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Effect of suicide gene therapy in combination with immunotherapy on antitumour immune response & tumour regression in a xenograft mouse model for head & neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the one of the most frequent cancers in the world (1) and is the dominant cancer among the males in India (2). Although a great deal of progress has been made in the conventional treatment modalities, the disease is still incurable. A promising alternate therapy is gene therapy that is still in an experimental stage. In this treatment modality, active DNA is used that codes for a functional, therapeutic protein.

Prodrug activation strategy, also known as 'Suicide' gene therapy is one of the most frequently used strategies for gene therapy of cancer. Viral Herpes simplex thymidine kinase (HSVtk) converts a nontoxic prodrug ganciclovir (GCV) into a toxic form thereby killing the cells expressing the enzyme. HSVtk phosphorylates GCV resulting in a monophosphate form of GCV, which is further phosphorylated to di- and triphosphate forms by the endogenous thymidine kinase. The tri-phosphate GCV gets incorporated into growing DNA chain, inhibits DNA polymerase resulting in cell death (3,4). Another interesting phenomenon called as 'Bystander effect' is observed where cells neighbouring those expressing HSVtk also are killed (5) thereby enhancing tumour cell kill. The HSVtk strategy is also reported to induce the systemic immune response (6) and enhance the NK cell killer activity in vivo (7).

Cancers of head and neck region have high recurrence rates. This may partly be due to failure of the mucosal immune system to generate effective immune response. Hence, modalities involving enhancement of the antitumour immune response could improve the outcome of treatment in these patients. Here we report the efficacy of combined therapy of retroviral HSVtk/ GCV and naked IL-2 DNA injection in a HNSCC xenograft mouse model. The IL-2 injection induced antitumour immune response comprising the NK and dendritic cells in the nude mouse model.

Material & Methods

Cell lines and xenografts: The cell lines used in the study included 293 (Human embryonic kidney cells), PA311 (amphotropic retroviral packaging cell line) and NIH3T3 (Mouse fibroblast cell line). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO BRL, USA) with 10 per cent foetal calf serum (FCS) (GIBCO BRL, USA). NT8e, a cell line established from human head and neck cancer by our group at Cancer Research Institute, Mumbai was also maintained in the same medium (8). In order to obtain xenograft tumours 1x[10.sup.7] NT8e tumour cells in 100 [micro]l Hanks balanced salt solution (HBSS) were injected subcutaneously on the dorsal right flank of the nude mouse as described earlier (8,9).

Plasmid DNA constructs: The IL-2 plasmid, pCMV-IRES-IL2-neo (6.8 kb), was obtained as a kind gift from Dr F. Rosenthal (Freiberg, Germany). The IL-2 plasmid construct was cut with Hind III to obtain a 4.9 kb empty vector DNA construct (10) which was used as vector control without IL-2.

Construction of recombinant retroviral vectors and retroviral transduction: The recombinant Moloney murine leukaemia virus based retroviral vector pLTKSN was constructed as described earlier (11). The orientation of HSV-tk gene was confirmed using a double digest of Ssp I and Bgl II. The construct along with the ecotropic helper virus pEcoLandau (kind gift from Dr Robert Naviaux, Salk Institute, USA) was transfected in 293 cells and selected on G418 (GIBCO BRL, USA). The culture supernatant from stable clone number 41 was used to infect amphotropic packaging cell line PA311 along with polybrene. The infected PA317 cells were selected on 800 [micro]g/ml G418. Single cell-derived clones were isolated and expanded. One of the stable clones obtained by this ping pong method (12) was named as PLTK41.1 and used as virus producing cells (VPCs) in this study. Viral titre was determined by infection of NIH3T3 cells with virus containing supernatants from PLTK47.1 as described below.

