An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations.

Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumor regression but ultimately fail to elicit cures. As a result, there is an intense interest in how to best combine targeted therapies with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumors with the host immune system are inadequate, in part due to the low tumor mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumor mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumor mutational burden and autochthonous tumors remained refractory to immunotherapy. However, establishing clonal cell lines from these tumors enabled the generation of an immunogenic syngeneic transplantation model of KRAS-mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, antitumor immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C -expressing tumors was markedly potentiated by the adaptive immune system, highlighting the importance of using immunocompetent models for evaluating targeted therapies. Overall, this model provides a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer. SIGNIFICANCE: This study develops a mouse model of immunogenic KRAS-mutant lung cancer to facilitate the investigation of optimal combinations of targeted therapies with immunotherapies.

Early clinical trials of Toca 511 and Toca FC show a promising novel treatment for recurrent malignant glioma.

INTRODUCTION: Glioblastoma and anaplastic astrocytoma are two of the most aggressive and common glioma malignancies in adults. These high-grade gliomas (HGG) universally recur despite aggressive treatment modalities and have a median overall survival (mOS) of approximately 14 months from initial diagnosis. Upon recurrence, there is no standard of care and these patients have a dismal prognosis of around 9 months at time of recurrence. Areas covered: In this article, we assess the newly published phase I data of Toca 511 and Toca FC, a two-drug combination therapy for recurrent HGG (rHGG) tumors, for effectiveness and safety. Expert opinion: These early studies provide very encouraging results for Toca 511 and Toca FC in rHGG. This therapy had a response rate of 11.3% and a mOS of 11.9 months in 56 patients, an improvement compared to historical controls. Furthermore, all responders were complete responses after extended follow-up. The drug is well tolerated for most patients. Responders tended to be young and have high-performance scores prior to beginning therapy, but more studies are necessary to understand the patient profile that receives the most benefit. Randomized-controlled trials are warranted for Toca 511 and Toca FC to confirm drug efficacy.

Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme.

BACKGROUND: Chemotherapy is an effective option to treat recurrent or metastatic cancer but its debilitating side-effects limit the dose and time of exposure. Prodrugs that can be activated locally by an activating enzyme can minimize collateral damage from chemotherapy. We previously demonstrated the efficacy of a poly-L-lysine-based theranostic nanoplex containing bacterial cytosine deaminase (bCD) that locally converted 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil in MDA-MB-231 primary tumor xenografts. MATERIALS AND METHODS: Here we used a more effective variant of bCD to target metastatic red fluorescence protein expressing MDA-MB-435 cells in the lungs. We used an intravenous injection of tumor cells and monitored tumor growth in the lungs for 5 weeks by which time metastatic nodules were detected with optical imaging. The animals were then treated with the bCD-nanoplex and 5-FC. RESULTS: We observed a significant decrease in metastatic burden with a single dose of the enzyme-nanoplex and two consecutive prodrug injections. CONCLUSION: These results are a first step towards the longitudinal evaluation of such a strategy with multiple doses. Additionally, the enzyme can be directly coupled to imaging reporters to time prodrug administration for the detection and treatment of aggressive metastatic cancer.

Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma.

PSMA-specific theranostic nanoplex for combination of TRAIL gene and 5-FC prodrug therapy of prostate cancer.

Metastatic prostate cancer causes significant morbidity and mortality and there is a critical unmet need for effective treatments. We have developed a theranostic nanoplex platform for combined imaging and therapy of prostate cancer. Our prostate-specific membrane antigen (PSMA) targeted nanoplex is designed to deliver plasmid DNA encoding tumor necrosis factor related apoptosis-inducing ligand (TRAIL), together with bacterial cytosine deaminase (bCD) as a prodrug enzyme. Nanoplex specificity was tested using two variants of human PC3 prostate cancer cells in culture and in tumor xenografts, one with high PSMA expression and the other with negligible expression levels. The expression of EGFP-TRAIL was demonstrated by fluorescence optical imaging and real-time PCR. Noninvasive (19)F MR spectroscopy detected the conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by bCD. The combination strategy of TRAIL gene and 5-FC/bCD therapy showed significant inhibition of the growth of prostate cancer cells and tumors. These data demonstrate that the PSMA-specific theranostic nanoplex can deliver gene therapy and prodrug enzyme therapy concurrently for precision medicine in metastatic prostate cancer.

