Long-Term Survival of Cellulose Sulphate-Encapsulated Cells and Metronomic Ifosfamide Control Tumour Growth in Pancreatic Cancer Models-A Prelude to Treating Solid Tumours Effectively in Pets and Humans.

BACKGROUND: The use of encapsulated cells for the in vivo delivery of biotherapeutics is a promising new technology to potentiate the effectiveness of cell-based therapies for veterinary and human application. One use of the technology is to locally activate chemotherapeutics to their short-lived highly active forms. We have previously shown that a stable clone of HEK293 cells overexpressing a cytochrome P450 enzyme that has been encapsulated in immunoprotective cellulose sulphate beads can be implanted near solid tumours in order to activate oxazaphosphorines such as ifosfamide and cyclophosphamide to the tumour-killing metabolite phosphoramide mustard. The efficacy of this approach has been shown in animal models as well as in human and canine clinical trials. In these previous studies, the oxazaphosphorine was only given twice. An analysis of the Kaplan-Meier plots of the results of the clinical trials suggest that repeated dosing might result in a significant clinical benefit. AIMS: In this study, we aimed to (i) demonstrate the stable long-term expression of cytochrome P450 from a characterized, transfected cell clone, as well as (ii) demonstrate that one implanted dose of these encapsulated cytochrome P450-expressing cells is capable of activating multiple doses of ifosfamide in animal models. METHODOLOGY: We initially used cell and molecular methods to show cell line stability over multiple passages, as well as chemical and biological function in vitro. This was followed by a demonstration that encapsulated HEK293 cells are capable of activating multiple doses of ifosfamide in a mouse model of pancreatic cancer without being killed by the chemotherapeutic. CONCLUSION: A single injection of encapsulated HEK293 cells followed by multiple rounds of ifosfamide administration results in repeated anti-tumour activity and halts tumour growth but, in the absence of a functioning immune system, does not cause tumour regression.

The Role of a Betaretrovirus in Human Breast Cancer: Enveloping a Conundrum.

Most of the evidence that a human betaretrovirus (HBRV/HMTV) highly related to mouse mammary tumour virus (MMTV) has an etiological role in breast cancer has been summarized in a recent comprehensive Special Issue of "Viruses" entitled "Human Betaretrovirus (HBRV) and Related Diseases". Shortly after publication of this special issue, a detailed analysis of aligned env sequences was published and concluded that (i) MMTV and HBRV/HMTV cannot be distinguished on the basis of aligned env sequences and (ii) more sequence data covering the full-length env or HBRV/HMTV genomes from multiple isolates is needed. Although productive infection of human cells by MMTV (and presumably HBRV/HMTV) has been shown, it is imperative that the receptor(s) enabling HBRV/HMTV to infect human cells are defined. Moreover, there is currently no compelling data for common integration sites, in contrast to MMTV induced mammary tumorigenesis in mice, suggesting that other mechanisms of tumorigenesis are associated with HBRV/HMTV infection. These issues need to be resolved before a clear link between MMTV/HBRV/HMTV and human breast cancer can be concluded.

Release characteristics of cellulose sulphate capsules and production of cytokines from encapsulated cells.

The size and speed of release of proteins of different sizes from standard cellulose sulphate capsules (Cell-in-a-Box®) was investigated. Proteins with molecular weights of up to around 70kD can be released. The conformation, charge and concentration of the protein being released play a role in the release kinetics. Small proteins such as cytokines can be easily released. The ability to produce cytokines at a sustained and predefined level from encapsulated cells genetically engineered to overexpress such cytokines and implanted into patients may aid immunotherapies of cancer as well as infectious and other diseases. It will also allow allogeneic rather than autologous cells to be used. We show that cells encapsulated in polymers of cellulose sulphate are able to release cytokines such as interleukin-2 (IL-2) in a stimulated fashion e.g. using phorbol 12-myristate 13-acetate (PMA) plus ionomycin. Given the excellent documented safety record of cellulose sulphate in patients, these data suggest that clinical usage of the technology may be warranted for cancer treatment and other diseases.

Recent developments linking retroviruses to human breast cancer: infectious agent, enemy within or both?

Evidence is accumulating that one or more beta-retrovirus is associated with human breast cancer. Retroviruses can exist as an infectious (exogenous) virus or as a part of the genetic information of cells due to germline integration (endogenous). An exogenous virus with a genome that is highly homologous to mouse mammary tumour virus is gaining acceptance as possibly being associated with human breast cancer, and recently furnished evidence is discussed in this article, as is the evidence for involvement of an endogenous human beta-retrovirus, HERV-K. Modes of interaction are also reviewed and linkages to the APOBEC3 family are suggested.

