Dynamic contrast-enhanced and diffusion-weighted MRI for early detection of tumoral changes in single-dose and fractionated radiotherapy: evaluation in a rat rhabdomyosarcoma model.

We aimed to examine different intratumoral changes after single-dose and fractionated radiotherapy, using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE) MRI in a rat rhabdomyosarcoma model. Four WAG/Rij rats with rhabdomyosarcomas in the flanks received single-dose radiotherapy of 8 Gy, and four others underwent fractionated radiotherapy (five times 3 Gy). In rats receiving single-dose radiotherapy, a significant perfusion decrease was found in the first 2 days post-treatment, with slow recuperation afterwards. No substantial diffusion changes could be seen; tumor growth delay was 12 days. The rats undergoing fractionated radiotherapy showed a similar perfusion decrease early after the treatment. However, a very strong increase in apparent diffusion coefficient occurred in the first 10 days; growth delay was 18 days. DW-MRI and DCE-MRI can be used to show early tumoral changes induced by radiotherapy. Single-dose and fractionated radiotherapy induce an immediate perfusion effect, while the latter induces more intratumoral necrosis.

Allogeneic bone marrow transplantation and donor lymphocyte infusion in a mouse model of irradiation-induced myelodysplastic/myeloproliferation syndrome (MD/MPS): evidence for a graft-versus-MD/MPS effect.

The role of graft-versus-malignancy reactivity in the effects of allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion (DLI) for myelodysplastic syndromes is as yet not well established. Clinical data are limited and animal models are scarce. Here, we report on the effects of allogeneic bone marrow transplantation (alloBMT) and DLI in a novel model of irradiation-induced murine myelodysplastic/myeloproliferation syndrome (MD/MPS). Total body irradiation with 8.5 Gy in SJL/J mice gave rise to a lethal wasting syndrome in 60% of mice, characterized by 1 degrees normocellular bone marrow with dysplastic features in erythroid, myeloid and megakaryocytic cell lineages, 2 degrees lymphosplenomegaly with spleens harboring a prominent extramedullary hematopoiesis with erythroid, myeloid and megakaryocytic lineages exhibiting dysplastic features, and foci of dysplastic hematomyelopoiesis in the liver, 3 degrees peripheral thrombocytopenia and 4 degrees evidence of disseminated infection or leukemic transformation in selected animals. This clinicopathological picture was consistent with a murine form of MD/MPS. Syngeneic or allogeneic (BALB/c) T cell-depleted BMT could not prevent the occurrence of lethal MD/MPS. In contrast, DLI at weeks 2-4 after BMT led to restoration of the dysbalanced hematomyelopoiesis. However, severe DLI-induced acute graft-versus-host disease occurred, precluding a survival advantage. We present evidence of the existence of a post-alloBMT DLI-induced graft-versus-MD/MPS effect in murine irradiation-induced MD/MPS.

CTLA-4 blockade in murine bone marrow chimeras induces a host-derived antileukemic effect without graft-versus-host disease.

We studied the effect of CTLA-4 blockade on graft-versus-leukemia and graft-versus-host responses in a mouse model of minor histocompatibility-mismatched bone marrow transplantation. Early CTLA-4 blockade induced acute graft-versus-host disease. Delayed CTLA-4 blockade resulted in a lethal condition with lymphosplenomegaly, but with stable mixed T-cell chimerism, unchanged alloreactive T-cell frequencies and absent anti-host reactivity in vitro. In contrast, multiorgan lymphoproliferative disease with autoimmune hepatitis and circulating anti-DNA auto-antibodies were documented. Splenic lymphocytes exhibited ex vivo spontaneous proliferation and a marked proliferative response against host-type dendritic cells pulsed with syngeneic (host-type) tissue-peptides. Both phenomena were exclusively mediated by host and not donor T cells, supporting an autoimmune pathogenesis. Selectively host-derived T-cell immune reactivity was equally documented against leukemia-peptide-pulsed dendritic cells, and this was paralleled by a strong in vivo antileukemic effect in anti-CTLA-4-treated and subsequently leukemia-challenged chimeras. In conclusion, delayed CTLA-4 blockade induced a host-derived antileukemic effect, occurring in the context of an autoimmune syndrome and strictly separated from graft-versus-host disease. Both antileukemic and autoimmune responses depended on the allogeneic component, as neither effect was seen after syngeneic bone marrow transplantation. Our findings reveal the potential of using CTLA-4 blockade to establish antileukemic effects after allogeneic hematopoietic stem cell transplantation, provided autoimmunity can be controlled.

