Cytokine 71 (2015) 318–326

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Suppression of proliferation, tumorigenicity and metastasis of lung cancer cells after their transduction by interferon-beta gene in baculovirus vector Alexandra A. Lykhova a,⇑, Yuri I. Kudryavets a, Ludmila I. Strokovska b, Natalia A. Bezdenezhnykh a, Nadiia I. Semesiuk a, Inna N. Adamenko a, Olga V. Anopriyenko b, Ada L. Vorontsova a a b

R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, 45 Vasilkivska St., 03022 Kyiv, Ukraine Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,150 Zabolotnogo Str., 03680 Kyiv, Ukraine

a r t i c l e

i n f o

Article history: Received 13 February 2014 Received in revised form 6 August 2014 Accepted 28 October 2014

Keywords: Interferon-beta Recombinant baculovirus Lewis lung carcinoma cells Tumorigenicity Metastasis

a b s t r a c t Background: Gene therapy represents an interesting alternative treatment for cancers. Interferon-beta is well known as a multifunctional cytokine that provides antiviral, antiproliferative, antiangiogenic and immunomodulating effects. For this reason introduction of this cytokine gene in baculovirus vector is seen as a rather promising tool for anticancer therapy. Aim: Investigation of biological behavior in vitro and in vivo of lung cancer cells modified by interferonbeta gene which was introduced into the cells in vitro with baculovirus vector. Materials and Methods: Studies were performed on mouse Lewis lung carcinoma cells as the tumor model (LL cell line). Transductions of cells by recombinant baculoviruses, in vitro and in vivo analysis of tumor cell biology and immunocytochemical method have been used. Results: The study of various in vitro biological parameters of LL cancer cells transduced by recombinant baculovirus with interferon gene demonstrated that the transduction of cells is accompanied by significant inhibition of their proliferation and ability to form colonies in semisolid agar. Also, transduction of LL cells with interferon gene inhibited their tumorigenicity, i.e. the ability to cause formation of tumors and metastases in lungs of mice in vivo. Anti-tumor activity of recombinant interferon is realized via high level of its local production in tumors, induced by LL carcinoma cells, transduced with recombinant interferon-beta gene. Recombinant baculovirus without interferon gene did not influence significantly on tumorigenicity and metastatic ability of lung cancer cells. Conclusions: Introduction of interferon-beta gene in Lewis lung carcinoma cells in vitro in recombinant baculovirus leads to inhibition of their proliferation potential and malignant behavior in vitro, tumorigenicity and metastatic activity in vivo. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Lung cancer remains to be the leading cause of cancer-related mortality, demonstrating the limited efficacy of traditional surgical Abbreviations: IFN-beta, interferon-beta; BV, baculovirus vector; rBV, recombinant baculovirus vector without interferon-beta gene; rBV/IFN-beta, recombinant baculovirus vector with interferon-beta gene; plaque forming unit, pfu; NCS, newborn calf serum; MOI, multiplicity of infection; VSV, vesicular stomatitis virus; colony forming activity, CFA; IU, international units; i/m, intramuscularly; MHC, major histocompatibility complex; DCs, dendritic cells; AcMNPV, Autographa californica multiple nuclear polyhedrosis virus; PBS, phosphate buffered saline; EMT, epithelial–mesenchymal transition; TLR, Toll-like receptor; MSCs, mesenchymal stem cells. ⇑ Corresponding author. http://dx.doi.org/10.1016/j.cyto.2014.10.029 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.

and cytotoxic chemotherapy in these patients. The development of new and effective approaches for therapy of patients with advanced lung cancer remains a major health imperative [1]. Gene therapy presents a promising alternative treatment for cancers, a number of studies have demonstrated that gene therapy can effectively reduce tumor growth in animal models [2–4]. Viral vectors are one of the most effectual means for genetic modification of the most of somatic cells in vitro and in vivo [3]. Recombinant baculoviruses with a strong expression promoter have been stated as a new generation of gene therapy vehicles holding a great promise [5,6]. The baculoviruses have a large genome and thus can accommodate big transgene or a few transgenes. In addition, they are easy to accumulate and obtain high levels of