Redesigned Escherichia coli cytosine deaminase: a new facet of suicide gene therapy.

BACKGROUND: The Escherichia coli cytosine deaminase (CD)/5-fluorocytosine (5-FC) approach emerges as a potential aid for suicide gene therapy in the field of modern cancer treatment. However, the poor binding affinity of CD towards 5-FC compared to the natural substrate cytosine limits its application for successful suicide gene therapy. Redesigning a bacterial mutant CD with site-directed mutagenesis showed higher potency compare to wild-type CD (wtCD) in vitro. In the present study, we conducted a comparative analysis of F186W mutant and wtCD in a human lung cancer cell line (A549). METHODS AND RESULTS: A comparative investigation was initiated with cell viability analyses by MTT and trypan blue dye exclusion assays on A549 cells transfected with wtCD and F186W genes. The mode of cell death was confirmed by acridine Orange/ethidium Bromide dual staining. Furthermore, flow cytometric assessments were performed by cell cycle analysis and caspase 3 assay. The experimental results showed a drug dependent decrease in cell viability; interestingly, mutant (F186W) reached IC50 at a much lower concentration of prodrug (5-FC) than wtCD. Cell cycle analysis showed that G1 arrest of a larger population of 5-FC treated F186W transfected cells, in contrast to that of wtCD under similar conditions. The caspase 3 assay revealed progression and execution of apoptosis. CONCLUSIONS: We report a novel bacterial CD mutant that provided a superior alternate to the wtCD suicide gene. The F186W mutant required a much lower dose of 5-FC to reach its IC50 , thus minimizing the systemic side effects of large doses of 5-FC as required for wtCD.

Genetic immunotherapy for hepatocellular carcinoma by endothelial progenitor cells armed with cytosine deaminase.

Endothelial progenitor cells (EPCs) serve as cellular vehicles for targeting cancer cells and are a powerful tool for delivery of therapeutic genes. Cytosine deaminase (CD), a kind of frequent suicide gene which can kill carcinoma cells by converting a non-poisonous pro-drug 5-flucytosine (5-FC) into a poisonous cytotoxic 5-fluorouracil (5-FU). We combined super-paramagnetic iron oxide (SPIO) nanoparticles labeled EPCs with CD gene to treat grafted liver carcinomas and tracked them with 7.0 T Magnetic resonance imaging (MRI). Results showed that the therapeutic EPCs loaded with CD plus 5-Fc provided stronger carcinoma growth suppression compared with treatment using CD alone. The CD/5-Fc significantly inhibited the growth of endothelial cells and induced carcinoma cells apoptosis. These results indicate that EPCs transfected with anti-carcinoma genes can be used in carcinoma therapy as a novel therapeutic modality.

A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells.

The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB­MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P<0.05). Compared with GCV or 5-FC alone, the growth inhibition rate (GIR) was significantly higher in the combined treatment (F=85.35, P<0.05). The reconstructed MSCs/tk+CD+ vectors were capable of slowing down the growth of human SKOV3 cells in the presence of prodrugs in vitro. The use of combination chemotherapy exhibited a more significant inhibitory effect than using a single prodrug.

Combined alloreactive CTL cellular therapy with prodrug activator gene therapy in a model of breast cancer metastatic to the brain.

PURPOSE: Individual or combined strategies of cellular therapy with alloreactive CTLs (alloCTL) and gene therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain. EXPERIMENTAL DESIGN: AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant. RESULTS: AlloCTL preparations were cytotoxic, proliferated, and produced IFN-γ when coincubated with target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy-treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50-83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles. CONCLUSION: The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain.