Efficacy of gene therapy-delivered cytosine deaminase is determined by enzymatic activity but not expression.

The potential utility of tumour-selective 5-fluorouracil treatment using attenuated Salmonella serovar typhimurium recombinant for cytosine deaminase (TAPET-CD) has been documented in experimental settings. The present data demonstrate that in vivo (19)F-magnetic resonance spectroscopy measurements allow the outcome prediction of this prokaryotic-based therapy, demonstrating the necessity of non-invasive real-time imaging techniques for treatment monitoring.

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Evaluation of the larynx for tumour recurrence by diffusion-weighted MRI after radiotherapy: initial experience in four cases.

Radiotherapy-induced changes in the soft tissues of the neck hamper the early detection of persistent or recurrent tumour by clinical examination and imaging procedures. Diffusion-weighted (DW) MRI is a non-invasive technique capable of probing tissue properties by measuring the movement of water. The purpose of the ongoing study is to examine the usefulness of DW-MRI for differentiation of persistent or recurrent tumour from post-radiotherapeutic sequelae or complications. Four patients, suspected of tumour recurrence after radiotherapy for laryngeal squamous cell carcinoma, were examined using a DW-MRI sequence on a clinical 1.5 T MR system prior to surgery. In two patients, the DW-MRI images showed an asymmetric hyperintense lesion on b1000 images with low apparent diffusion coefficient (ADC)-value, compatible with tumour on histopathology. All surrounding tissue presented high ADC values and absent signal on the b1000 images, histopathologically correlating to post-radiotherapeutic changes. The images of the third and fourth patient showed absent or minimal symmetric hyperintensity of the laryngeal soft tissues on the b1000 images and high ADC-values. In these cases, the histopathological diagnosis of radionecrosis was made and no tumour was found. In all four cases, differentiation of tumoral tissue from radiotherapy-induced tissue alterations was possible with DW-MRI.

In vivo antitumour effect of combretastatin A-4 phosphate added to fractionated irradiation.

BACKGROUND: The study aimed at evaluating the potential benefit from a combination of fractionated ionising radiation with the vascular-targeting compound combretastatin A-4 phosphate (CA-4-P). MATERIALS AND METHODS: Syngenic rat rhabdomyosarcoma (R1), growing subcutaneously, was treated at 2 different sizes: either small (2 +/- 0.5 cm3) or large (10.94 +/- 0.6 cm3). Localised fractionated irradiation of the tumours (5 x 3 Gy) in 5 days was followed 1 day later by an intraperitoneal CA-4-P treatment (25 mglkg). RESULTS: The combined treatment of only large tumours resulted in a small additional growth delay when compared with radiotherapy only. CONCLUSION: The present data indicate a size-dependent increase in tumour growth delay from combining fractionated irradiation with CA-4-P.

Evaluation of hypoxia in an experimental rat tumour model by [(18)F]fluoromisonidazole PET and immunohistochemistry.

