together, these data indicate that both tumor growths of PC14PE6-RFP lung adenocarcinomas as well as formation of ME was efficiently controlled by treatment with oncolytic rVACV GLV-1h108 encoding for the scAb against VEGF in mice.
Late-stage cancer is often accompanied by the formation of ME, which are often resistant to conventional anti-neoplastic therapy and thus represent a major challenge in oncology. In this study, we showed that virotherapy using Vaccinia virus as an oncolytic agent and as a drug-delivering vehicle may become a promising therapeutic strategy for the treatment of lung adenocarcinoma-associated ME. So far, using a Measles virus strain as oncolytic agent, only Iankov et al. [32, 33] reported therapeutic effectiveness of oncolytic virotherapy in a malignant pleural effusion breast cancer model. In addition, we showed for the first time that oncolytic virotherapy decreases tumor-derived hVEGF levels, leads to viral infection of TCCBVs, and inhibits tumor cell invasion of the peritumoral tissue, thereby preventing formation of ME. Interestingly, VACV treatment did not lead to destruction of the tumor vasculature. Therefore, therapy of ME was not dependent on virus-mediated vessel destruction. Finally, the use of the recombinant Vaccinia virus strain GLV-1h108 encoding for a scAb against VEGF significantly enhanced the oncolytic mono-therapy.
In the present study, we found that the formation of ME in the PC14PE6 lung adenocarcinoma model was independent of the local pleural microenvironment. After generation of subcutaneous lung xenografts on the flank of mice we observed tumor-associated ME, elevated intratumoral VEGF levels, enlarged blood vessels and tumor cells invading the peritumoral space in 100% of the mice. These findings are in accordance with previous findings for the orthotopic model . Despite of the “artificial” character of the subcutaneous tumor ME model several aspects contribute to the direct relevance of this model to MPE pathogenesis in cancer patients. First of all, in the subcutaneous tumor-ME model as well as in patients ME are the results of fluid accumulation in body cavities due to increased fluid formation and/or reduced drainage. Second, in both situations VEGF and the invasive potential of tumor cells are main contributors to increased fluid accumulation and leakage in nearby body cavities. Finally, the main differences of MPE in patients and ME in the subcutaneous tumor-ME model are the location of the body cavities where the exudates accumulate – the pleura versus the “cavity” between the subcutis and the peritoneum.
In contrast to orthotopic MPE, subcutaneous ME at the flank are non-invasively detectable due to hematoma formation in the skin and/or exudates accumulation in the groin. Thus, for monitoring of ME there is no need to sacrifice the mice or need for special instrumentation such as MRI scanners. Therefore, we propose that this subcutaneous model of ME may be an easier-to-handle model with comparable characteristics of the published orthotopic model and is especially suited for the screening of therapeutic interventions for tumor-associated ME.
VEGF is a well established mediator of vascular permeability and can be found in significant higher concentrations in effusions of malignant compared to non-malignant origin [34–36]. Several groups have already shown that the anti-VEGF-based therapy using Bevacizumab profoundly counteracts effusion formation in patients with various solid tumors [37, 38]. Therefore, VEGF seems to be one of the important factors contributing to local hyperpermeability leading to tumor-associated ME. Several cell types such as leukocytes, mesothelial cells, and cancer cells may serve as a potential source for vasoactive VEGF. By the use of human tumor cells in a murine host context, we were able to differentiate between the hVEGF produced by tumor cells and the mVEGF produced by host cells such as leukocytes. As oncolytic virotherapy specifically reduces hVEGF and not mVEGF accompanied by resolution of ME, the results indicate that the most important sources for VEGF were the tumor cells itself. Furthermore, the results demonstrated that mVEGF-producing cells were not efficiently infected and lysed by oncolytic virotherapy using rVACV. The important role of tumor cell-derived VEGF in this model of ME was further demonstrated by the use of a rVACV strain encoding for therapeutic antibodies against VEGF (GLAF-1) in the direct local proximity to VEGF-producing cancer cells, which significantly enhances the therapeutic effect on ME formation.