Encapsulated cells expressing a chemotherapeutic activating enzyme allow the targeting of subtoxic chemotherapy and are safe and efficacious: data from two clinical trials in pancreatic cancer.

Despite progress in the treatment of pancreatic cancer, there is still a need for improved therapies. In this manuscript, we report clinical experience with a new therapy for the treatment of pancreatic cancer involving the implantation of encapsulated cells over-expressing a cytochrome P450 enzyme followed by subsequent low-dose ifosfamide administrations as a means to target activated ifosfamide to the tumor. The safety and efficacy of the angiographic instillation of encapsulated allogeneic cells overexpressing cytochrome P450 in combination with low-dose systemic ifosfamide administration has now been evaluated in 27 patients in total. These patients were successfully treated in four centers by three different interventional radiologists, arguing strongly that the treatment can be successfully used in different centers. The safety of the intra-arterial delivery of the capsules and the lack of evidence that the patients developed an inflammatory or immune response to the encapsulated cells or encapsulation material was shown in all 27 patients. The ifosfamide dose of 1 g/m2/day used in the first trial was well tolerated by all patients. In contrast, the ifosfamide dose of 2 g/m2/day used in the second trial was poorly tolerated in most patients. Since the median survival in the first trial was 40 weeks and only 33 weeks in the second trial, this strongly suggests that there is no survival benefit to increasing the dose of ifosfamide, and indeed, a lower dose is beneficial for quality of life and the lack of side effects. This is supported by the one-year survival rate in the first trial being 38%, whilst that in the second trial was only 23%. However, taking the data from both trials together, a total of nine of the 27 patients were alive after one year, and two of these nine patients were alive for two years or more.

Phase I/II clinical trial of encapsulated, cytochrome P450 expressing cells as local activators of cyclophosphamide to treat spontaneous canine tumours.

Based upon promising preclinical studies, a clinical trial was performed in which encapsulated cells overexpressing cytochrome P450 enzyme isoform 2B1 were implanted around malignant mammary tumours arising spontaneously in dogs. The dogs were then given cyclophosphamide, one of the standard chemotherapeutic agents used for the treatment of mammary tumours. The dogs were assessed for a number of clinical parameters as well as for reduction in tumour size. The treatment was well tolerated with no evidence of adverse reactions or side effects being associated with the administration of the encapsulated cells. Reductions in tumour size of more than 50% were observed for 6 out of the 11 tumours analysed while 5 tumours showing minor responses, i.e. stable disease. In contrast, the tumours that received cyclophosphamide alone showed only stable disease. Taken together, this data suggests that encapsulated cytochrome P450 expressing cells combined with chemotherapy may be useful in the local treatment of a number of dog mammary tumours and support the performance of further clinical studies to evaluate this new treatment.

Revisiting a role for a mammary tumor retrovirus in human breast cancer.

There remains great controversy as to whether mouse mammary tumor virus (MMTV), the etiological agent of mammary cancer in mice, or a closely related human retrovirus, plays a role in the development of breast cancer in humans. On one hand, retroviruses such as human T-cell lymphotropic virus and human immunodeficiency virus (HIV) are known causative agents of cancer (in the case of HIV, albeit, indirectly), but attempts to associate other retroviruses with human cancers have been difficult. A recent, high profile, example has been the postulated involvement of another mouse virus, xenotropic murine leukemia virus-related virus, in human prostate cancer, which is now thought to be due to contamination. Here, we review some of the more recent evidence for and against the involvement of MMTV in human breast cancer and suggest future studies that may allow a definitive answer to this conundrum.

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.

The 5' leader sequence of mouse mammary tumor virus enhances expression of the envelope and reporter genes.

Mouse mammary tumor virus (MMTV) is a complex betaretrovirus, which utilizes a Rev-like auxiliary protein Rem to export the unspliced viral RNA from the nucleus. MMTV env mRNA appears to be exported via a distinct, Rem-independent, mechanism. Here, we analysed the effect of an extensively folded region coinciding with the 5' leader sequence on env gene expression. We found that the presence of the 5' leader stimulates expression of the envelope protein. Enhanced Env production was accompanied by increased cytoplasmic levels of env mRNA. The 5' leader promotes nucleocytoplasmic translocation and increases stability of env mRNA. The region responsible for this effect was mapped to the distal part of the 5' leader. Furthermore, the 5' leader inserted in the sense orientation into a heterologous luciferase expression construct increased luciferase activity.

