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. 2010 Nov 13;60(2):273–281. doi: 10.1007/s00262-010-0941-y

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery

Gregory Driessens 1,✉, Lise Nuttin 1, Alain Gras 1, Julie Maetens 1, Stephane Mievis 1, Marylène Schoore 1, Thierry Velu 2, Liliane Tenenbaum 1, Véronique Préat 3, Catherine Bruyns 1
PMCID: PMC11029469  PMID: 21076828

Abstract

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an “all in vivo” strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful “all in vivo” vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-010-0941-y) contains supplementary material, which is available to authorized users.

Keywords: Cancer vaccines, GM-CSF, Dendritic cells, Irradiation, Adeno-associated virus

Introduction

New therapeutic strategies against cancer aim to combine different approaches able to induce tumor-specific effector and memory T cell responses that might control tumor outgrowth. Among these, combining classical treatments like radiotherapy or chemotherapy with immunotherapy appears very promising [1]. In our laboratory, we have previously developed and validated, in two different murine tumor models, a successful therapeutic strategy associating dendritic cell (DC) and GM-CSF secreting tumor cell vaccines [2, 3]. We have also shown that the main difference between cured and uncured animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlated with tumor rejection [4]. Nevertheless, engineering individual GM-CSF secreting tumor cell lines could represent a tedious step limiting the application of such vaccinations for cancer patients’ treatment. We therefore aimed to develop an “all in vivo” strategy of combined vaccines using the in vivo-treated tumor as a source of tumor antigens for DC vaccines injected locally and an exogenous source of GM-CSF.

DC are the most potent antigen-presenting cells for stimulation of primary immune responses, representing thereby an attractive adjuvant for cancer immunotherapy [5]. Indeed, many animal studies and human clinical trials reported the induction of tumor-specific CD8+ and CD4+ T cell responses by DC vaccines [6]. DC are sufficient for, and probably exclusive in mediating MHC-class I cross-presentation of exogenous tumor antigens to CD8+ T cells [7, 8]. In fact, an optimal cross-presentation process requires the phagocytosis of apoptotic tumor cells by immature DC, leading to MHC I and II peptide presentation, and a DC maturation signal provided by the release of inflammatory stimuli by surrounding necrotic cells [9]. In the absence of danger signals, the DC remain immature and induce T cell tolerance rather than T cell activation [10].

Radiation therapy is one of the primary treatments for cancer along with surgery and chemotherapy. In vivo local γ-irradiation induces the apoptosis [11] and necrosis [12] of tumor cells, affording two types of cell death needed for an efficient DC-mediated induction of strong immune responses [9]. The association of local radiotherapy and DC vaccination was tested in different murine tumor models, resulting in increased antitumor immune responses and longer mice survival than separate treatments [13, 14].

DC vaccines for cancer certainly hold promises but definitely need improvements, especially for enhancing tumor-specific responses able to eradicate preexisting tumors. Granulocyte–macrophage colony-stimulating factor (GM-CSF) has become an attractive vaccine adjuvant able to enhance antitumor immune response by inducing proliferation, maturation, and migration of DC as well as expansion and differentiation of B and T lymphocytes [15]. Numerous preclinical and clinical studies have demonstrated the dose-dependent immunostimulating effects of GM-CSF in a variety of cancer vaccine approaches [16]. Such tumor cells genetically engineered to express GM-CSF induce tumor-specific immunity through the recruitment of DC to the site of inoculation with subsequent increased presentation of tumor antigens [17]. Different methods have been tested to deliver the cytokine directly in vivo such as the intradermal administration of recombinant GM-CSF or GM-CSF DNA [18, 19], the injection of GM-CSF-encapsulated biodegradable microspheres [20], or the use of recombinant adenoviral vectors [21]. Adeno-associated-virus (AAV)-based vectors represent another class of well-developed vectors for gene delivery not encoding any viral gene and being less immunogenic than adenoviral vectors [22]. AAV vectors have already shown their potential for in vivo antitumor therapies [23]. To avoid the problem of virus dissemination from the tumor during and after the infusion, a biocompatible thermoreversible gel (Pluronic F127-Poloxamer 407) was used to encapsulate the vectors, resulting in increased transgene expression [24, 25].