Moreover, the invasive potential of PC14PE6 tumor cells seems to be critical for the formation of ME . This is due to the disruption of the tumor capsule, which facilitates the leakage of interstitial fluid into the peritumoral tissue or body cavities. Interestingly, oncolytic virotherapy seems to alter the invasive potential of PC14PE6 tumor cells hence VACV-treated tumors revealed defined tumor capsules.
Yano and colleagues established the orthotopic PC14PE6 malignant effusion lung adenocarcinoma model and reported in elegant publications the presence of an abnormal tumor vasculature in PC14PE6 tumors [6, 31]. Nowadays, it is widely accepted that the abnormalities of the tumor vasculature contribute significantly to the disturbed microenvironment of solid tumors. This may finally result in a highly disturbed blood flow which does not follow a constant, unidirectional path in tumors resulting in local hyperpermeability or zones of ischaemia . Furthermore, Yano et al. reported that a large proportion of blood vessels exhibited large lumina with transluminal bridges of endothelial processes , which are hallmarks of non-sprouting angiogenesis . In accordance, we observed the existence of enlarged blood vessels with transluminal bridges in PC14PE6 tumors. Moreover, by the use of RFP-expressing PC14PE6 tumor cells, mouse-specific anti-CD31 antibodies, and Vaccinia virus infection, we were able to identify tumor cells within these enlarged vessels (TCCBVs) forming transluminal bridges. Our data furthermore indicated that the invasive tumor cells did not contribute to distant metastases formation, however, we do not know whether TCCBVs were the result of the abnormal high intratumoral VEGF level or other microenvironmental abnormalities. However, a study from Dvorak et al.  indicated that VEGF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels. Furthermore, it was reported that the chemotactic action of VEGF enables tumor cell-trans-endothelial migration  and tumor cells may more easily penetrate a retracted endothelial monolayer caused by VEGF than a tightly arranged monolayer. This suggests that the vascular permeability activity of VEGF contributes to an “offensive ability” of the tumor cells, allowing them to penetrate blood vessels . Taken together, we speculate that TCCBVs may be the result of an abnormal high VEGF accumulation in blood vessels of tumors followed by migration and invasion of the endothelium by tumor cells.
Besides the strong effect of the rVACV-mediated inhibition of VEGF on ME formation other mechanisms such as the VACV-induced inflammatory host response may contribute to the resolution of ME. Reports of using bacterial cell-derived immunomodulatory preperations such as OK-432 as successful therapeutics in patients with MPE indicate the importance of the host immune response to therapy outcome [44–46]. In addition, recently published data showed that an immunostimulatory transgene encoded by oncolytic measles virus significantly enhanced the anti-tumor effect in xenograft models of metastatic cancer, including malignant pleural effusion model. To elucidate whether the VACV-mediated host immune response contributes to the resolution of ME is the focus of ongoing research.
In conclusion, this study showed that oncolytic virotherapy of lung adenocarcinomas resolved and prevented formation of ME mainly via decreasing tumoral VEGF production, infection of TCCBVs and inhibition of invasion of the peritumoral space. The significantly enhanced therapeutic efficacy using rVACV encoding for scAb against VEGF further highlights the promising application of recombinant oncolytic Vaccinia viruses encoding for specific immuno-therapeutic antibodies enabling targeted therapy.
Days post infection
Days post implantation
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Fetal bovine serum
Green fluorescent protein
Malignant pleural effusion
Magnetic resonance imaging
Multi spin echo
Plaque forming unit
Red fluorescent protein
Recombinant Vaccinia virus strain
Signal to noise ratio
Tumor cell-containg blood vessel
Vascular endothelial growth factor.
The authors thank Franziska Neubert, Jason Aguilar and Sabine Voll for excellent technical assistance and Saskia Wittber for animal care.
- Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Canc J Clin. 2012, 62: 10-29. 10.3322/caac.20138.View ArticleGoogle Scholar
- Uzbeck MH, Almeida FA, Sarkiss MG, Morice RC, Jimenez CA, Eapen GA, Kennedy MP: Management of malignant pleural effusions. Adv Ther. 2010, 27: 334-347. 10.1007/S12325-010-0031-8.View ArticlePubMedGoogle Scholar
- Bennett R, Maskell N: Management of malignant pleural effusions. Curr Opin Pulm Med. 2005, 11: 296-300.PubMedGoogle Scholar
- Lombardi G, Zustovich F, Nicoletto MO, Donach M, Artioli G, Pastorelli D: Diagnosis and treatment of malignant pleural effusion: a systematic literature review and new approaches. Am J Clin Oncol. 2010, 33: 420-423. 10.1097/COC.0b013e3181aacbbf.View ArticlePubMedGoogle Scholar
- Grove CS, Lee YCG: Vascular endothelial growth factor: the key mediator in pleural effusion formation. Curr Opin Pulm Med. 2002, 8: 294-301. 10.1097/00063198-200207000-00009.View ArticlePubMedGoogle Scholar
- Yano S, Shinohara H, Herbst RS, Kuniyasu H, Bucana CD, Ellis LM, Fidler IJ: Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells. Am J Pathol. 2000, 157: 1893-1903. 10.1016/S0002-9440(10)64828-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Yano S, Nokihara H, Hanibuchi M, Parajuli P, Shinohara T, Kawano T, Sone S: Model of malignant pleural effusion of human lung adenocarcinoma in SCID mice. Oncol Res. 1997, 9: 573-579.PubMedGoogle Scholar
- Hamed EA, El-Noweihi AM, Mohamed AZ, Mahmoud A: Vasoactive mediators (VEGF and TNF-alpha) in patients with malignant and tuberculous pleural effusions. Respirology. 2004, 9: 81-86. 10.1111/j.1440-1843.2003.00529.x.View ArticlePubMedGoogle Scholar
- Yano S, Herbst RS, Shinohara H, Knighton B, Bucana CD, Killion JJ, Wood J, Fidler IJ: Treatment for malignant pleural effusion of human lung adenocarcinoma by inhibition of vascular endothelial growth factor receptor tyrosine kinase phosphorylation. Clin Canc Res. 2000, 6: 957-965.Google Scholar
- Verheul HM, Hoekman K, Jorna AS, Smit EF, Pinedo HM: Targeting vascular endothelial growth factor blockade: ascites and pleural effusion formation. Oncologist. 2000, 5 (Suppl 1): 45-50.View ArticlePubMedGoogle Scholar
- Matsumori Y, Yano S, Goto H, Nakataki E, Wedge SR, Ryan AJ, Sone S: ZD6474, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, inhibits growth of experimental lung metastasis and production of malignant pleural effusions in a non-small cell lung cancer model. Oncol Res. 2006, 16: 15-26.PubMedGoogle Scholar
- Kobold S, Hegewisch-Becker S, Oechsle K, Jordan K, Bokemeyer C, Atanackovic D: Intraperitoneal VEGF inhibition using bevacizumab: a potential approach for the symptomatic treatment of malignant ascites?. Oncologist. 2009, 14: 1242-1251. 10.1634/theoncologist.2009-0109.View ArticlePubMedGoogle Scholar
- Van TT, Hanibuchi M, Goto H, Kuramoto T, Yukishige S, Kakiuchi S, Sato S, Sakaguchi S, Dat LT, Nishioka Y: SU6668, a multiple tyrosine kinase inhibitor, inhibits progression of human malignant pleural mesothelioma in an orthotopic model. Respirology. 2012, 17 (6): 984-990. 10.1111/j.1440-1843.2012.02193.x. AugView ArticlePubMedGoogle Scholar
- Bourke MG, Salwa S, Harrington KJ, Kucharczyk MJ, Forde PF, de Kruijf M, Soden D, Tangney M, Collins JK, O’Sullivan GC: The emerging role of viruses in the treatment of solid tumours. Canc Treat Rev. 2011, 37: 618-632. 10.1016/j.ctrv.2010.12.003.View ArticleGoogle Scholar
- Urruticoechea A, Alemany R, Balart J, Villanueva A, Vinals F, Capella G: Recent advances in cancer therapy: an overview. Curr Pharm Des. 2010, 16: 3-10. 10.2174/138161210789941847.View ArticlePubMedGoogle Scholar
