Tumor hypoxia: a target for selective cancer therapy

Tumor hypoxia has been considered to be a potential therapeutic problem because it renders solid tumors more resistant to sparsely ionizing radiation (IR) and chemotherapeutic drugs. Moreover, recent laboratory and clinical data have shown that tumor hypoxia is also associated with a more malignant phenotype and poor survival in patients suffering from various solid tumors. Therefore, selective targeting of hypoxic tumor cells has been explored, and since severe hypoxia (pO₂<0.33%, 2.5 mmHg) does not occur in normal tissue, tumor hypoxia could be exploited for therapeutic advantage. However, the following three characteristics of hypoxic tumor regions present obstacles in targeting hypoxic cells. First, it is difficult to deliver a sufficient amount of drug to a region that is remote from blood vessels. Second, one must specifically target hypoxic tumor cells while sparing normal well-oxygenated tissue from damage. Finally, the severely hypoxic tumor cells to be attacked have often stopped dividing. Therefore, high delivery efficiency, high specificity and selective cytotoxicity are all necessary to target and combat hypoxic tumor cells. The current review describes progress on the biological aspects of tumor hypoxia and provides a compilation of the recent molecular approaches used to target hypoxic tumors. These approaches include our work with a unique hypoxia-targeting protein drug, TOP3, with which we have sought to address the above three difficulties.     

Nanoscale delivery systems for multiple drug combinations in cancer

Although drug-delivery systems have been developed to improve drug biodistribution and efficiency in cancer therapy, some limitations still hinder successful drug targeting and delivery. Multiple drugs in combination seems a promising strategy for cancer therapy. It enables drugs to be delivered to multiple targets and exhibits the additive or synergistic effects of drugs. Physiological barriers are known to be the main obstacles of insufficient drug efficacy and delivery in tumors, but they are likely to be potential targets in combination therapy as well. This article discusses some general considerations for optimizing multiply drug delivery, including drug-release profiles and loading strategies.     

Targeting pediatric malignancies for T cell-mediated immune responses

Successful immune targeting of malignancies hinges upon the ability to activate specific T-cell populations to recognize and attack tumor but spare normal vital tissues. Investigators in the field of tumor immunology are currently utilizing at least three distinct approaches toward this goal. In the first approach, molecular targets of cytolytic T cells which spontaneously develop in tumorbearing patients have been identified and are subsequently used as immunogens in immunotherapy trials. Whereas this approach originally focused upon the identification of tumor antigens in the immune-responsive tumors malignant melanoma and renal cell carcinoma, it surprisingly led to the identification of a variety of molecules that are now known to be expressed in other common pediatric and adult tumors. In the second approach, tumor-specific molecules (eg, mutant p53 and chromosomal translocations) that have been identified in individual tumors during the study of neoplastic transformation are used as immunogens. Because chromosomal translocations are common in pediatric tumors, such targets may be of particular interest in pediatric oncology. In the third approach, immunization with whole tumor cell components is undertaken with the assumption that the most immunogenic molecules within the tumor will dominate the immune response induced. The benefits and limitations for each approach, particularly as it pertains to the development of immunotherapy for pediatric tumors, are discussed in this article.     

Molecular-targeted nanotherapies in cancer: enabling treatment specificity

Chemotherapy represents a mainstay and powerful adjuvant therapy in the treatment of cancer. The field has evolved from drugs possessing all-encompassing cell-killing effects to those with highly targeted, specific mechanisms of action; a direct byproduct of enhanced understanding of tumorigenic processes. However, advances regarding development of agents that target key molecules and dysregulated pathways have had only modest impacts on patient survival. Several biological barriers preclude adequate delivery of drugs to tumors, and remain a formidable challenge to overcome in chemotherapy. Currently, the field of nanomedicine is enabling the delivery of chemotherapeutics, including repositioned drugs and siRNAs, by giving rise to carriers that provide for protection from degradation, prolonged circulation times, and increased tumor accumulation, all the while resulting in reduced patient morbidity. This review aims to highlight several innovative, nanoparticle-based platforms with the potential of providing clinical translation of several novel chemotherapeutic agents. We will also summarize work regarding the development of a multistage drug delivery strategy, a robust carrier platform designed to overcome several biological barriers while en route to tumors.     