In the present study, by developing a successful “all in vivo” vaccination strategy combining tumor radiotherapy with DC vaccines and exogenous GM-CSF supply we have generated a protocol easily translatable to clinic and accessible to a much bigger number of cancer patients.

Materials and methods

Animals

Male inbred Fischer 344 rats, purchased from Charles River Laboratories (l’Arbresle, France) were housed at the Animal Facility of Université Libre de Bruxelles, Faculty of Medicine, in accordance with European Community guidelines and used at the age of 10–12 weeks.

Cell lines

The 9L and 9LmGM-CSF subline previously generated in our laboratory [2] were cultured in complete medium at 37°C in a humidified incubator with 5% CO2.

Generation of dendritic cells

Rat immature DC were generated as described previously [2]. Briefly, bone marrow cell suspensions were first depleted in FcR positive and plastic adherent cells and then cultured in presence of 0.5 ng/mL mGM-CSF for 8 days in so-called DC medium, i.e., RPMI 1640 supplemented with 25 mM HEPES, 2 mM l-glutamine, 10% FCS, 1% sodium-pyruvate, 5 × 10−5 M 2-mercaptoethanol, 50 μg/ml gentamicin.

In vivo induction and detection of tumor apoptosis

Rats were anesthetized and the tumor site was positioned in front of a hole made in a lead pipe protecting the rest of the body. Tumors from irradiated and control rats were excised 24 h post treatment and analyzed for apoptosis induction by two independent tests (see on-line supplementary methods and Fig. 1).

Tumor transduction with recombinant AAV2/1-mGM-CSF vectors

Construction of the AAV2/1-mGM-CSF vectors

pTR-mGM-CSF plasmid containing an optimized gene coding for murine GM-CSF under the control of the CMV promoter was constructed by inserting the CMV-mGM-CSF cassette between the AAV2 terminal repeats in pTR-UF2. The CMV-mGM-CSF fragment (1.4 kb) was obtained from p-orMuGM-CSF plasmid (Geneart, Germany) after SpeI-PvuII digestion. This fragment was inserted at the SmaI-XbaI sites of pUC19 to generate pUC-GM-CSF plasmid, which was used in a second step to generate the pTR-mGM-CSF plasmid. CMV-mGM-CSF fragment was obtained by Eco RI-SalI digestion compatible with same restriction sites in pTR-UF2 plasmid that contains AAV2 terminal repeats and bovine growth hormone (bGH) poly(A) signal. pTR-mGM-CSF plasmid was provided to the “Laboratoire de Thérapie Génique” (INSERM U649, Nantes, France) which produced the AAV2/1-mGM-CSF vectors. Viral stock: 2.8 × 1010 Vg/ml (Vg: viral genomes).

In vitro and in vivo 9L transduction with recombinant AAV vectors

For in vitro transduction assays, 9L cells were plated and transduced the next day with AAV2/1-mGM-CSF or GFP vectors at different multiplicities of infection (MOI) at 37°C. After 2 h of incubation, complete medium was added. At different time points post-transduction, the cells were analyzed by flow cytometry for eGFP expression or by ELISA for GM-CSF production (e-Biosciences).

For in vivo transduction assays, 105 9L cells were implanted in the flanks of Fischer 344 rats. When a tumor was palpable, the rats were anesthetized, shaved, and 2 × 107 viral particles AAV2/1-mGM-CSF (25 μl) were injected within the tumor. At several time points following virus inoculation, rats were killed and tumors were excised and weighted before being crushed mechanically at 4°C in PBS supplemented with a cocktail of protease inhibitors (Roche Diagnostics). Tumor lysates were centrifuged at 13,000 rpm/min for 5 min and supernatants were measured by ELISA for GM-CSF content (E-Biosciences).