- Vaha-Koskela MJ, Heikkila JE, Hinkkanen AE: Oncolytic viruses in cancer therapy. Canc Lett. 2007, 254: 178-216. 10.1016/j.canlet.2007.02.002.View ArticleGoogle Scholar
- Parato KA, Senger D, Forsyth PA, Bell JC: Recent progress in the battle between oncolytic viruses and tumours. Nat Rev Canc. 2005, 5: 965-976.View ArticleGoogle Scholar
- Liu TC, Galanis E, Kirn D: Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat Clin Pract Oncol. 2007, 4: 101-117. 10.1038/ncponc0736.View ArticlePubMedGoogle Scholar
- Rowan K: Oncolytic viruses move forward in clinical trials. J Natl Canc Inst. 2010, 102: 590-595. 10.1093/jnci/djq165.View ArticleGoogle Scholar
- Russell SJ, Peng KW, Bell JC: Oncolytic virotherapy. Nat Biotechnol. 2012, 30: 658-670. 10.1038/nbt.2287.View ArticlePubMedGoogle Scholar
- Zhang Q, Yu YA, Wang E, Chen N, Danner RL, Munson PJ, Marincola FM, Szalay AA: Eradication of solid human breast tumors in nude mice with an intravenously injected light-emitting oncolytic vaccinia virus. Canc Res. 2007, 67: 10038-10046. 10.1158/0008-5472.CAN-07-0146.View ArticleGoogle Scholar
- Zhang Q, Liang C, Yu YA, Chen N, Dandekar T, Szalay AA: The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of F14.5L inactivation. Mol Genet Genomics. 2009, 282: 417-435. 10.1007/s00438-009-0475-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Yu Z, Li S, Brader P, Chen N, Yu YA, Zhang Q, Szalay AA, Fong Y, Wong RJ: Oncolytic vaccinia therapy of squamous cell carcinoma. Mol Canc. 2009, 8: 45-View ArticleGoogle Scholar
- Gentschev I, Muller M, Adelfinger M, Weibel S, Grummt F, Zimmermann M, Bitzer M, Heisig M, Zhang Q, Yu YA: Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68. PLoS One. 2011, 6: e22069-10.1371/journal.pone.0022069.PubMed CentralView ArticlePubMedGoogle Scholar
- Gentschev I, Donat U, Hofmann E, Weibel S, Adelfinger M, Raab V, Heisig M, Chen N, Yu YA, Stritzker J, Szalay AA: Regression of human prostate tumors and metastases in nude mice following treatment with the recombinant oncolytic vaccinia virus GLV-1h68. J Biomed Biotechnol. 2010, 2010: 489759-PubMed CentralView ArticlePubMedGoogle Scholar
- Advani SJ, Buckel L, Chen NG, Scanderbeg DJ, Geissinger U, Zhang Q, Yu YA, Aguilar RJ, Mundt AJ, Szalay AA: Preferential replication of systemically delivered oncolytic vaccinia virus in focally irradiated glioma xenografts. Clin Canc Res. 2012, 18: 2579-2590. 10.1158/1078-0432.CCR-11-2394.View ArticleGoogle Scholar
- Weibel S, Raab V, Yu YA, Worschech A, Wang E, Marincola FM, Szalay AA: Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection. BMC Canc. 2011, 11: 68-10.1186/1471-2407-11-68.View ArticleGoogle Scholar
- Frentzen A, Yu YA, Chen N, Zhang Q, Weibel S, Raab V, Szalay AA: Anti-VEGF single-chain antibody GLAF-1 encoded by oncolytic vaccinia virus significantly enhances antitumor therapy. Proc Natl Acad Sci USA. 2009, 106: 12915-12920. 10.1073/pnas.0900660106.PubMed CentralView ArticlePubMedGoogle Scholar
- Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, Winkler F: Real-time imaging reveals the single steps of brain metastasis formation. Nat Med. 2010, 16: 116-122. 10.1038/nm.2072.View ArticlePubMedGoogle Scholar
- Gentschev I, Adelfinger M, Josupeit R, Rudolph S, Ehrig K, Donat U, Weibel S, Chen NG, Yu YA, Zhang Q: Preclinical evaluation of oncolytic vaccinia virus for therapy of canine soft tissue sarcoma. PLoS One. 2012, 7: e37239-10.1371/journal.pone.0037239.PubMed CentralView ArticlePubMedGoogle Scholar