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recombinant gene expression [7]. However, in spite of a good understanding of all these attractive advantages of baculovirus, the virus-based gene therapy is still in its infancy, no practical application in cancer therapy is reported, even preclinical animal studies have been scarcely trialed [8]. Tumor-cell-targeted cytokine gene therapy has recently received a special attention as a possible alternative to systemic administration of cytokines in the cancer therapy [9]. The possibility to contribute improved cytokine production at the tumor cell site has opened the new ways to determining more natural and selective antitumor therapies with cytokines. It is becoming evident that antitumor activity of any given cytokine consistently expressed by tumor cells and the mechanisms elicited by this process in the host are determined not only by the intrinsic biological activities of the cytokine itself but also by the characteristics of the specific tumor model. For this reason, it is particularly important to conduct studies on the effects exerted by different cytokines expressed in the tumor cells [10]. Effects of the type I IFNs include direct antiproliferative impact on a number of tumor targets [11]. In various comparative studies of growth-inhibitory activities in melanoma cells, IFN-beta exhibited a more pronounced growth inhibition activity than IFN-alpha [12,13]. Notably, studies of IFN-beta gene therapy of human and mouse malignancies conducted on mouse models revealed that IFN-beta induces both direct antiproliferative and apoptotic effects, as well as systemic immunity against the tumor targets [14]. Type I IFNs regulate MHC class I expression and enhancement of cytotoxic T-lymphocyte activity and T-helper cell functions. Additionally, type I IFNs will activate natural killer cells and induce macrophage activity [15]. Type I IFNs can induce maturation of DCs marked by enhancement of MHCII and CD86 activities and facilitate migration of Langerhans cells. Regulation of DCs by type I IFNs contributes to induction of MHC I cross processing and CD8+ responses as well as the induction of CD4+ responses [16]. All these findings are viewed as a compelling evidence that type I IFNs influence numerous immune responses [17]. Studies of the pharmacokinetics of IFN-beta shown that this protein has a very short period of half-life in the organism after parenteral protein administration [18,19]. These data suggest that the low efficiency of IFNs in cancer trials may have been caused by either deficient delivery or a lack of steady delivery of the protein to the tumor site. Assuming that local gene therapy has at least a potential to overcome these limitations, we have tested the IFNbeta gene delivery by a recombinant baculovirus vector. In the present study, we have used a recombinant baculovirus vector encoding the IFN-beta gene and tested the effect of transduction by that virus on proliferation and tumorigenicity of Lewis lung carcinoma cells in vitro and in vivo. Our findings indicate that mouse lung carcinoma cells (LL cell line) are sensitive to baculovirus infection and baculovirus-mediated IFN-beta gene can be used in the treatment of lung cancer. Since IFN-beta is secreted by cells, one could expect both autocrine and paracrine effects to be responsible for the observed significant effect. We present here an evidence that IFN-beta gene delivery into a tumor LL cells using the recombinant baculovirus can effectively inhibit tumor growth.

2. Materials and methods

319

under the control of strong CAG promoter cassette (rBV/IFN-beta) was used as the IFN-beta expressing vehicle [20,21]. Vector without the ifn-b gene (rBV) was used as a control. Concentration of virus vectors in the initial solutions was 2.3–4  108 pfu/ml for rBV/IFN-beta and 4  108 pfu/ml for rBV. 2.2. Cell lines Cells of murine Lewis lung carcinoma (LL cell line) [22] and L929 cells were granted by Bank of Cell Lines of R.E. Kavetsky Institute of Experimental pathology, oncology and radiobiology, NAS of Ukraine (IEPOR NASU). LL cell line were maintained in plastic flask (TPP, Italy) in DMEM with 4 mmol/l L-glutamine (PAA, Austria) with 10% of newborn calf serum (NCS) (PAA, Austria) and 40 mcg/ml gentamicin (Sigma, USA). L929 cells were maintained in RPMI-1640 (Sigma, USA) supplemented with 10% NCS (PAA, Austria) and 40 mcg/ml gentamicin (Sigma, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Cells were detached with EDTA solution (Sigma, USA) and performed by the standard method [23]. Cell density was counted with hemocytometer and viability was determined by exclusion tests with 0.04% solution of trypan blue dye (Bio-Rad, USA). 2.3. Transduction method Cellular transduction was performed at a multiplicity of infection (MOI = number pfu virus per cell) of recombinant baculovirus 100 and 200. The LL cells suspension in RPMI-1640 medium mixed with virus specimens in the PBS at a 1:1 ratio was incubated for 2 h at room temperature. After that a complete culture medium was added and cells were incubated 24–96 h at 37 °C and 5% CO2. The LL cells in the PBS without rBV were used as a control. The culture medium from LL cells transduced with recombinant baculovirus was harversted and stored at 20 °C. Titer of mouse IFN-beta was determined by standard microtitered method of evaluation of antiviral activity. Cell line L929 and vesicular stomatitis virus (VSV) were used as a test-system [24]. VSV(1 x 108 pfu/ml) was obtained from the Bank of Cell Line IEPOR NASU. The dilution of the preparation that protected 50% of L929 cell monolayer from cytopathic effect of VSV(2 x 104 pfu per well) was taken as a unit of IFN activity. IFN titer was expressed in the international units (IU) using the mouse IFN-beta (Cat. No 19032 Sigma, USA) as a standard. 2.4. Definition of proliferative activity of cells After transduction the cell suspensions (1  104 cells/well) were seeded into 96-well culture plates in DMEM (PAA, Austria) with 10% NCS (PAA, Austria) and 40 mg/ml gentamicin (Sigma, USA) and incubated for 24–96 h at 37 °C in the presence of 5% CO2. Then the medium was removed and the cells were counted by colorimetric assay with crystal violet (Sigma, USA) [25]. All experiments were performed in triplicate and the results were expressed as inhibitory rate (IR):

IR ¼ ð1  A540 ðexperimentÞ=A540 ðcontrolÞÞ  100% Besides, the cells in appropriate wells were detached with EDTA solution (Sigma, USA) and counted with hemocytometer. Their viability was determined by exclusion tests with 0.04% solution of trypan blue dye (Bio-Rad, USA).