Use of Poloxamer 407 to protect AAV2/1-mGM-CSF vectors or recombinant GM-CSF

Poloxamer 407 (Pluronic F-127) is a non-toxic polymer of polyoxyethylene and polyoxypropylene. Hydrogels show thermoreversible properties, being liquid at temperatures <15°C and solid at temperatures >15°C. A 12.5% solution of poloxamer was prepared with 375 mg of powder in 3 mL of PBS. For homogenization the solution was shaken during 3 h at 4°C. Mixtures of 10 μL poloxamer solution and 10 μL AAV2/1-mGM-CSF vectors or recombinant mGM-CSF were prepared just before the intratumoral injection and kept at 4°C all along the procedure.

In vivo combined therapeutic vaccinations

At day 0, 105 9L cells were inoculated s.c. in one flank of Fischer 344 rats. As shown in Fig. 1, the tumor (site) was locally irradiated (10 Gy, 137Cs irradiator) on days 3, 10, and 17 while total rat body was shielded in a specially designed lead apparatus. An exogenous source of mGM-CSF was provided intratumorally on same days consisting in injections of recombinant mGM-CSF alone or in poloxamer 407 (Pluronic F-127), or continuously delivered by osmotic pumps (Alzet, model 2004) or by injections of AAV2/1-mGM-CSF vectors. Finally, 3 × 106 DC were injected on days 4, 11, and 18 at the tumor site. Tumor size was measured weekly and rats were killed when tumor size was greater than 9 cm2. Several independent experiments were performed with reproducible outcomes, using individual treatment groups of 4–6 rats. Rats that were cured by such vaccinations were re-challenged after 2 or 4 months with 105 9L tumor cells s.c. in one flank.

Fig. 1.

Fig. 1

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The “all in vivo” strategy of vaccination. On day 0 Fischer 344 rats were injected subcutaneously with 105 9L tumor cells in one flank. On days 3, 10, and 17 the tumor site was locally γ-irradiated (10 Gy) before being injected with an exogenous source of GM-CSF (AAV2/1-mGM-CSF vectors or recombinant protein complexed or not with poloxamer 407). On days 4, 11, and 18 the rats were vaccinated with 3 × 106 DC injected at the tumor site

Statistics

Statistical analysis of means comparison was done using the unpaired t test with Welch correction if needed. Survival data were compared using the log-rank test.

Results

Vaccinations combining DC with local in vivo irradiation and “classical” GM-CSF delivery show weak therapeutic effect

With the aim to simplify the successful model of vaccination we previously described, combining three essential elements we previously identified as strictly required to get a high therapeutic effect, [i.e., injections of DC and apoptotic tumor cells in vitro engineered to secrete GM-CSF (2)], we tested the association of DC vaccines with an in vivo local tumor irradiation and GM-CSF delivery, as illustrated in Fig. 1. The induction of tumor apoptosis by a local irradiation at 10 or 20 Gy was first monitored (see on-line supplementary methods and Fig. 1). Regarding the strong inhibition of 9L tumor growth with the higher dose, 10 Gy was chosen for further experiments.

Figure 2 shows that the rats which underwent tumor irradiation alone (n = 6) had a prolonged survival when compared with control non-treated animals, one animal being even completely cured. Combining tumor irradiation with DC vaccines (n = 10) did not significantly increase the survival. Moreover, providing exogenous GM-CSF, locally at tumor site, by injecting repeatedly the recombinant cytokine (n = 11) or by implanting osmotic pumps delivering GM-CSF continuously for 28 days (n = 9) had no additional therapeutic effect as compared with irradiation alone or the association of irradiation and DC vaccines. None of these conditions reached the 60% survival score we obtained in previous studies with DC vaccines in vitro mixed with irradiated GM-CSF secreting 9L cells (Fig. 2, inserted graph).

Fig. 2.