Determination of viral titers for PLTK47.1: NIH3T3 cells were plated at a density of 1x[10.sup.5] in a 60 mm petri dish. After 24 h, cells were infected with culture supernatant from PLTK47.1 at different dilutions along with 1 [micro]l (8 [micro]g/ml) polybrene in DMEM. After 6 h, medium was replaced with DMEM containing 10 per cent FCS. Next day the cells were harvested and cultured in 100 mm petri dishes with 800 [micro]g/ml G418. After 14 days on selection, cells were fixed in 10 per cent neutral buffered formalin (NBF) and stained with 0.5 per cent methylene blue in methanol (13). Dilutions used for infection were 1:100, 1:1000, 1:10000, and 1:100000. The number of colonies were counted and the viral titre was calculated as follows:

Viral titre (cfu/ml) = No. of colonies x Split ratio x Dilution factor

GCV sensitivity of PLTK47.1 infected cells: The sensitivity of PLTK41.1 VPC to ganciclovir (GCV, Cymevene, F. Hoffmann-La Roche, Basel, Switzerland) was assessed using sulpho-rhodamine B (SRB) assay (14) as well as flow cytometry. For SRB assay, 5000 PLTK41.1 cells per well were seeded in a 96 well plate and for flow cytometry 1x[10.sup.5] PLTK41.1 cells were plated in 60 mm petri dishes. 24 h later, different concentrations of GCV were added and cells incubated for 3 or 6 days. The cells were collected by trypsinization, fixed in 70 per cent alcohol, resuspended in 500 [micro]l PBS, stained with 20 [micro]g propidium iodide (Sigma-Aldrich, USA) and acquired on flow cytometer (BDFACScan, BD Biosciences, USA). The data were analyzed using Cell Quest software (BD Biosciences, USA).

In vivo suicide gene therapy - antitumour effect of PLTK47.1 and IL-2: NT8e cells (1x[10.sup.7]) were injected subcutaneously in 100 [micro]l HBSS on the right flank of NMRi nude mice. Treatment schedules began after the tumour reached 5x5 mm, which was within 10-15 days of NT8e injection. The tumour bearing mice were divided into 1 treatment groups (Table), and 1x[10.sup.7] PTLK41.1 VPC in 100 [micro]l were injected intratumourally where indicated. Four days post VPC injection, intraperitoneal injection of GCV (100 mg/kg body weight) was administered once daily for 14 days. Fifty microgram of IL-2 plasmid DNA or empty vector DNA was injected intramuscularly in hind limb muscles one day prior to VPC injection. The tumour volumes were calculated using the formula (a x [b.sup.2])/2, where a, b are two longest perpendicular diameters. The results were expressed as mean ratio of tumour volume before and after completion of therapy. Mice were sacrificed 2 wk after completion of GCV injections or earlier if they appeared weak. Tumour tissues were collected for histology at the end of the experiment, stained with hematoxylin and eosin (HE) and were analyzed independently by a pathologist who was unaware of treatment groups. All animal studies had prior approval from Institute Animal Ethics Committee.

Analysis of immune cells by flow cytometry: Mice were sacrificed after completion of the treatment, spleen collected and a single cell suspension made from spleen. After taking a viable cell count using Trypan blue dye, the cells were resuspended at a density of 2x[10.sup.7] cells/ml in FACS staining buffer (PBS+ 1% FCS + 0.1% sodium azide). To 50 [micro]l of this cell suspension 5 [micro]l of CD11cPE antibody (Pharmingen, San Diego, CA, USA) for staining dendritic cells and 1 [micro]l of CD49bFITC antibody for staining Pan NK cells (DX5, Pharmingen, San Diego, CA, USA) were added. The cell suspensions were incubated on ice for 45 min in dark after which they were washed thrice with 2 ml FACS staining buffer. Finally, the cells were resuspended in 400 [micro]l FACS staining buffer. A total of 10,000 gated cells were acquired per sample on flow-cytometer (BDFACScan, USA) and data analyzed using CELLQUEST software (BD Biosciences, USA).

Immunostaining for caspase-3: Tumour tissue sections were incubated with anti active caspase-3 antibody (Promega, Madison, WI, USA) at 4[degrees]C for 12 h. Detection was carried out using Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA, USA). The slides were developed using 3,3'- diaminobenzidine (DAB) staining procedure.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay: TUNEL assay was performed using the DeadEnd Fluorometric TUNEL System kit as instructed by the manufacturer (Promega, Madison, WI, USA). Tumour tissue sections were fixed in 4 per cent paraformaldehyde and digested with Proteinase K. Terminal deoxynucleotidyl transferase (TdT) enzyme mix was added and the slides incubated at 37[degrees]C for 1 h in dark. The TUNEL reaction was terminated by washing slides with 2x sodium chloride and sodium citrate solution (SSC) for 15 min at RT. The sections were counterstained with propidium iodide. The extent of cell death between treated and untreated animal groups was analyzed using a Laser Confocal Microscope (LSM 510 Meta, Zeiss, Germany).