2.1. Recombinant vector 2.5. Immunocytochemical and immunohistochemical assay Recombinant baculovirus (BV) vectors based on Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) were obtained using baculovirus expression system Bac-to-Bac (Invitrogen,USA). Recombinant vector, containing mouse ifn-b gene

The levels of proliferative activity and synthesis of recombinant murine IFN-beta in LL cells transduced with BVs were determined by immunocytochemical evaluation of cells expressing Ki-67

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protein (marker of DNA replication) and IFN-beta protein. Immediately after transduction cells were planted on the coverslip and, 48 h later, analysed by the standard method [26] using rabbit monoclonal antibodies against Ki-67 or against mouse IFN-beta (Thermo Scientific, USA). The antigen/antibody reaction was visualized with Ultra Vision LPValue Detection system. DAB Plus (3,3-diaminobenzidine) (Thermo Scientific, USA) was used as a chromogen. Immunoexpression of Ki-67 and IFN-beta was determined in LL cells and evaluated semiquantitatively using H-score system (registering the number of positive cells and the intensity of staining). The results were calculated at a magnification 400 in 5 randomly selected different fields of view. The percentage of the positive cells was multiplied by the score number corresponding to the staining intensity (0 – none, 1 – low, 2 – middle, 3 – high). With 4 intensity levels, the resulting score ranged from 0 (no staining cells) to 300 (diffuse intense staining of cells). The average percentage of positive cells was calculated using the following formula: H-Score = (% of cells stained at intensity 1  1) + (% of cells stained at intensity 2  2) + (% of cells stained at intensity 3  3) [27]. For immunohistochemical assay, 3 lm thick paraffin embedded tissue sections were placed on glass slides (Superfrost Plus, Menzel-Glaser, Thermo Scientific, Germany). The samples were deparaffinised with xylene for 40 min, rehydrated in 96% ethanol for 20 min and washed in deionised water. After that samples had been incubated in a citrate buffer at 90 °C for 20 min. After the incubation the samples were cooled at room temperature and washed in a PBS. To block nonspecific background staining Hydrogen Peroxide block and Protein block (Ultra Vision Quanto Detection system, Thermo Scientific, USA) were used. Further sections were incubated at room temperature for 1 h with the primary rabbit polyclonal antibodies anti-IFN-beta (Thermo Scientific, Pierce, USA) diluted 1:200. Immunoexpression of antigen was visualized by Ultra Vision Quanto Detection system (Thermo Scientific, USA). DAB Quanto (3,3-diaminobenzidine) (Thermo Scientific, USA) was used as a chromogen. The samples were counterstained with hematoxylin solution according to Mayer (BioChemika, Sigma). Nonimmune rabbit serum (X0902; Dako) was added instead of primary antibodies and used as negative controls. Immunoexpression of IFN-beta was evaluated in tumor tissue. The level of cytokine expression was evaluated semiquantitatively, using 1000 magnifications and classified according to the scores: 0 – no staining (76%) [28]. Pictures of immunostained LL cells and Lewis lung carcinoma tissue was made by the light microscope Axiostar plus (Carl Zeiss, Germany) equipped with digital camera (Canon Power Shot G5, UK) at 1000 magnification. 2.6. Semisolid agar colony assay The assay was conducted in parallel wells of 6-well plates. Base layers of 4 ml of 0.5% agar (Difco, USA) were prepared on RPMI1640 medium with 10% NCS. A 2 ml overlayer of 0.33% agar containing LL cells (2  103) in DMEM medium with 10% NCS and 40 mcg/ml gentamicin was then applied and the plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2. After 14 days of the incubation, the plating colonies were stained with 0.2% solution methylthiasoliletetrazolium (Sigma, USA) or 0.4% solution of neutral red dye (Sigma, USA) at 37 °C for 3 h. After that, the number and the size of colonies were counted in each well using binocular magnifiers BM-51-2 (LOMO, Russia). The size of colony was defined as small (10–30 cells), middle (31–50 cells) and large (more than 50 cells). Images of live colonies was made