New treatment strategies for malignant gliomas

Malignant gliomas are the most prevalent type of primary brain tumor in adults. Despite progress in brain tumor therapy, the prognosis of malignant glioma patients remains dismal. The median survival of patients with glioblastoma multiforme, the most common grade of malignant glioma, is 10-12 months. Conventional therapy of surgery, radiation and chemotherapy is largely palliative. Essentially, tumor recurrence is inevitable. Salvage treatments upon recurrence are palliative at best and rarely provide significant survival benefit. Therapies targeting the underlying molecular pathogenesis of brain tumors are urgently required. Common genetic abnormalities in malignant glioma specimens are associated with aberrant activation or suppression of cellular signal transduction pathways and resistance to radiation and chemotherapy. Several low molecular weight signal transduction inhibitors have been examined in preclinical and clinical malignant glioma trials. The efficacy of these agents as monotherapies has been modest, at best; however, small subsets of patients who harbor specific genetic changes in their tumors may display favorable clinical responses to defined small molecule inhibitors. Multitargeted kinase inhibitors or combinations of agents targeting different mitogenic pathways may overcome the resistance of tumors to single-agent targeted therapies. Well designed studies of small molecule kinase inhibitors will include assessment of safety, drug delivery, target inhibition and correlative biomarkers to define mechanisms of response or resistance to these agents. Predictive biomarkers will enrich for patients most likely to respond in future clinical trials. Additional clinical studies will combine novel targeted therapies with radiation, chemotherapies and immunotherapies.     

Prodrug cancer gene therapy

There is no effective treatment for late stage and metastatic cancers of colorectal, prostate, pancreatic, breast, glioblastoma and melanoma cancers. Novel treatment modalities are needed for these late stage patients because cytotoxic chemotherapy offers only palliation, usually accompanied with systemic toxicities and poor quality of life. Gene directed enzyme prodrug therapy (GDEPT), which concentrates the cytotoxic effect in the tumor site may be one alternative. This review provides an explanation of the GDEPT principle, focusing on the development, application and potential of various GDEPTs. Current gene therapy limitations are in efficient expression of the therapeutic gene and in tumor-specific targeting. Therefore, the current status of research related to the enhancement of in situ GDEPT delivery and tumor-specific targeting of vectors is assessed. Finally, GDEPT versions of stem cell based gene therapy as another potential treatment modality for progressed tumors and metastases are discussed. Combinations of traditional, targeted, and stem cell directed gene therapy could significantly advance the treatment of cancer.     

Strategies to improve delivery of anticancer drugs across the blood–brain barrier to treat glioblastoma

Glioblastoma (GBM) is a lethal and aggressive brain tumor that is resistant to conventional radiation and cytotoxic chemotherapies. Molecularly targeted agents hold great promise in treating these genetically heterogeneous tumors, yet have produced disappointing results. One reason for the clinical failure of these novel therapies can be the inability of the drugs to achieve effective concentrations in the invasive regions beyond the bulk tumor. In this review, we describe the influence of the blood–brain barrier on the distribution of anticancer drugs to both the tumor core and infiltrative regions of GBM. We further describe potential strategies to overcome these drug delivery limitations. Understanding the key factors that limit drug delivery into brain tumors will guide future development of approaches for enhanced delivery of effective drugs to GBM.     

Malignancy in neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) represents a major risk factor for development of malignancy, particularly malignant peripheral nerve sheath tumors (MPNST), optic gliomas, other gliomas, and leukemias. The oncologist will see NF1 patients referred for treatment of malignancy, and should be alert to the possibility of undiagnosed NF1 among patients with cancer. Brain tumors tend to have a more indolent course in NF1 than in the general population, and hence are best managed conservatively. MPNST, in contrast, do not respond to standard chemotherapy or radiation therapy. The most effective treatment of MPNST appears to be early diagnosis and surgery, but early diagnosis is hampered by frequent occurrence within preexisting large tumors, making new growth or change difficult to detect. New insights into pathogenesis now offer hope of development of specific methods of treatment with reduced toxicity and more precise molecular targeting. There is an urgent need, however, to develop methods to measure tumor growth and monitor outcomes, develop preclinical drug screening systems, and further explore the pathogenesis of the disorder to determine whether mechanisms other than Ras regulation may be important in pathogenesis.     