This study aimed to evaluate tumour hypoxia by comparing [(18)F]Fluoromisonidazole uptake measured using positron emission tomography ([(18)F]FMISO-PET) with immunohistochemical (IHC) staining techniques. Syngeneic rhabdomyosarcoma (R1) tumour pieces were transplanted subcutaneously in the flanks of WAG/Rij rats. Tumours were analysed at volumes between 0.9 and 7.3 cm(3). Hypoxic volumes were defined using a 3D region of interest on 2 h postinjection [(18)F]FMISO-PET images, applying different thresholds (1.2-3.0). Monoclonal antibodies to pimonidazole (PIMO) and carbonic anhydrase IX (CA IX), exogenous and endogenous markers of hypoxia, respectively, were used for IHC staining. Marker-positive fractions were microscopically measured for each tumour, and hypoxic volumes were calculated. A heterogeneous distribution of hypoxia was observed both with histology and [(18)F]FMISO autoradiography. A statistically significant correlation (P<0.05) was obtained between the hypoxic volumes defined with [(18)F]FMISO-PET and the volumes derived from the PIMO-stained tumour sections (r=0.9066; P=0.0001), regardless of the selected threshold between 1.4 and 2.2. A similar observation was made with the CA IX staining (r=0.8636; P=0.0006). The relationship found between [(18)F]FMISO-PET and PIMO- and additionally CA IX-derived hypoxic volumes in rat rhabdomyosarcomas indicates the value of the noninvasive imaging method to measure hypoxia in whole tumours.

Combretastatin A-4 phosphate enhances CPT-11 activity independently of the administration sequence.

We evaluated the effect of different intervals and sequences of the vascular targeting agent combretastatin A-4 disodium phosphate (CA4DP) and CPT-11 administration on tumour growth delay and intratumoral uptake of CPT-11 using a syngeneic rhabdomyosarcoma tumour model. Irrespective of the administration sequence, the combination of CA4DP and CPT-11 significantly increases tumour growth delay in comparison with both drugs alone (P<0.001). Intratumoral CPT-11 concentration generally decreased (up to 5-fold) in the combination groups, while SN-38, the active metabolite of CPT-11, increased up to 9-fold. However, the increased amount of intratumoral SN-38 trapping after CA4DP injection did not correlate with the observed tumour growth delay. In conclusion, CA4DP significantly enhances the antitumour effect of CPT-11, which is not greatly influenced by the administration sequence, and which lacks a correlation with the intratumoral trapping of CPT-11 or SN-38. Mechanisms other than trapping are likely to be involved in the chemosensitising capacity of CA4DP.

Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours.

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase.

Effect of antivascular endothelial growth factor treatment on the intratumoral uptake of CPT-11.

Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an in vivo study to investigate the relation between VEGF-blocking therapy, tumoral blood vessels, and intratumoral uptake of anticancer drugs. Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.

Vascular targeting: a potential additional anti-cancer treatment.

Our preclinical in vivo investigations were aimed to evaluate the potential of selectively targeting the tumour vasculature as an additional anti-cancer strategy. Using a clinical angiography method and the tumour growth delay assay, the efficacy of the vascular targeting compound combretastatin A-4 phosphate was demonstrated in rat rhabdomyosarcomas: specifically, an inverse efficacy as compared to radio- or chemotherapy was measured when comparing small and large tumours. The combination of this vascular targeting compound with ionising radiation indicated, depending on the timing and the sequence, a potential benefit. Within the limits of our experiments, no significant increase in tumour growth delay was measured when TNP-470 anti-angiogenesis was given after the combretastatin A-4 phosphate treatment. The use of the vascular targeting agent did advance the in vivo application of a non-apathogenic anaerobe Clostridium transfer system of therapeutic proteins. A strong improvement of the selective expression of cytosine deaminase in the tumour microenvironment was observed, even with very small tumours. In summary, the present preclinical results demonstrate several advantages from the introduction of vascular targeting next to classical and novel anti-cancer therapies.

Tumor-specific gene delivery using genetically engineered bacteria.