by inverted microscope Axiovert 25 (Carl Zeiss, Germany) equipped with digital camera (Canon Power Shot A640, UK) at 200 magnification. Colony-forming activity (CFA) of LL cells was determined by the formula: CFA = (A/B)  100%, wherein A – the number of colonies per well, B – the number of cells planted in semisolid agar per well. 2.7. Tumorigenicity and metastasis in vivo assays Tumorigenic and metastatic potentials of LL cells transduced with rBV and rBV/IFN-beta were determined in experiments in vivo using male C57BL/6 mice aged 7–8 weeks and weight 20–22 g wiring vivarium of IEPOR, NASU. Mice were kept under standard conditions with granulated feed and water ad labidum. The animal welfare and experimental procedures were approved by the Animal Ethics Committee. Efforts were conducted to minimize the animals’ suffering. Animals were euthanaised using either anesthesia. The suspensions of control and transduced LL cells (at a dose 3  105 cells per mouse in 0.1 ml of saline solution) were injected to animals intramuscularly (i/m). Ten mice in each group were used in all in vivo experiments, which were repeated thrice. The dynamics of growth of i/m tumors in mice investigated by measuring the diameter of tumors in two projections every 2 days. On the fourth day after the injections of tumor cells, tissue samples were isolated, fixed in 4% PBS-formalin solution and embedded in paraffin. To count metastases mice were euthanized on the 38th day after the i/m injections of cells. The lungs were isolated and fixed in 4% PBS-formalin solution. The number of metastases and their diameter were determined using the dental spatulas (1–3 mm) and binocular magnifier BL-51-2 (Lomo, Russia). The volume of tumors and metastases were determined in mm3 by the formula V = D3  0.52, where V – volume of metastases, D – diameter of a single tumor or metastatic nodule. 2.8. Statistical analysis Statistical data processing was performed by the Student t-test or nonparametric U-criterion Villcockson–Mann–Whitney using STATISTICA 6.0. Statistical significance was set at p 6 0.05. 3. Results 3.1. Expression of recombinant interferon-beta and proliferative activity cells transduced by baculoviruses Transduction of LL cells by rBV and rBV/IFN-beta is followed with some cytopathogenic effect at MOI 100 pfu/cell and more. The virus without the inserted genes reduces the number of living cells on the next day after the transduction by 10–25%. Expression of recombinant IFN-beta in the cells trancduced by rBV/IFN inhibited their proliferation by 50% compared to untreated cells (Table 1). The possibility of rBV/IFN-beta-transduced LL cells to produce IFN-beta into the growth medium was studied using different multiplicity of BV infection in vitro. It was found that the level of IFN production directly depends on the infection dose of rBV/IFN up to MOI 100 pfu/cell, but increasing of viral dose (MOI 200 pfu/cell, data not shown) did not influence the IFN-beta levels significantly. Compared to cells of another tumor models transduced by rBV/ IFN-beta, LL cells produce low quantity of recombinant IFN (100– 300 IU/ml only). For example, cells of mouse melanoma MM-4 cell line produce – 3000–5000 IU/ml at 50,000 transduced cells [4] which might be explained by higher sensitivity of LL cells to growth inhibition induced by IFN than that of MM-4 cells, thus

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A.A. Lykhova et al. / Cytokine 71 (2015) 318–326 Table 1 Proliferative activity and IFN-beta production of LL cells 48 h after their transduction by rBV and rBV/IFN-beta. Virus

MOI (pfu/cell)

IFN-beta (IU/ml)

The level of expression of Ki-67 protein on a H-score system (points)

Number of Ki-67-positive cells (%)

Control (untreated cells) rBV rBV/IFN-beta

0 100 100

0 0 150 ± 8

250 ± 15 250 ± 23 70 ± 5.5⁄⁄

100 100 50 ± 5.5⁄

Proliferative activity of LL cells transduced with BVs was assessed by immunocytochemical analysis of the percentage of Ki-67 positive cells and the intensity of their staining (H-score system). The level of recombinant IFN-beta was determined in culture medium from LL cells transduced with BVs by standard microtitered method of evaluation of antiviral activity. ⁄ p < 0.005. ⁄⁄ p < 0.001 compared to control.