Fig. 2

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The therapeutic efficacy of DC vaccines combined with in vivo irradiation and delivery of recombinant GM-CSF is weak. Tumor inoculation and rat’s vaccination were performed as described in Fig. 1. Exogenous GM-CSF was provided next to the tumor site either by osmotic pumps delivering 50 ng/day, continuously for 28 days or by injecting 650 ng of recombinant cytokine on days 3, 10, and 17. Survival data are pooled from three independent experiments. **p < 0.01 versus CTRL. By comparison, the therapeutic efficacy of in vitro prepared DC vaccines combined with irradiated 9LmGM-CSF tumor cells is illustrated in the inserted graph

AAV serotype 1 has the highest efficiency to transduce 9L cells

From our previous results we suspected that, to be efficient in vivo, the GM-CSF had to be directly secreted by the tumor cells. We therefore decided to test the efficiency of AAV vectors to transduce 9L tumors in vivo. In a first step, we compared in vitro, at different multiplicities of infection (MOI), the capacity of different AAV serotypes (1, 2 and 5) containing the eGFP marker gene to transduce 9L cells. Figure 3a and b illustrate representative flow cytometry data showing that the AAV2/1-eGFP vectors were the most efficient for transducing 9L cells. Indeed, both the proportion of eGFP+ cells and the Mean Fluorescence Intensity (MFI) were the highest with AAV serotype 1 as compared with other serotypes tested. Moreover, we have also observed that AAV1 vectors were more efficient than adenoviral vectors to transduce 9L cells in vitro and that the irradiation of 9L cells before or after their transduction by AAV2/1-eGFP vectors had no modulatory effect on the GFP expression (data not shown).

Fig. 3.

Fig. 3

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AAV1 vectors efficiently transduce 9L cells. 9L cells were transduced in vitro with different AAV serotypes coding for the GFP. 48 h post transduction, cells were analyzed by flow cytometry for the proportion of GFP positive cells (a) and the intensity of GFP expression, MFI (b). Data are from one representative experiment out of 3. 9L cells were transduced in vitro and in vivo with an AAV1 coding for the mGM-CSF protein. c Culture supernatants of transduced cells were analyzed by ELISA, 24 h post-transduction, for the secretion of mGM-CSF by comparison with 9L and 9LmGM-CSF cells. *p < 0.05; **p < 0.01; ***p < 0.001. d Palpable 9L tumors were transduced in vivo by intratumoral injection of AAV2/1-mGM-CSF vectors. 24 h and 4 days post-transduction, the infected tumors were removed, crushed, and tumor lysates were analyzed for GM-CSF content. ***p < 0.001 versus 9L tumors

In a second step, we constructed the pTR-mGM-CSF plasmid and tested the AAV2/1-mGM-CSF vectors produced by the “Laboratoire de Thérapie Génique” (Nantes, France) for 9L transduction. ELISA assay on supernatants from in vitro transduced 9L cells, 24 h after infection, showed a dose-dependent increase in GM-CSF secretion which, at high MOI, was equal to the secretion by the 9LmGM-CSF cell line (Fig. 3c). For the in vivo assay, palpable 9L tumors were injected in vivo with AAV2/1-mGM-CSF vectors, and the GM-CSF content within tumor lysates was measured 24 h and 4 days after infection. Data illustrated in Fig. 3d confirm the in vivo efficacy of AAV2/1-mGM-CSF vectors to transduce efficiently 9L tumors.

The in vivo intratumoral delivery of GM-CSF with AAV1 vectors highly improves the therapeutic efficacy of DC vaccines combined with in vivo tumor irradiation

On the basis of previous results showing a good efficacy of AAV2/1-mGM-CSF vectors to transduce 9L tumor cells in vitro and in vivo, we tested these vectors within the same “all in vivo” combined vaccination protocol priorly designed (Figs. 1, 2). AAV1 vectors coding for the GFP protein or an irrelevant protein (GDNF) were used as negative controls. In contrast to the different unsuccessful methods we previously tested for an in vivo exogenous delivery of GM-CSF, the intratumoral injection of AAV2/1-mGM-CSF vectors allowed to cure 60% of the rats from a pre-implanted 9L tumor (Fig. 4). Moreover, the rats treated by DC vaccines combined with in vivo local tumor irradiation and transduction with AAV2/1-mGM-CSF vectors showed a statistical increase in survival as compared with animals identically treated but receiving AAV2/1-CTRL vectors (p < 0.05) or with animals receiving DC vaccines and AAV2/1-mGM-CSF vectors but whose tumors were not irradiated (p < 0.001).