A triple suicide gene strategy that improves therapeutic effects and incorporates multimodality molecular imaging for monitoring gene functions.

Gene-directed enzyme prodrug therapy (GDEPT), or suicide gene therapy, has shown promise in clinical trials. In this preclinical study using stable cell lines and xenograft tumor models, we show that a triple-suicide-gene GDEPT approach produce enhanced therapeutic efficacy over previous methods. Importantly, all the three genes (thymidine kinase, cytosine deaminase and uracil phosphoribosyltransferase) function simultaneously as effectors for GDEPT and markers for multimodality molecular imaging (MMI), using positron emission tomography, magnetic resonance spectroscopy and optical (fluorescent and bioluminescent) techniques. It was demonstrated that MMI can evaluate the distribution and function/activity of the triple suicide gene. The concomitant expression of these genes significantly enhances prodrug cytotoxicity and radiosensitivity in vitro and in vivo.

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.

[Cytotoxicity of cytosine deaminase and herpes simplex virus thymidine kinase genes in melanoma cells is independent on promoter strength].

In preparation of the therapeutic genetic constructs aimed to the gene-programmed enzymatic transformation of the non-toxic prodrug into toxin within cancer cells the right choice of regulatory elements (promoters and enhancers) is essential. This is widely accepted that the efficiency of the gene therapy constructions is dependent, in particular, on the strength of promoters driving the expression of the therapeutic genes. In this work we demonstrated, using the melanoma-specific promoters and enhancers of human melanoma inhibitory activity and mouse tyrosinase gene, that for the development of cytotoxic effect the promoter strength is not of primary importance. In the case of HSVtk, coding for the herpes simplex virus thymidine kinase, and FCU1, coding for cytosine deaminase/uracil phosphoribosyltransferase hybrid protein genes, their cytotoxic activity was determined by the quantity of the added prodrug.

Targeted tumor therapy with a fusion protein of an antiangiogenic human recombinant scFv and yeast cytosine deaminase.

In adults, endothelial cell division occurs only in wound healing, during menstruation, or in diseases such as wet age-related macular degeneration or development of benign or malignant tissues. Angiogenesis is one of the major requirements to supply the fast developing tumor tissue with oxygen and nutrients, and enables it to spread into other tissues far from its origin. We selected the extradomain B (ED-B), a splice variant of fibronectin, which is exclusively expressed in ovaries, uterus, during wound healing, and in tumor tissues, as a target for the development of an innovative antiangiogenic, prodrug-based targeted tumor therapy approach. We designed a fusion protein termed L19CDy-His, consisting of the antibody single chain fragment L19 for targeting ED-B and yeast cytosine deaminase for the conversion of 5-fluorocytosine into cytotoxic 5-fluorouracil. We purified high amounts of the fusion protein from Pichia pastoris that is stable, enzymatically active, and retains 75% of its activity after incubation with human plasma for up to 72 hours. The binding of L19CDy-His to ED-B was confirmed by an enzyme-linked immunosorbent assay and quantified by surface plasmon resonance spectroscopy determining a KD value of 81±7 nM. L19CDy-His successfully decreased cell survival of the murine ED-B-expressing teratocarcinoma cell line F9 upon addition of the prodrug 5-fluorocytosine. Our data demonstrate the suitability of targeting ED-B by L19CDy-His for effective prodrug-based tumor therapy.

Evaluation of a gene-directed enzyme-product therapy (GDEPT) in human pancreatic tumor cells and their use as in vivo models for pancreatic cancer.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems. METHODS: Here, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs. RESULTS: Tumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively. CONCLUSION: These data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy.

Emerging implications of nonmammalian cytosine deaminases on cancer therapeutics.