LL control

LL/rBV

LL/rBV/IFN-beta

120

Number of live cells, %

recombinant IFN-beta in LL cells may inhibit their own biosynthesis. These results correlate with that observed earlier [24] about high sensitivity of LL cells to exogenous IFN-beta (50 IU/ml for IC50). Apart from the antiviral activity in culture medium transduced by rBV/IFN-beta cells, it is also important to determine how other biological properties of the produced recombinant IFN-beta, including its antiproliferative activity, correspond to such natural mouse IFN. In order to compare the quality of the mouse IFN we used IFN induced in L929 cells by Newcastle disease virus and murine cell line with a known sensitivity to the antiproliferative activity of IFN – Lewis lung carcinoma (LL cell line). Studies have shown that the sensitivity of cells to recombinant IFN-beta, obtained from the culture medium transduced by rBV/IFN-beta cells, coincides with their sensitivity to natural IFN induced in murine cells by virus [24]. Indeed, production of recombinant IFN-beta by LL cells resulted in a significant dose-dependent inhibition of cell growth compared to control. In this case, the increase of doubling time of cells, decrease of mitotic cells number and the levels of proliferation antigen Ki-67 expression were observed (Fig. 1, Table 1). Our results showed that the expression of Ki-67 protein in the untreated LL cells and transduced by rBV was detected at the same levels in 100% of the cells. After the transduction by rBV/IFN-beta Ki-67 expression in LL cells was detected only in 50% cells (Fig. 1). These data indicate that the expression of transduced IFN-beta gene in LL cells leads to significant suppression the proliferative potential of these tumor cells. Besides these experimental findings, inhibition of cell proliferation activity was confirmed in another series of experiments by staining alive cells with crystal violet or trypan blue dye exclusion tests. The results showed that transduction of LL cells with rBV/ IFN-beta leads to inhibition of cell growth on 50% 48 h after transduction (Fig. 2). These date correlate with the results which showed inhibition of Ki-67 expression in studied cells (Table 1). These studies have also shown that the number of living cells after 72 and 96 h of transduction is lower the control levels by 80% and 90%, respectively (Fig. 2). Immunocytochemical analysis of LL cells transduced with rBV and rBV/IFN-beta in vitro has shown that expression of IFN-beta was observed in 75 ± 5% (87 ± 5 points, on the H-score system) of LL cells transduced rBV/IFN-beta, but the level of IFN synthesis in

100 80

**

* ***

60 40

*** ***

20 0 48 h

72 h

96h

Time of cell culvaon Fig. 2. Antiproliferative activity of recombinant IFN-beta in LL cells. The number of alive LL cells after their transduction with BVs at MOI = 100 pfu/cell was determined by staining live cells with crystal violet dye or staining dead cells with trypan blue dye. The results obtained using these two methods were similar. Therefore, figure presented the data obtained by colorimetric assay with crystal violet dye. ⁄ p < 0.002; ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 compared to control.

various rBV/IFN-beta transduced cells in population was different. The number of IFN high producing cells reaches about 5–7% (Figs. 3 and 4). The characteristic feature of the IFN producing cells is that they released cell membrane ‘‘blebs’’ with recombinant IFN-beta to react with anti-IFN antibody (Fig. 4). Control rBV did not induce endogenous IFN-beta in tumor cells (0 points, on the H-score system) (Fig. 3). Immunohistochemical staining with antibody against murine IFN-beta of tumor samples that were taken at 4th day after LL cell injection (6th day after their transduction in vitro) revealed that IFN-beta cytokine expression was observed only in small groups or single tumor cells of tumor tissue formed by LL/rBV/IFN-beta cells (Fig. 5C, D). Interesting that in these samples, compared to controls, was observed mostly stromal tissue with a single or small groups of tumor cells. The level of cytokine expression was low because only 22 ± 4% of these cells expressed IFN-beta (Fig. 5A–D). 3.2. Colony-forming activity It is well known that anchorage-independent growth is one of the features of malignant cell transformation, which is considered

Fig. 1. Immunocytochemical analysis the expression of the Ki-67 protein in nuclei of LL cells transduced by rBV and rBV/IFN-beta (MOI = 100 pfu/cell). Immediately after transduction LL cells (A, B, C) were planted on the coverslip, 48 h later, analysed by the immunocytochemical assay with anti-Ki-67 antibodies and photographed (magnification 1000): A – LL control – untreated cells, B – LL/rBV, C –LL/rBV/IFN-beta.

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Fig. 3. Immunocytochemical analysis the expression of recombinant murine IFN-beta in the LL cells transduced by rBV and rBV/IFN (MOI = 100 pfu/cell). Immediately after transduction LL cells (A, B, C) were planted on the coverslip, 48 h later, analysed by the immunocytochemical assay with anti-murine IFN-beta antibodies and photographed (magnification 1000). A – LL control – untreated cells, B – LL/rBV (control), C – LL/rBV/IFN-beta.

Fig. 4. Immunocytochemical analysis the features of the expression of recombinant IFN-beta in LL cells. This panel of images shows one of the mechanisms of synthesis and producing recombinant IFN-beta in LL cells. Transduction of cells by rBV/IFN-beta leads to formation blebs with recombinant cytokine, which react with anti-IFN-beta antibody (") (magnification 1000).