NCI Image-Guided Drug Delivery Summit

On April 17, 2010, scientists from academia, the National Cancer Institute (NCI), and the Food and Drug Administration (FDA) assembled at "The NCI Image Guided Drug Delivery Summit," in Washington D.C., to discuss recent advances, barriers, opportunities, and regulatory issues related to the field. The meeting included a scientific session and an NCI/FDA session, followed by a panel discussion of speakers from both sessions. Image-guided drug delivery (IGDD) in cancer is a form of individualized therapy where imaging methods are used in guidance and monitoring of localized and targeted delivery of therapeutics to the tumor. So, a systematic approach to IGDD requires mechanisms for targeting, delivery, activation, and monitoring of the process. Although the goal in IGDD is to optimize the therapeutic ratio through personalized image-guided treatments, a major challenge is in overcoming the biological barriers to the delivery of therapeutics into tumors and cells. Speakers discussed potential challenges to clinical translation of nano-based drug delivery systems including in vivo characterization of nanocarriers, preclinical validation of targeting and delivery, studies of biodistribution, pharmacokinetics, pharmacodynamics, and toxicity as well as scale-up manufacturing of delivery systems. Physiologic and quantitative imaging techniques may serve as enabling tools that could potentially transform many existing challenges into opportunities for advancement of the field.     

Antibodies in the therapy of colon cancer

Clinical research in antibody-based cancer therapy has been driven for many years by the prospect of identifying cell-surface antigens with sufficiently restrictive tissue expression patterns to allow the specific targeting of antibody to tumor tissue. Few if any such antibodies capable of targeting rapidly and efficiently to solid tumors have been identified. The main reasons for this are based on the inherent pharmacokinetics and physiology of IgG, the immunoglobulin G molecule. Factors that may limit targeting potential include accessibility of tumor antigen, and antibody affinity, molecular size, and metabolism. Immunoglobulins have evolved to optimally protect an organism from foreign invaders rather than to act as an efficient carrier molecule for therapeutic reagents. Despite these potential limitations, our growing understanding of the biologic and physiologic principles that underlie targeted therapy has led to the development of a generation of novel reagents and the first "positive" clinical trials. Recent strategies for therapeutic use of antibodies in colon cancer have focused on (I) unmodified mouse IgG; (2) immune globulin as carrier for targeted delivery of radioisotopes; toxins, and therapeutic molecules; (3) genetically engineered antibody constructs redesigned for specific uses; (4) humanized, nonimmunogenic IgG structures; and (5) novel antigen targets in tumors. Genetically engineered antibody constructs provide an exciting approach to address and subsequently overcome some of the problems identified for unmodified IgG. These new constructs should increase the dose fraction localized in tumors versus normal tissue and thereby improve the delivery capacity. In contrast, strategies such as immune-mediated cytotoxicity are less dependent on the quantitative difference between the antibody fraction localized in tumor and the nonlocalized fraction. Because antibodies, which direct host cytotoxic mechanisms, become activated in the tumor only when bound to antigen, one would not expect nonspecific toxic effects from nonlocalized antibody. The hypothesis that antibodies alone can destroy tumor tissue solely by directing immune cytotoxic mechanisms is just now being tested in clinical trials evaluating a new generation of humanized antibodies.     