Fig. 4.

Fig. 4

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In vivo tumor transduction with AAV2/1-mGM-CSF vectors highly improves the therapeutic efficacy of combined vaccines. Tumor inoculation and rat’s vaccination were performed as described in Fig. 1. Exogenous GM-CSF was provided after in vivo tumor irradiation by injecting AAV2/1-mGM-CSF vectors at the tumor site. Survival curves represent a pool of 2 (square; diamond) or 3 (circle; triangle) independent experiments. *p < 0.05; ***p < 0.001 versus CTRL or AAV2/1-mGM-CSF + DC (no tumor irradiation); **p < 0.01 versus CTRL

The controlled delivery of high concentration of recombinant GM-CSF in poloxamer 407 has a similar therapeutic efficacy as AAV2/1-mGM-CSF vectors

With the aim to increase the curative efficacy of the AAV2/1-mGM-CSF vectors, we have experienced their controlled release with a thermosensitive polymer, the poloxamer 407. As shown in Fig. 5, this strategy did not increase the rat’s survival as compared with the uncoated AAV2/1-mGM-CSF vectors. But, unexpectedly, the use of poloxamer 407 to deliver high dose of recombinant mGM-CSF allowed getting a notable therapeutic efficacy when combined with DC vaccines and local tumor irradiation. Indeed, almost 60% of such treated rats were completely cured from a pre-implanted tumor, as referred to 25% of the rats with low dose of cytokine. By using a straightforward supply of exogenous GM-CSF, we thus developed a successful “all in vivo” therapeutic vaccination protocol combining tumor radiotherapy with DC vaccines, easily translatable to clinic.

Fig. 5.

Fig. 5

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Controlled release of high concentration of GM-CSF recapitulates the therapeutic efficacy obtained with AAV1-GM-CSF vectors. Tumor inoculation and rat’s vaccination were performed as described in Fig. 1. Following tumor irradiation, the exogenous GM-CSF was delivered at the tumor site by injecting AAV2/1-mGM-CSF vectors or recombinant mGM-CSF, both complexed with poloxamer 407. Survival curves represent a pool of at least two independent experiments. Vaccines of DC combined with local in vivo tumor irradiation and injection of high doses of mGM-CSF (650 ng/injection, diamond) or AAV2/1-mGM-CSF (triangle) in poloxamer show a slight statistical increase in survival as compared with low GM-CSF (125 ng/injection, square) conditions with p = 0.06 and p = 0.03, respectively. There is no significant difference between the injections of AAV2/1-mGM-CSF and high mGM-CSF in poloxamer

Long-term survivor rats are resistant to a rechallenge with 9L cells

Rats that were cured by DC vaccines combined with in vivo tumor irradiation and local injection of AAV2/1-mGM-CSF vectors or recombinant mGM-CSF capped in poloxamer were challenged after 2–4 months with parental 9L tumor cells. Tumor growth was monitored once a week for several weeks, comparatively with same tumor implantation in naïve controls. As shown in Fig. 6, both vaccinated groups were highly protected against the tumor rechallenge, indicating the emergence of anti-tumor memory immune responses in those animals.

Fig. 6.