the most precise and convincing in vitro assay correlated with tumorigenic and metastatic potential of tumor cells [29,30]. Our investigation has shown that transduction of LL cells by rBV/ IFN-beta (MOI = 200 pfu/cell) has resulted in significant inhibition of ability of cells to form colonies in semisolid agar that indicated on inhibition of their autonomy and tumorigenicity in vitro (Figs. 6 and 7). Untreated LL cells exhibited 11.4 ± 0.4% colonies in agar and only 1 ± 0.15% (p < 0.005) was observed after rBV/IFN-beta transduction of LL cells. Interestingly, that rBV also inhibited LL cell colony forming activity (CFA) up to 3.2 ± 0.4% (P < 0.01) (Fig. 7A). The latest results demonstrate that the rBV inhibited CFA of LL cells in semisolid agar by 3.5 times only, but after rBV/IFN-beta transduction the CFA of LL tumor cells was suppressed significantly by 11 times compared to control (p < 0.005) (Fig. 7A). Our data show that rBV and rBV/IFN-beta reduce colony formation in agar by between 65% and 90%, indicating a reduction in the degree of anchorage independence of these cells in the presence of recombinant IFN-beta (Fig. 7A and B). Transduction of LL cells by control virus without IFN-beta gene suppressed the formation of large and medium-sized colonies by 3 times and small colonies by 2 times. At the same time, transduction of LL cells by rBV/IFN-beta provides inhibition of large colonies formation by 16 times, medium-sized colonies by 19 times and the number of small colonies by 4 times (Fig. 7B). These results indicate that transduction of LL cells by recombinant baculoviruses significantly reduced formation of mainly large and medium-sized colonies, i.e. colonies of the most malignant clones. Thus, these data suggest that the transplantation of IFN-producing tumor cells transduced by rBV/IFN-beta in vivo can lead to increase of local IFN-beta concentration in the area of the injection

of these cells. Apart from that, one could expect changes in the properties of tumor cells which affect the nature of tumor growth: inhibition of proliferative activity and tumorigenicity of cells and other immunophenotypic changes: decrease of Twist and Slug EMT protein expression, inhibition of nuclear p14ARF expression and increase of nuclear p21Waf1 expression (manuscript in preparation). 3.3. Tumor growth kinetics and metastases formation In the next series of experiments we have studied the effect of rBV/IFN-beta on tumor growth and metastasis of LL cells after i/m administration of transduced cells. To investigate whether baculovirus gene delivery could be effective in inhibiting tumorigenicity, we separately transduced LL tumor cells with rBV and rBV/IFN-beta at a MOI of 100pfu/cell. C57Bl/6 mice were implanted intramuscularly with an equal number of rBV or rBV/IFN-beta infected cells and untreated control cells. Tumor growth was monitored starting from 7th day following the tumor induction. In all of mice implanted with uninfected cells or cells transduced by rBV tumors were registered on 12th day after the injection. At the same time, no tumors were observed in mice that received rBV/IFN-infected cells (Fig. 8). On the 18th day after LL cells injection tumor growth was absent only in mice, which were injected with rBV/IFN-beta-transduced cells. It is important to note, that in this group, on the 21st day after injection of LL cells, tumors developed in 80% animals; however, the size of these tumors was significantly smaller than those in the control mice or mice, which were injected with cells transduced by rBV. On the 38th day in 10% of mice with rBV/IFN-

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323

Fig. 5. Positive immunostaining of tumor cells with murine IFN-beta in the tissue of Lewis lung carcinoma induced by LL cells transduced in vitro with rBV/IFN-beta (MOI = 100 pfu/cell). Four days after the i/m administration of LL control, LL/rBV and LL/rBV/IFN-beta cells (6 days after their transduction in vitro with BVs) in mice, paraffin embedded tissue sections were processed for immunohistochemistry with anti-IFN-beta antibodies. Positive cells were counted in 5 view fields under microscope at magnification 1000. A – tumor tissue of Lewis lung carcinoma induced by LL control cells, B – tumor tissue of Lewis lung carcinoma induced by LL/rBVcells, C – group of IFNbeta positive cells in tumor stroma, D – single cells expressing IFN-beta.

Fig. 6. A series of images shows the ability of LL cells to form colonies in semisolid agar after their transduction with recombinant baculoviruses. LL control, LL/rBV and LL/ rBV/IFN-beta cells (2  103 per well) were planted on 6-well plates in semisolid agar, cultured for 14 days and photographed. Demonstrated types of colonies met in study groups (A, B, C) most frequently. A – untreated LL cells; B – LL/rBV; C – LL/rBV/IFN-beta (magnification 200).

beta transduced cells injected, tumors were not grown up. These data suggest that the local presence of a relatively low quantity of IFN-producing LL cells (level of recombinant IFN-beta production is 150 IU/50,000 cells) resulted in a significant inhibition of tumor growth (Fig. 8).