The loco-regional control of cancer remains a major contributor to the treatment outcome for many cancer patients prescribed conventional radiotherapy or chemotherapy. Failure of treatment coupled with the realisation that cancer is essentially a genetic disease has led to development of many clinical protocols based on gene therapy. In this review, we will describe an alternative gene delivery system based on the use of non-pathogenic bacteria. Tumor regressions have been reported long ago in patients with bacterially infected tumors, suggesting that bacteria could target tumors and have local anti-tumor effects. The basis of this phenomenon is attributable to the unique properties of the tumor micro-environment. The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localise within and cause cell lysis of experimental animal tumors. One of the most important strains in that context is Clostridium. Other bacteria have also been implicated in experimental anti-cancer settings. Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising. We will discuss the potential advantages and the pitfalls of this alternative delivery approach. We will emphasize the importance of hypoxia in solid tumors and discuss the potential of radiation-inducible promoters and combined treatment modalities, involving vascular targeting and radiotherapy. We believe that this approach will act in a complementary way to current radiotherapy and chemotherapy treatments of solid tumors.

Clostridium spores for tumor-specific drug delivery.

Insufficient blood supply of rapidly growing tumors leads to the presence of hypoxia, a well-known feature in solid tumors. Hypoxia is known to decrease the efficiency of currently used anti-cancer modalities like surgery, chemotherapy and radiotherapy. Therefore, hypoxia seems to be a major limitation in current anti-cancer therapy. The use of non-pathogenic clostridia to deliver toxic agents to the tumor cells takes advantage of this unique physiology. These strictly anaerobic, Gram-positive, spore-forming bacteria give, after systemic administration, a selective colonization of hypoxic/necrotic areas within the tumor. Moreover, they can be genetically modified to secrete therapeutic proteins like cytosine deaminase or tumor necrosis factor-alpha. The specificity of this protein delivery system can be further increased when expression is controlled by the use of a radio-inducible promoter, leading to increased spatial and temporal regulation of protein expression. This approach of bacterial vector systems to target protein expression to the tumor can be considered very safe since bacteria can be eliminated at any moment by the addition of proper antibiotics. The Clostridium-based delivery system thus presents an alternative therapeutic modality to deliver anti-tumor agents specifically to the tumor site. This high selectivity offers a major advantage in comparison with the classical gene therapy systems.

Hypoxia as a target for combined modality treatments.

There is overwhelming evidence that solid human tumours grow within a unique micro-environment. This environment is characterised by an abnormal vasculature, which leads to an insufficient supply of oxygen and nutrients to the tumour cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxia has been shown to be an independent prognostic indicator of poor outcome in prostate, head and neck and cervical cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angiogenesis and metastasis. Several years ago, scientists realised that the unique properties within the tumour micro-environment could provide the basis for tumour-specific therapies. Efforts that are underway to develop therapies that exploit the tumour micro-environment can be categorised into three groups. The first includes agents that exploit the environmental changes that occur within the micro-environment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxia-specific gene delivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promoter elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the various properties ascribed to tumour hypoxia and the current efforts underway to exploit hypoxia for improving cancer treatment will be discussed.

BOLD contrast fMRI of whole rodent tumour during air or carbogen breathing using echo-planar imaging at 1.5 T.

The aim of this study was to evaluate the feasibility of functional MR imaging (fMRI) at 1.5 T, exploiting blood oxygenation level-dependent (BOLD) contrast, for detecting changes in whole-tumour oxygenation induced by carbogen (5% CO2+95% O2) inhalation of the host. Adult WAG/Rij rats with rhabdomyosarcomas growing subcutaneously in the lower flank were imaged when tumours reached sizes between 1 and 11 cm3 (n=12). Air and carbogen were alternatively supplied at 2 l/min using a snout mask. Imaging was done on a 1.5-T MR scanner using a T2*-weighted gradient-echo, echo-planar imaging (GE-EPI) sequence. Analysis of the whole-tumour EPI images was based on statistical parametric maps. Voxels with and without signal intensity changes (SIC) were recorded. Significance thresholds were set at p<0.05, corrected for multiple comparisons. In continuous air breathing condition, 3 of 12 tumours showed significant negative SIC and 1 tumour had a clear-cut positive SIC. The remaining tumours showed very little or no change. When switching to carbogen breathing, the SIC were significantly positive in 10 of 12 tumours. Negative SIC were present in 4 tumours, of which three were simultaneously characterised by positive SIC. The overall analysis indicated that 6 of the 12 tumours could be considered as strong positive responders to carbogen. Our research demonstrates the applicability of fMRI GE-EPI at 1.5 T to study whole-tumour oxygenation non-invasively. The observed negative SIC during air condition may reflect the presence of transient hypoxia during these measurements. Selection of tumours on the basis of their individual response to carbogen is possible, indicating a role of such non-invasive measurements for using tailor-made treatments.