Fig. 6

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Long-term survivor rats are resistant to a rechallenge with 9L cells. The rats that were cured by DC vaccines combined to in vivo tumor irradiation and local injection of AAV2/1-mGM-CSF vectors (n = 12) or recombinant mGM-CSF in poloxamer (n = 12) were rechallenged after 2–4 months with 105 9L tumor cells, subcutaneously in a flank. Tumor growth was monitored once a week for several weeks, comparatively with same tumor implantation in naïve controls. Data represent the percentages of tumor-bearing rats in each group. ***p < 0.001 versus CTRL

Discussion

The present study demonstrates for the first time the great curative potential of an original “all in vivo” vaccination protocol combining local tumor radiation therapy with DC vaccines and a straightforward supply of GM-CSF. Indeed, by using in vivo tumor irradiation as a source of tumor antigens for DC vaccines injected locally with exogenous supply of recombinant GM-CSF previously coated in a biocompatible thermoreversible hydrogel (poloxamer 407), we were able to cure 60% of the rats with pre-implanted tumors. The therapeutic efficacy of the in vivo vaccines reproduces the high survival score we previously reported with in vitro prepared vaccines associating DC and irradiated GM-CSF secreting 9L cells [2]. In addition, the immune response induced by such vaccinations revealed strong CTL responses against 9L cells in both cured and non-cured animals. The presence of splenic IFN-γ secreting CD8+ and CD4+ T cells was also increased in vaccinated cured rats while non-cured animals presented CD8+ IFN-γ+ T cells but very low levels of CD4+ IFN-γ+ T cells (on-line supplementary methods and Fig. 2), a pattern totally identical to the one described extensively for the in vitro prepared vaccines [2, 4]. Moreover, long-term survivor rats developed anti-tumor memory immune responses. Importantly, this new strategy avoids the need to engineer a GM-CSF secreting tumor cell line or the use of viral vectors which could represent a limiting and tedious step for the application of such vaccinations on cancer patients.

DC are the critical decision-making cells in the immune system [5]. However, despite successful application on animal models, active immunization with ex vivo generated and antigen-pulsed DC have shown limited efficacy in clinical trials arguing for the development of combined strategies that could finally lead to efficient treatments in clinic [26]. In vivo loading DC with irradiated tumor cells avoids the use of defined peptides limited to certain HLA types and to known tumor antigens and allows the presentation of unknown internalized antigens both on MHC I (for cross-priming of naïve CD8+ T cells) and MHC II (for tumor-specific help) molecules. Moreover, DC can receive maturation signals through different pathways including proinflammatory cytokines (TNF-α, Il-6, Il-1β, and PGE2) and products of dying cells, i.e., damage-associated molecular pattern molecules (DAMPs). Our data corroborate the work of den Brok et al. showing that in situ tumor ablation creates an efficient antigen source for the generation of antitumor immunity [27]. Interestingly, even if DNA damage has long been considered to be the main mechanism for tumor reduction observed after irradiation, our results showing a growth delay but no regression of the tumor and the presence of few apoptotic/necrotic tumor cells suggest additional mechanisms of action for the radiotherapy in this model. First, the in vivo irradiation treatment plays an important role in slowing down the growth of the tumor to give more time to the immune system to target it and second, it contributes to the efficacy of the vaccination strategy by providing a source of apoptotic/necrotic tumor antigens that help to induce adaptive immune response. These results are supported by recent reports showing that local tumor radiation could inhibit tumor growth by inducing tumor-specific CTL [28] and that different radiation-induced bystander responses could also contribute to the therapeutic response [29].

Numerous murine models and preliminary clinical trials have revealed the potent ability of GM-CSF to enhance antitumor immunity by a coordinated humoral and CD4+/CD8+ mediated cellular response with a broad cytokine profile [15]. However, if GM-CSF gene-engineered tumor cell vaccines were quite efficient to protect animals from a parental tumor injection, they were poorly effective against established tumors [30]. But, as shown for tumor cells genetically modified to secrete GM-CSF, in vivo intratumoral GM-CSF gene delivery leads to in situ recruitment and activation of DC which, as mature DC, can initiate antitumor immune responses at the tumor site and later by trafficking to the spleen [17, 18, 21]. Since the majority of DC injected as vaccines die early by apoptosis and necrosis, by supplying GM-CSF locally at irradiated tumor site, our strategy might favor the recruitment of endogenous DC and ensure their effective migration to the T cell areas in lymph nodes, thereby facilitating a possible exchange of tumor antigens between the injected and the endogenous DC which is optimal for in vivo expansion of the T cell responses as previously reported [31]. Interestingly, by inducing strong systemic cytotoxic (on-line supplementary Fig. 2) and memory responses, this in vivo vaccination strategy that directly targets tumors in vivo could also be efficient at targeting distant micrometastasis, an hypothesis that could not been tested in the encapsulated 9L tumor model.