To study the metastatic potential of tumor cells transduced by rBV/IFN-beta we used a model of spontaneous metastasis. Control and BVs transduced LL cells were injected to C57Bl/6 mice and 38 days after the rate of tumors growth and spontaneous metastasis were studied. The study showed that the transduction of LL lung

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(B)

5 4.5

12 10 8 6

**

4

*

2

Colony-forming acvity, %

Colony-forming acvity, %

(A) 14

2 1

4 3.5

3

3 2.5 2

3***

1.5

1** 2*

1

3*** 1* 2*

0.5 0

0 LL control

LL/rBV

LL/rBV/IFN-beta

Experimental groups

LL control

LL/rBV

LL/rBV/IFN-beta

Experimental groups

Fig. 7. (A) Colony-forming activity of LL cells transduced with rBV or rBV/IFN-beta at MOI = 200 pfu/cell in semisolid agar. LL control, LL/rBV and LL/rBV/IFN-beta cells (2  103 per well) were planted in semisolid agar on 6-well plates, cultured for 14 days and stained with MTT solution or neutral red dye solution. CFA of LL cells was determined as the ratio of planted cells and formed colonies and presented in %. ⁄p < 0.005; ⁄⁄p < 0.01 compared to control. (B) Comparative analysis of the size of the colonies formed LL cells transduced with recombinant baculoviruses at MOI = 200 pfu/cell in semisolid agar. 1. – Large colonies (>50 cells), 2. – Medium colonies (31–50 cells), 3. – small colonies (10–30 cells). ⁄p < 0.001; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.05 compared to control.

carcinoma cells by rBV/IFN-beta inhibited not only tumor growth, but also spontaneous metastasis: volume of lung nodules in mice was decreased by 70% compared to that in the animals of the control group (4.3 ± 1.7 mm3 vs 68.7 ± 18 mm3 in control mice, p < 0.02) (Fig. 9). The results show that the process of spontaneous dissemination of carcinoma and growth of lung metastases were significantly inhibited in mice with lung carcinoma which contains IFN-producing tumor cells. 4. Discussion Gene therapy is considered a hopeful therapeutic strategy for cancer treatment and has been successfully introduced in animal models using various types of viral vector, gene expression regulation elements, and supposed antitumor genes. Autographa californica multiple nuclear polyhedrosis virus -based vector is tested as a new type of delivery vehicle for transgene expression in mammalian cells [2]. Currently recombinant baculovirus has been widely used for transgenic expression of different proteins in mammalian cells [31]. Thus, a wide range of opportunities of application of baculovirus vector as immunomodulator and delivery vehicles of transgene, are making it an attractive tool for the in vitro system, but it also offers a convenient way to deliver genes in vivo.

Volume of tumor, mm3

LL control

LL/rBV

LL/rBV/IFN-beta

2000 1800 1600 1400 1200 1000 800 600 400 200 0 12 day 15 day 18 day 21 day 23 day 26 day 29 day 34 day 38 day

Day aer injecon of tumor cells Fig. 8. Suppression of tumor growth of Lewis lung carcinoma in mice. LL cells transduced in vitro by rBV and rBV/IFN-beta at a MOI 100 pfu/cell were challenged i/m in C57Bl/6 mice (3  105 cells per mouse). Tumor volumes 40 days after LL cells transduction with IFN-beta gene are presented.

Baculoviruses are known as not replicable in mammalian cells, but in response to infection by baculovirus through Toll-like receptors, several cytokines including IFN can be activated in cells. It should be said that the data on the ability of baculoviruses to induce IFN or other cytokines in mammalian cells are disputable. These contradictions may be linked to the dose of virus, the type of cells, which interact with baculovirus, or other methodological nuances. Previously it was shown that baculovirus in mesenchymal stem cells (MSCs) induced IL-6 and IL-8 production, but did not induce IFN [32]. In other studies, however, it was shown that baculovirus can induce in vitro in immunocompetent cells or in MSCs various cytokines including IFN (alpha, beta, gamma) [33]. Nevertheless, the properties of tumor cells and their production of endogenous cytokines in response to transduction of recombinant baculoviruses were not investigated. Also, biological and phenotypic characteristics of tumor cells in the cases of introduction in them of recombinant baculovirus with genes of cytokines, including IFN have not been studied properly. IFN-beta, as a well known multifunctional cytokine, can provide various effects that can be grouped into major four: antiviral, antiproliferative, antiangiogenic and immunomodulating. For this reason, introduction of this cytokine gene in baculovirus vector may be a rather promising tool for anticancer therapy. Tumor cells transduced with recombinant baculovirus, especially producing transgenic IFN, may significantly change their biological and antigenic characteristics, performing the role of peculiar transplant cancer vaccines. Therefore, we considered it important to determine the behavior of tumor cells in vitro or in vivo in the tumor as a whole in a situation of long-term presence in the population of tumor specific IFN-producing insiders which carry out permanent cytokine supply like «drip irrigation». The present study is primarily aimed to explore the mouse Lewis lung carcinoma response to transduction by recombinant baculovirus with IFN-beta gene and to investigate the tumorigenicity and metastatic ability of these cells after rBV/IFN-beta transduction in vitro and in vivo. In order to observe the inhibitory effect of rBV/IFN-beta on tumor growth, the LL cell line obtained from a Lewis lung carcinoma [22] was used as a model both in vitro and in vivo. The extent of baculovirus-mediated recombinant gene transfer was evaluated by measuring the IFN production in culture medium of cells and protein expression of the IFN-beta gene in LL cells by immunocytochemical and immunohistochemical assays.