Insertion or deletion of the Cheo box modifies radiation inducibility of Clostridium promoters.

Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter. In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.

Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation.

One of the major problems with gene therapy today is the lack of tumour specificity. The use of anaerobic apathogenic clostridia as a gene transfer system can target anoxic areas within the tumour. These bacteria can be genetically modified to express therapeutic proteins such as TNFalpha locally in the tumour. As shown in our results, ionising irradiation can be used in clostridia to activate genes encoding cytotoxic agents under control of a radiation-inducible promoter. A 44% significant increase (P < 0.05) in TNFalpha secretion was seen 3.5 h after a single dose of 2 Gy. A second dose of 2 Gy was also capable of repeating gene activation and gave a significant increase of TNFalpha production of 42% (P < 0.05). These results provide evidence that spatial and temporal control of gene expression can be achieved using a radio-inducible promoter. Repetitive gene activation was feasible with a second dose of 2 Gy, indicating that fractionated radiotherapy could lead to repeated gene induction resulting in prolonged and enhanced protein expression. Gene targeting by ionising radiation could thus provide a new means of increasing the therapeutic ratio in cancer treatment.

Increasing specificity of anti-tumor therapy: cytotoxic protein delivery by non-pathogenic clostridia under regulation of radio-induced promoters.

BACKGROUND: Pathogenic clostridia, genetically engineered to express therapeutic genes, will specifically target hypoxic regions in tumors. This specificity can be further improved if expression of these genes is controlled by a radio-induced promoter, leading to spatial and temporal control of gene expression. MATERIALS AND METHODS: Following administration of Clostridium spores to tumor bearing rats, normal tissue and tumoral specimens were compared for colonization. Clostridium was genetically modified to express tumor necrosis factor a or cytosine deaminase. Expression of these proteins was assayed. Northern blot hybridizations were used to detect genes which are radio-induced. RESULTS: Clostridium gave a selective colonization of tumors. The recombinant clostridia expressed in vitro and in vivo TNF alpha and cytosine deaminase. Clostridial SOS-repair genes were induced at a dose of 2 Gy. CONCLUSIONS: Pathogenic Clostridium can be used for tumor specific delivery of therapeutic genes. The specificity can be improved via radio-induced promoters. Overall, this new gene delivery system can lead to an increase of the therapeutic ratio in cancer treatment.

Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum.

The presence of severe hypoxia and necrosis in solid tumors offers the potential to apply an anaerobic bacterial enzyme/prodrug approach in cancer treatment. In this context the apathogenic C. acetobutylicum was genetically engineered to express and secrete E. coli cytosine deaminase (CDase). Considerable levels of functional cytosine deaminase were detected in lysates and supernatants of recombinant C acetobutylicum cultures. After administration of the recombinant Clostridium to rhabdomyosarcoma bearing rats used as a model, cytosine deaminase could be detected at the tumor site. Moreover, following administration of the vascular targeting agent combretastatin A-4 phosphate significantly increased levels of cytosine deaminase were detected at the tumor site as a consequence of enlarged tumor necrosis and subsequently improved growth of C. acetobutylicum. The results provide evidence for the potential application of Clostrisdium-based therapeutic protein transfer to tumors in anticancer therapy.