Viral vectors, especially adenoviral vectors, are commonly used for intratumoral cytokine gene delivery into solid tumors [21, 32] even in combination with DC vaccines [33]. Adeno-associated-virus (AAV)-based vectors represent another class of well-developed vectors having the advantages to be non-pathogenic, replication defective, and to have a weak immunogenicity and toxicity [22, 23]. Our data showing that among three different serotypes (1,2 and 5), AAV1 vectors were the most efficient to transduce 9L cells/tumors are in agreement with other studies reporting the AAV serotype 1 is more efficient than AAV2 for cancer gene therapy [34, 35]. In the “all in vivo” combined vaccination protocol we have developed, the association of intratumoral injection of AAV2/1-mGM-CSF vectors with irradiation and DC vaccines allowed recapitulating the therapeutic efficacy (60% curative score) obtained with the in vitro prepared vaccines. In agreement with Shi et al. [36] but in contradiction with Kurane et al. [37], we observed that GM-CSF protein expression by tumor cells was a superior antitumor immune stimulant than exogenous GM-CSF delivered in the tumor microenvironment by repeated injections or osmotic mini-pumps [38]. This could be explained in part by the short half-life (4-6 h) of recombinant GM-CSF [39]. To avoid viral vector dissemination decreasing the efficacy of gene therapy, Wang and collaborators have successfully tested a biocompatible polymer (poloxamer 407) which could significantly increase the viscosity of the virus suspension when increasing the temperature from 4 to 37°C [24]. When using same method for intratumoral injection of AAV2/1-mGM-CSF vectors, we did not increase the rat’s survival as compared with uncoated AAV2/1-mGM-CSF vectors. But, unexpectedly, the use of poloxamer 407 to coat recombinant GM-CSF allowed getting a notable dose-related increase in therapeutic efficacy when combined with DC vaccines and local tumor irradiation. Poloxamer 407 was previously shown to enhance adenovirus and lentivirus-mediated gene delivery [40], but combining an AAV vector with pluronic-based gel for in vivo intratumoral delivery has not been reported. Same is true for recombinant GM-CSF or GM-CSF plasmid formulated with poloxamer 407.

The combination of conventional anti-tumor therapy (radiotherapy or chemotherapy) with immunotherapy (DC, cytokines …) has opened new avenues in cancer treatment, showing synergistic effects superior to the treatments taken separately [1, 13, 41]. Interestingly, in an original phase II trial, JJ Mulé reported recently the induction of anti-tumor responses in pancreatic cancer patients who received an adenovector-mediated intratumoral injection of TNF-α with radiotherapy and the intratumoral administration of immature DC [42]. With strong and encouraging results in a therapeutic murine model, our original strategy, combining in vivo tumor radiation therapy with DC vaccines and a straightforward supply of GM-CSF, appears as one of the most promising for being translated to human cancer trials, particularly considering the relative failure of late clinical trials with DC vaccines or GM-CSF-secreting tumor cell vaccines and the need for further clinical development of these vaccines [43, 44].

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 92 kb) (92.3KB, pdf)

Acknowledgments

We thank Catherine Melas for excellent technical assistance and the “Laboratoire de thérapie génique de Nantes” for the production of rAAV vectors”. G. Driessens was fellow of the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture and Télévie. This work was supported by the Belgian State Prime Minister’s office, Service for Science, Technology, and Culture, the Fonds National de la Recherche Scientifique, the Fonds de la Recherche Scientifique Médicale, the Actions de Recherche Concertées of the Communauté Française de Belgique.

Footnotes

G. Driessens and L. Nuttin equally contributed to this work.

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