Selective targeting of liver cancer with the endothelial marker CD146

Hepatocellular carcinomas are well-vascularized tumors; the endothelial cells in these tumors have a specific phenotype. Our aim was to develop a new approach for tumor-specific drug delivery with monoclonal antibody targeting of endothelial ligands. CD146, a molecule expressed on the endothelial surface of hepatocellular carcinoma, was identified as a promising candidate for targeting. In the present study, endothelial cells immediately captured circulating anti-CD146 (ME-9F1) antibody, while antibody binding in tumors was significantly higher than in hepatic endothelium. Macroscopically, after intravenous injection, there were no differences in the mean accumulation of anti-CD146 antibody in tumor compared to liver tissue, due to a compensating higher blood vessel density in the liver tissue. Additional blockade of nontumoral epitopes and intra-arterial administration, improved selective antibody capture in the tumor microvasculature and largely prevented antibody distribution in the lung and liver. The potential practical use of this approach was demonstrated by imaging of radionuclide-labeled ME-9F1 antibody, which showed excellent tumor-selective uptake. Our results provide a promising principle for the use of endothelial markers for intratumoral drug delivery. Tumor endothelium–based access might offer new opportunities for the imaging and therapy of hepatocellular carcinoma and other liver malignancies.     

Anti-angiogenic gene therapy for cancer (review)

Angiogenesis is a prerequisite for the growth and metastasis of solid tumors. Studies have confirmed that in the absence of angiogenesis, tumors rarely have the ability to develop beyond a few millimeters in diameter. Tumor-associated endothelium is activated by the production of soluble factors by tumor and stromal cells. Tumor-associated endothelial cells are physiologically homogeneous and divide more frequently. Thus, targeting the proliferation of tumor neovasculature will form an effective anti-cancer therapy. With the identification of drug and biological molecules that inhibit the growth of tumor endothelium, several attempts have been made in preclinical and clinical research to evaluate the potential of anti-angiogenic therapy. Although several drugs and purified proteins have shown promise, their wide-spread application is limited by half-life, side effects and cost involved. Gene therapy approaches, on the other hand, have the potential to overcome these limitations. Further, genetic transfer of anti-angiogenic genes can be combined with other treatments including radiation therapy or chemotherapy for synergistic effects. In this review, we provide a comprehensive account of factors and mechanisms underlying tumor angiogenesis, the potential and limitations of available gene therapy vectors for anti-angiogenesis and current status of preclinical and clinical gene therapy studies targeting tumor angiogenesis.     

Anti-tumor gene therapy

Gene therapy as an anti-tumor strategy is becoming a powerful tool for cytokine delivery to inhibit the growth of many tumors. Several delivery systems are being utilized and designed for the expression of specific genes to achieve a therapeutic result. Liposomes, retroviral vectors, and adenoviral vectors have all been used and eventual clinical application may depend on the type of tumor, the location, the specific gene carried, and the patients health status. Novel expression vectors may eventually achieve tissue-specific targeting and low immuno-reactivity. Inactivation of mutated oncogenes, such as ras, or re-expression of inactive suppressor genes, such as p53 have been used as strategies for anti-tumor therapy. Additionally, exogenous genes, su ch as viral thymidine kinase that metabolize chemotherapeutic agents to achieve local cytotoxicity have also been employed. Neuro-endocrine tumors are targets of these genetic strategies since they are often difficult to treat by conventional methods bec ause of their location (brain tumors) or because they have spread from the primary tumor (melanoma). Further advances in the design of these vectors may achieve safe targeting of a variety of malignant tumors.     

Combining Targeted Therapies: Practical Issues to Consider at the Bench and Bedside