- Yano S, Shinohara H, Herbst RS, Kuniyasu H, Bucana CD, Ellis LM, Davis DW, McConkey DJ, Fidler IJ: Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis. Canc Res. 2000, 60: 4959-4967.Google Scholar
- Iankov ID, Msaouel P, Allen C, Federspiel MJ, Bulur PA, Dietz AB, Gastineau D, Ikeda Y, Ingle JN, Russell SJ, Galanis E: Demonstration of anti-tumor activity of oncolytic measles virus strains in a malignant pleural effusion breast cancer model. Breast Canc Res Treat. 2010, 122: 745-754. 10.1007/s10549-009-0602-z.View ArticleGoogle Scholar
- Iankov ID, Allen C, Federspiel MJ, Myers RM, Peng KW, Ingle JN, Russell SJ, Galanis E: Expression of immunomodulatory neutrophil-activating protein of Helicobacter pylori enhances the antitumor activity of oncolytic measles virus. Mol Ther. 2012, 20: 1139-1147. 10.1038/mt.2012.4.PubMed CentralView ArticlePubMedGoogle Scholar
- Cheng D, Rodriguez RM, Perkett EA, Rogers J, Bienvenu G, Lappalainen U, Light RW: Vascular endothelial growth factor in pleural fluid. Chest. 1999, 116: 760-765. 10.1378/chest.116.3.760.View ArticlePubMedGoogle Scholar
- Yanagawa H, Takeuchi E, Suzuki Y, Ohmoto Y, Bando H, Sone S: Vascular endothelial growth factor in malignant pleural effusion associated with lung cancer. Canc Immunol Immunother. 1999, 48: 396-400. 10.1007/s002620050592.View ArticleGoogle Scholar
- Strizzi L, Catalano A, Vianale G, Orecchia S, Casalini A, Tassi G, Puntoni R, Mutti L, Procopio A: Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma. J Pathol. 2001, 193: 468-475. 10.1002/path.824.View ArticlePubMedGoogle Scholar
- Gerber HP, Ferrara N: Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Canc Res. 2005, 65: 671-680.Google Scholar
- Pichelmayer O, Gruenberger B, Zielinski C, Raderer M: Bevacizumab is active in malignant effusion. Ann Oncol. 1853, 2006: 17-Google Scholar
- Nagy JA, Chang SH, Dvorak AM, Dvorak HF: Why are tumour blood vessels abnormal and why is it important to know?. Br J Canc. 2009, 100: 865-869. 10.1038/sj.bjc.6604929.View ArticleGoogle Scholar
- Risau W: Mechanisms of angiogenesis. Nature. 1997, 386: 671-674.View ArticlePubMedGoogle Scholar
- Dvorak HF, Sioussat TM, Brown LF, Berse B, Nagy JA, Sotrel A, Manseau EJ, Van de Water L, Senger DR: Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels. J Exp Med. 1991, 174: 1275-1278. 10.1084/jem.174.5.1275.View ArticlePubMedGoogle Scholar
- Prager GW, Lackner EM, Krauth MT, Unseld M, Poettler M, Laffer S, Cerny-Reiterer S, Lamm W, Kornek GV, Binder BR: Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab. Mol Oncol. 2010, 4: 150-160. 10.1016/j.molonc.2010.01.002.View ArticlePubMedGoogle Scholar
- Lee TH, Avraham HK, Jiang S, Avraham S: Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability. J Biol Chem. 2003, 278: 5277-5284. 10.1074/jbc.M210063200.View ArticlePubMedGoogle Scholar
- Hayashi Y, Torisu M: New approach to management of malignant ascites with streptococcal preparation OK-432. III. OK-432 attracts natural killer cells through a chemotactic factor released from activated neutrophils. Surgery. 1990, 107: 74-84.PubMedGoogle Scholar
- Katano M, Torisu M: New approach to management of malignant ascites with a streptococcal preparation, OK-432. II. Intraperitoneal inflammatory cell-mediated tumor cell destruction. Surgery. 1983, 93: 365-373.PubMedGoogle Scholar
- Torisu M, Katano M, Kimura Y, Itoh H, Takesue M: New approach to management of malignant ascites with a streptococcal preparation, OK-432. I. Improvement of host immunity and prolongation of survival. Surgery. 1983, 93: 357-364.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.