Nonmammalian cytosine deaminases (CDs) have been investigated for last 30 years in the context of cancer therapy. The therapeutic effect of CD is based on its ability to catalyze the conversion of nontoxic prodrug 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU) by deamination of the number 4 carbon of 5FC. This deamination property of CD has been explored to develop innovative therapeutic approach for treatment of cancer. A general overview is needed for the identification of efficient cytosine deaminases for potential use in cancer therapy. In this review, we have discussed about nonmammalian CDs for a variety of prodrug gene/enzyme therapy applications with several recent examples. Finally, we have provided a prospective on the future aspects of CDs and their applications in cancer therapy.

Single agent- and combination treatment with two targeted suicide gene therapy systems is effective in chemoresistant small cell lung cancer cells.

BACKGROUND: Transcriptional targeted suicide gene (SG) therapy driven by the insulinoma-associated 1 (INSM1) promoter makes it possible to target suicide toxin production and cytotoxicity exclusively to small cell lung cancer (SCLC) cells and tumors. It remains to be determined whether acquired chemoresistance, as observed in the majority of SCLC patients, desensitizes SCLC cells to INSM1 promoter-driven SG therapy. METHODS: A panel of SCLC cell lines resistant to clinically relevant chemotherapeutics was characterized regarding the expression of proteins involved in response to chemotherapy and regarding INSM1 promoter activity. Sensitivity towards INSM1 promoter-driven SG therapy was tested using different systems: Yeast cytosine deaminase-uracil phosphoribosyl transferase (YCD-YUPRT) in combination with the prodrug 5-fluorocytosine (5-FC) or Escherichia coli nitroreductase (NTR) together with the bromomustard prodrug SN27686. RESULTS: The chemoresistant cell lines displayed heterogeneous expression profiles of molecules involved in multidrug resistance, apoptosis and survival pathways. Despite this, the INSM1 promoter activity was found to be unchanged or increased in SCLC chemoresistant cells and xenografts compared to chemosensitive variants. INSM1 promoter-driven SG therapy with YCD-YUPRT/5-FC or NTR/SN27686, was found to induce high levels of cytotoxicity in both chemosensitive and chemoresistant SCLC cells. Moreover, the combination of INSM1 promoter-driven YCD-YUPRT/5-FC therapy and chemotherapy, as well as the combination of INSM1 promoter-driven YCD-YUPRT/5-FC and NTR/SN27686 therapy, was observed to be superior to single agent therapy in chemoresistant SCLC cells. CONCLUSIONS: Collectively, the present study demonstrates that targeted SG therapy is a potent therapeutic approach for chemoresistant SCLC patients, with the highest efficacy achieved when applied as combination SG therapy or in combination with standard chemotherapy.

Antitumor activity of mutant bacterial cytosine deaminase gene for colon cancer.

AIM: To evaluate bacterial cytosine deaminase (bCD) mutant D314A and 5-fluorocytosine (5-FC) for treatment of colon cancer in a mouse model. METHODS: Recombinant lentivirus vectors that contained wild-type bCD gene (bCDwt), and bCD mutant D314A gene (bCD-D314A) with green fluorescence protein gene were constructed and used to infect human colon carcinoma LoVo cells, to generate stable transfected cells, LoVo/null, LoVo/bCDwt or LoVo/bCD-D314A. These were injected subcutaneously into Balb/c nude mice to establish xenograft models. Two weeks post-LoVo cell inoculation, PBS or 5-FC (500 mg/kg) was administered by intraperitoneal (i.p.) injection once daily for 14 d. On the day after LoVo cell injection, mice were monitored daily for tumor volume and survival. RESULTS: Sequence analyses confirmed the construction of recombinant lentiviral plasmids that contained bCDwt or bCD-D314A. The lentiviral vector had high efficacy for gene delivery, and RT-PCR showed that bCDwt or bCD-D314A gene was transferred to LoVo cells. Among these treatment groups, gene delivery or 5-FC administration alone had no effect on tumor growth. However, bCDwt/5-FC or bCD-D314A/5-FC treatment inhibited tumor growth and prolonged survival of mice significantly (P < 0.05). Importantly, the tumor volume in the bCD-D314A/5-FC-treated group was lower than that in the bCDwt/5-FC group (P < 0.05), and bCD-D314A plus 5-FC significantly prolonged survival of mice in comparison with bCDwt plus 5-FC (P < 0.05). CONCLUSION: The bCD mutant D314A enhanced significantly antitumor activity in human colon cancer xenograft models, which provides a promising approach for human colon carcinoma therapy.