Comparative evaluation of preclinical in vivo models for the assessment of replicating retroviral vectors for the treatment of glioblastoma.

Despite impressive improvements in neurosurgical techniques, radiation and chemotherapy during the past few years, little progress has been made in the treatment of malignant gliomas. Recently, the efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including gliomas. In this study, we have thus critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to RCR virus vector spread, sensitivity towards the cytosine deaminase (CD)/5-flurocytosine (5-FC)/5-flurouracil (5-FU) suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD(50) values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types.

Encapsulated cells to focus the metabolic activation of anticancer drugs.

One of the first strategies for cancer gene therapy was the use of suicide gene/prodrug combinations, originally delivered to tumor cells using viral vectors. A major limitation of this approach was the inefficiency of suicide gene delivery. An alternative strategy, in which the suicide genes are physically juxtaposed to the tumor, involves the implantation of encapsulated, genetically modified cells. Cell encapsulation technologies were originally developed for the treatment of acquired and genetic diseases, such as diabetes. In the application of this technology for the treatment of tumors, cells that are genetically modified to overexpress suicide genes are encapsulated and implanted near solid tumors; this process is then followed by systemic prodrug administration. This review discusses the various cells types, suicide genes and prodrugs that have been used in preclinical and clinical trials, as well as the data that have been obtained from these studies. Future improvements for the production of second-generation approaches are also discussed.

Therapeutic application of cell microencapsulation in cancer.

Although cell encapsulation technologies were originally developed for the treatment of acquired and genetic diseases such as diabetes, they can also be applied to the treatment of a variety of solid tumours. There are a number of strategies aimed at treating tumours with encapsulated cells and most of these are reviewed in this chapter. Many of these strategies have shown promise in preclinical studies and clinical trials.

Human endogenous retroviral long terminal repeat sequences as cell type-specific promoters in retroviral vectors.

The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction.

Molecularly characterised xenograft tumour mouse models: valuable tools for evaluation of new therapeutic strategies for secondary liver cancers.

To develop and evaluate new therapeutic strategies for the treatment of human cancers, well-characterised preclinical model systems are a prerequisite. To this aim, we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours. Established xenograft tumours were patho- and immunohistologically characterised, and expression levels of cancer-relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying RT-PCR-based array technology. Most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained. No differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis. Interestingly, cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially. Thus, the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo.

Influence of vector design and host cell on the mechanism of recombination and emergence of mutant subpopulations of replicating retroviral vectors.

BACKGROUND: The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design. RESULTS: In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed. CONCLUSION: Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.

Identification of the Rem-responsive element of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has previously been shown to encode a functional homolog of the human immunodeficiency virus-1 (HIV-1) nuclear export protein Rev, termed Rem. Here, we show that deletion of the rem gene from a MMTV molecular clone interfered with the nucleo-cytoplasmic transport of genomic length viral mRNA and resulted in a loss of viral capsid (Gag) protein production. Interestingly, nuclear export of single-spliced env mRNA was only moderately affected, suggesting that this transcript is, at least to some extent, transported via a distinct, Rem-independent export mechanism. To identify and characterize a cis-acting RNA element required for Rem responsiveness (RmRE), extensive computational and functional analyses were performed. By these means a region of 490 nt corresponding to positions nt 8517-nt 9006 in the MMTV reference strain was identified as RmRE. Deletion of this fragment, which spans the env-U3 junction region, abolished Gag expression. Furthermore, insertion of this sequence into a heterologous HIV-1-based reporter construct restored, in the presence of Rem, HIV-1 Gag expression to levels determined for the Rev/RRE export system. These results clearly demonstrate that the identified region, whose geometry resembles that of other retroviral-responsive elements, is capable to functionally substitute, in the presence of Rem, for Rev/RRE and thus provide unequivocal evidence that MMTV is a complex retrovirus.

Rafts, anchors and viruses--a role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors.

Lipid rafts have been proposed as sites for the assembly of a number of viruses and are considered to play a major role in pseudotyping events. As a consequence, host glycosylphosphatidylinositol (GPI) anchored proteins commonly associated with lipid rafts can be found being incorporated into viral lipid envelopes with beneficial consequences for viral replication. In this review we will look at the link between lipid rafts, GPI-anchored proteins and retroviral particles and how these relationships can be exploited for the modification of enveloped viruses.