Statistical analysis: SPSS software, version 14 (Illinois, USA) was used for statistical analysis. Statistical significance was defined as P [less than or equal to] 0.05. Non parametric ANOVA was performed with Kruskal Wallis test. For flow cytometry data, the per cent positive cells for every type of population between treated and respective control groups were compared.


Construction of retroviral vector and determination of retroviral titre: After confirming orientation ofthe HSV-tk gene cloned in pLTKSN it was co-transfected into 293 cells with an ecotropic helper virus pEcoLandau to produce ecotropic viral particles. The culture supernatant from these co-transfected cells was used to infect an amphotropic packaging cell line PA311. Positive PA311 clones were selected on G418 in order to produce the amphotropic virus producing cell line (VPC) which were screened by RT-PCR to check for tk expression (data not shown). One of the positive clones PLTK41.1 was selected and used for further studies. Viral titre, as determined by infection of NIH3T3 cells with PLTK41.1 supernatants was 4.16 x [10.sup.5] cfu/ml (Fig. 1).

PLTK47.1 cell sensitivity to GCV: Sensitivity of PLKT41.1 cells to GCV was assessed for 3 days (Fig. 2A) as well as 6 days (Fig. 2B) by the SRB assay and flow cytometry. The results obtained from both the assays were almost identical. HSVtk conferred sensitivity to GCV to ~81 per cent PLTK41.1 cells when treated with 3 [micro]g/ml GCV for 3 days, while the cell kill was enhanced to 91 per cent when PLTK41.1 cells were incubated for 6 days at same concentration (Fig. 2B). Both the assays showed a GCV concentration dependent cell kill when incubated for 3 and 6 days separately.

In vivo combination gene therapy using IL-2 and HSVtk/GCV: The in vivo efficacy of PLTK47.1 along with GCV alone and in combination with IL-2 was evaluated in the HNSCC xenograft tumour model. Circulating levels of IL-2 have been reported earlier to be detectable by ELISA for 15 days after the injection reaching a peak on day 39. The treatment protocol (Fig. 3A) was started when the tumours reached ~5-1 mm in size. The ratio of tumour size before and at the end of experiment was compared with respective control groups (Fig. 3B). IL-2 receiving animals showed statistically significant reduction in the tumour ratio when compared to empty vector control (P<0.05). The HSVtk/ GCV group also showed effective tumour regression when compared to VPC alone or GCV alone (P<0.001) controls. The combination treatment group also showed significant tumour regression when compared to the control groups (P<0.05) but failed to show statistically significant regression when compared to suicide gene therapy alone or IL-2 alone (Fig. 3B).



Analysis of immune cells by flow cytometry: In order to study the effect of IL-2 DNA injection, the changes in immune cells were monitored by flow cytometry. Since the model used was a T cell deficient xenograft tumour model, the changes in the antigen presenting cells viz., the natural killer (NK) cells ([CD49b.sup.+], DX5) and dendritic cells (DC, [CD11c.sup.+]) were monitored in the splenic cell suspension (Fig. 4). Both the NK and DC cell numbers in splenic cell suspension were significantly higher in mice treated with IL-2 alone as compared to empty vector control (P<0.001). The suicide therapy alone or the combination therapy also induced both these cell types but the differences were not significant. The significant difference in the NK and DC populations was also reflected in slow tumour growth in the animals receiving IL-2 alone (Fig. 3).

Cell death in tumour tissue post-therapy: Histopathology of tumour tissues from animals treated with combination therapy showed increased differentiation and necrosis as compared to the various control groups (Fig. 5A). In order to elucidate the mechanism involved in tumour cell death, immunostaining for active caspase-3 was carried out on tumour tissue sections. The pathway leading to cell death was found to be caspase-3 dependent in our experimental model system for combination therapy as well as for both the individual therapies (Fig. 5B). The extent of cell death was also assessed by fluorometric TUNEL assay. Higher cell death was observed in tumour tissue sections from animals treated with combination therapy as compared to suicide therapy alone or IL-2 alone treated animal groups (Fig. 5C).