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9

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Fig. 9. Inhibitory effect of transduction LL cells by murine IFN-beta gene on spontaneous lung metastases in C57Bl/6 mice. Mice were injected i/m with (3  105 per mouse) untreated LL cells, LL/rBV or rBV/IFN-beta cells. The number of metastases (A) and volume of metastases (B) 40 days after LL cells transduction with IFN-beta gene are presented. ⁄p < 0.05; ⁄⁄p < 0.02 compared to control.

We have established that rBV does not cause pronounced cytotoxic effects in LL cells and do not change the expression in cells of the proliferation marker Ki-67 protein. It has been shown using biological and immunocytochemical methods, that in response to rBV transduction the LL cells do not synthesize and secrete endogenous interferons (alpha/beta/ gamma) or TNF. This actual finding suggests that tumor cells, in contrast to the immunocompetent or MSC cells, cannot respond, at least at the level of cytokine production, to TLR ligands like baculovirus. This may be due to the tissue nature of the cells, or defects in the TLR signaling system of these cells. At very least, malignant transformation of cells, for example immune cells (J774-mouse monocyte-like cell line), do not prevent activation of endogenous alpha/beta-IFN in these cells in response to baculovirus infection (our unpublished results). Nevertheless, tumor cells are able to actively support transgene expression and synthesis of recombinant IFN, as transduction of LL cells rBV/IFN-beta was accompanied by the synthesis and secretion of that cytokine: IFN titer in the culture medium reached 150 ± 8 IU/ml, and cytoplasm of 75 ± 5% cells contained the IFNbeta protein. The immunocytochemical analysis showed the distribution of immune complex antibodes/IFN-beta in the cytoplasm of LL cells and IFN releasing from the cells not only as soluble protein but also through formation blebs with IFN. Comparative analysis of the biological activity of IFN produced by transduced LL and MM-4 melanoma cells showed that it is antigenic, antivirus and antiproliferative properties correspond to natural murine type IFN [24]. IFN expression in LL cells, similarly to that in melanoma cells [4], also resulted in significant inhibition of cell proliferation: reduction of the total number of cells, including mitotic and Ki-67-positive cells in culture. Cancer cells are well known to have a high degree of autonomy, and the extent of its manifestation in vitro usually correlates with malignancy of cells in vivo [34]. We have shown that the properties of LL cells, transduced with rBV/IFN, have undergone major changes: colonies formation in semi-solid agar were sharply reduced, growth rate of the colonies was also hampered. The action of IFN reduced not only the total number of colonies, but also reduced their size. The ratio of large and small colonies in IFN-transduced cell cultures was rapidly shifting towards smaller colonies. These results demonstrate that the transduction of rBV/ IFN-beta into carcinoma cells suppresses their malignant phenotype and capacity to autonomous growth. It is also obvious that the antitumor activity of IFN-transgene expression was of paracrine nature and prolonged in time, as inhibition of clonal cell growth was observed for at least 2 weeks after the transduction

and antiproliferative effect of IFN-transgene spread to all cells in the culture, and not only on IFN-producing cells. Given that IFN-beta is a secreted protein, it is rather likely that it functions extracellularly as a regulator of cell growth and differentiation in an autocrine or paracrine manner. When the IFN gene is transduced into tumorigenic LL cells, their proliferative activity and anchorage-independent growth are reduced, and when these cells are inoculated into mice, baculovirus-mediated IFN gene expression significantly inhibited the growth of lung carcinoma and its metastases formation. The obtained results, which demonstrated the modification of the biological properties of lung carcinoma cells after transduction them in vitro by rBV/IFN-beta served as a justification for the in vivo experiments. Very important is the fact that the high antitumor and antimetastatic effects were obtained in the condition of relativity low levels of produced recombinant IFN-beta. These results indicate that presence of IFN-producing ‘‘insider’’ cells in tumors, especially highly sensitive to IFN, may provide a high antitumor effect. These results correlate with data [35], obtained earlier using adenovirus vector with IFN gene. 5. Conclusion Transduction of Lewis lung carcinoma cells by rBV/IFN-beta followed by synthesis and production of biologically active IFN-beta leads to inhibition of their proliferative and tumorigenic potential in vitro and in vivo. Baculovirus per se does not induce endogenous IFNs in LL carcinoma cells and does not inhibit their proliferation and tumorigenicity, but slightly inhibits anchorage-independent growth of these cells. Transduction of LL carcinoma cells with rBV/IFN-beta inhibits their ability to form solid tumors and spontaneous metastases in the lungs of mice. Acknowledgments This study was supported by the Grant N 11/2011 of President of the Ukraine for young scientists. References [1] Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, Chirieac LR, et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 2008;27:4702–11.

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