Stem cell based cancer gene therapy.

The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Suicide gene therapy using genetically engineered mesenchymal stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. This review provides an explanation of the stem cell-targeted prodrug cancer gene therapy principle, with focus on the choice of prodrug, properties of bone marrow and adipose tissue-derived mesenchymal stem and neural stem cells as well as the mechanisms of their tumor homing ability. Therapeutic achievements of the cytosine deaminase/5-fluorocytosine prodrug system and Herpes simplex virus thymidine kinase/ganciclovir are discussed. In addition, delivery of immunostimulatory cytokines, apoptosis inducing genes, nanoparticles and antiangiogenic proteins by stem cells to tumors and metastases is discussed as a promising approach for antitumor therapy. Combinations of traditional, targeted and stem cell-directed gene therapy could significantly advance the treatment of cancer.

Dual targeting of tumor angiogenesis and chemotherapy by endostatin-cytosine deaminase-uracil phosphoribosyltransferase.

Several antiangiogenic drugs targeting VEGF/VEGF receptor (VEGFR) that were approved by the Food and Drug Administration for many cancer types, including colorectal and lung cancer, can effectively reduce tumor growth. However, targeting the VEGF signaling pathway will probably influence the normal function of endothelial cells in maintaining homeostasis and can cause unwanted adverse effects. Indeed, emerging experimental evidence suggests that VEGF-targeting therapy induced less tumor cell-specific cytotoxicity, allowing residual cells to become more resistant and eventually develop a more malignant phenotype. We report an antitumor therapeutic EndoCD fusion protein developed by linking endostatin (Endo) to cytosine deaminase and uracil phosphoribosyltransferase (CD). Specifically, Endo possesses tumor antiangiogenesis activity that targets tumor endothelial cells, followed by CD, which converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the cytotoxic antitumor drug 5-fluorouracil (5-FU) in the local tumor area. Moreover, selective targeting of tumor sites allows an increasing local intratumoral concentration of 5-FU, thus providing high levels of cytotoxic activity. We showed that treatment with EndoCD plus 5-FC, compared with bevacizumab plus 5-FU treatment, significantly increased the 5-FU concentration around tumor sites and suppressed tumor growth and metastasis in human breast and colorectal orthotropic animal models. In addition, in contrast to treatment with bevacizumab/5-FU, EndoCD/5-FC did not induce cardiotoxicity leading to heart failure in mice after long-term treatment. Our results showed that, compared with currently used antiangiogenic drugs, EndoCD possesses potent anticancer activity with virtually no toxic effects and does not increase tumor invasion or metastasis. Together, these findings suggest that EndoCD/5-FC could become an alternative option for future antiangiogenesis therapy.

Imaging beyond the diagnosis: image-guided enzyme/prodrug cancer therapy.

The ideal therapy would target cancer cells while sparing normal tissue. However, in most conventional chemotherapies normal cells are damaged together with cancer cells resulting in the unfortunate side effects. The principle underlying enzyme/prodrug therapy is that a prodrug-activating enzyme is delivered or expressed in tumor tissue following which a non-toxic prodrug is administered systemically. Non-invasive imaging modalities can fill an important niche in guiding prodrug administration when the enzyme concentration is detected to be high in the tumor tissue but low in the normal tissue. Therefore, high therapeutic efficacy with minimized toxic effect can be anticipated. This review introduces the latest developments of molecular imaging in enzyme/prodrug cancer therapies. We focus on the application of imaging modalities including magnetic resonance imaging, position emission tomography and optical imaging in monitoring the enzyme delivery/expression, guiding the prodrug administration and evaluating the real-time therapeutic response in vivo.