The prodrug activation strategy for cancer has been gaining a lot of interest in the last few years. HSVtk, the enzyme used in prodrug activation, has been introduced into the tumour via retroviral vectors, by directly injecting VPC into the tumours. A number of clinical trials using suicide gene therapy have been undertaken using either retroviral VPC (15) or adenoviruses (16) or other gene delivery methods (17). In one of the clinical trials, patients with recurrent glioblastoma were treated by direct injection of HSV-1 tk retroviral-vector producing cells into the surgical cavity margins after tumour debulking (18). Overall median survival was 206 days, with 25 per cent of the patients surviving longer than 12 months, and 1 of the patients was free of detectable recurrence 2.8 yr after treatment (18). HSVtk-GCV treatment has proved to be non-toxic and safe in patients receiving intra-cerebral injections with no evidence of systemic spread of the retroviral vector (19). However, the HSVtk/GCV strategy alone is not sufficient to eradicate the tumour hence a combination with pleotropic cytokine like IL-2 was attempted in this study. HSVtk/GCV strategy is also shown to be successful when tried with other treatment modalities like irradiation (20,21), chemotherapy (22) and surgery (23).

In order to improve tumour cell kill, prodrug activation and immunotherapy have been used in gene therapy clinical trials (24-27). Simultaneous expression of HSVtk and IL-2 was shown to potentiate the induction of an immune response (28). In an experimental metastatic breast cancer model, a combination of HSVtk with IL-2 and granulocyte macrophage - colony stimulating factor (GM-CSF) resulted in a substantial reduction of tumour size as well as a significant reduction in lung metastasis (29). Similar results of augmentation of the effect of HSVtk therapy by IL-2 and GM-CSF together have been reported (30). In a study of 12 patients with recurrent glioblastoma multiforme the therapeutic potential of combining HSVtk and IL-2 using a bicistronic Moloney vector, was reported using retroviral VPC (25,31). However, with a bicistronic promoter the IL-2 gene would be lost once the tumour cell is killed.




In the present study we used a combination strategy with prodrug activation, for which we have constructed a retrovirus producing cell line pLTK41.1 carrying HSVtk, and immunotherapy using plasmid IL-2 DNA. Rather than using a bicistronic vector carrying both HSVtk and IL-2 genes, we have delivered HSVtk via retroviral VPC and IL-2 via direct intramuscular plasmid injection so that the IL-2 is in circulation longer (>10 days as reported earlier) (9). Our experiments failed to show a significant difference between combination therapy and other individual therapies, which may solely be attributed the lack of T cells in our experimental model. However, generation of memory T cells in immunocompetent individuals could help in a systemic long lived anti-tumour immune response which would prove very powerful for the treatment of metastatic cancers, and also for minimal residual disease.

This is perhaps the first report of combination therapy in a T cell deficient animal model, which puts us at an added advantage to look exclusively at antigen presenting cells (APCs). Our experimental approach provides an opportunity to have insight into the role of IL-2 and HSVtk in exclusively inducing the secondary target immune cells viz., NK, DCs (the primary target being T cells). We report that IL-2 alone significantly induces the proliferation of these secondary targets and restricts the tumour growth in vivo. We presented data from our preclinical studies using the HNSCC xenograft nude mouse model. This study may form the basis for further Phase 1 clinical trials in HNSCC patients.


The work was supported by a grant from the Department of Biotechnology, New Delhi, India (BT/PR/5491/Med/14/640/2004). The first author (AA) was a recipient of a fellowship from Advanced Centre for Treatment, Research & Education in Cancer, Mumbai. The authors thank Dr K. Ghosh, Director, NIIH, Mumbai and the staff members for help in flow cytometry.


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Aditya V. Ambade, Ganesh V. Joshi & Rita Mulherkar

Advanced Center for Treatment Research & Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, India

Received November 12, 2009

Reprint requests: Dr Rita Mulherkar, Advanced Center for Treatment Research & Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 210, India e-mail:
Table. Combination therapy--in vivo studies: Study design

Group NT8e VPC GCV IL-2 Empty Animals
 PLTK41.1 vector per group

I + + + + - 20
II + + + - - 21
III + - - + - 11
IV + + - - - 9
V + - + - - 31
VI + - - - + 28
VII + - - - - 4
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Author:Ambade, Aditya V.; Joshi, Ganesh V.; Mulherkar, Rita
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Oct 1, 2010
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