Numerous practical issues must be considered when combining targeted therapies in early clinical drug development. These include tumor resistance mechanisms, the existence of multiple, redundant signaling pathways, and the failure of single-agent therapies to achieve cures. The strategies adopted to examine combinatorial therapy include the goal of hitting more than one target by specifically inhibiting signal transduction cascades and suppressing specific mechanisms of action with the use of multitargeted kinase inhibitors made possible by high-throughput screening techniques, combinatorial chemistry, and chemoinformatics. Two complex considerations are: which agents to combine given the heterogeneity of tumors and their various underlying perturbations, including secondary mutations and feedback loops, and how to translate findings from the bench to the bedside or directly from the bedside. Another consideration is: When is there enough information to provide a rationale for instituting a phase I trial? Various strategies have been used in combining molecules, including targeting diverse pathways, inhibiting upstream and downstream signals, and adopting a synthetic lethality paradigm. Other issues are: determining appropriate target populations for treatment, how to combine therapeutics with diagnostics, and the frequency of targets in patients referred to clinical trials. Here, we review these issues and we propose various novel trial designs that are logical for determining the efficacy of a drug or drug combination for personalized treatment. A difficult issue that must be answered is how many and which drugs to combine. Recent technologies, such as multiplexed assay platforms and bioinformatics, will shape the future of clinical trials and help answer these questions surrounding combinatorial treatment.     

Salmonella promoters preferentially activated inside tumors

Salmonella enterica and avirulent derivatives prefer solid tumors over normal tissue in animal models. The identification of endogenous Salmonella promoters that are preferentially activated in tumors could further our understanding of this phenomenon. Toward this goal, a random library of S. enterica typhimurium 14028 genomic DNA was cloned upstream of a promoterless gene encoding the green fluorescent protein (GFP) TurboGFP. A population of Salmonella containing this library was injected i.v. into tumor-free nude mice and into human PC3 prostate tumors growing subcutaneously in nude mice. After 2 days, fluorescence-activated cell sorting was used to enrich for bacterial clones expressing GFP from spleens or tumors. The resulting libraries were hybridized to an oligonucleotide tiling array of the Salmonella genome. Eighty-six intergenic regions were found to be enriched in tumor samples but not in spleen. Twenty of these candidate promoters were also detected in the sequences of 100 random clones from a library enriched for expression in bacteria growing in tumors. Three candidate promoter clones were individually tested in vivo, and enhanced GFP expression in bacteria growing in tumor relative to spleen was confirmed. Two of the three clones (pflE and ansB promoter regions) are known to be induced in hypoxic conditions that pertain to many tumors. For many of the other candidate promoters preferentially induced in bacteria growing in tumors, regulatory mechanisms may not be related to hypoxia. The expression of therapeutics in Salmonella under the regulation of one or more promoters that are activated preferentially in tumors has the potential to improve the targeting of drug delivery.     

Nanotechnology-based drug delivery for cancer

Nanobiotechnologies have been applied to improve drug delivery and to overcome some of the problems of drug delivery in cancer. These can be classified into many categories that include use of various nanoparticles, nanoencapsulation, targeted delivery to tumors of various organs, and combination with other methods of treatment of cancer such as radiotherapy. Nanoparticles are also used for gene therapy for cancer. Some of the technologies enable combination of diagnostics with therapeutics which will be important for the personalized management of cancer. Some of the limitations of these technologies and prospects for future development are discussed.     

New treatment strategies for malignant gliomas

Malignant gliomas are the most prevalent type of primary brain tumor in adults. Despite progress in brain tumor therapy, the prognosis of malignant glioma patients remains dismal. The median survival of patients with glioblastoma multiforme, the most common grade of malignant glioma, is 10–12 months. Conventional therapy of surgery, radiation and chemotherapy is largely palliative. Essentially, tumor recurrence is inevitable. Salvage treatments upon recurrence are palliative at best and rarely provide significant survival benefit. Therapies targeting the underlying molecular pathogenesis of brain tumors are urgently required. Common genetic abnormalities in malignant glioma specimens are associated with aberrant activation or suppression of cellular signal transduction pathways and resistance to radiation and chemotherapy. Several low molecular weight signal transduction inhibitors have been examined in preclinical and clinical malignant glioma trials. The efficacy of these agents as monotherapies has been modest, at best; however, small subsets of patients who harbor specific genetic changes in their tumors may display favorable clinical responses to defined small molecule inhibitors. Multitargeted kinase inhibitors or combinations of agents targeting different mitogenic pathways may overcome the resistance of tumors to single-agent targeted therapies. Well designed studies of small molecule kinase inhibitors will include assessment of safety, drug delivery, target inhibition and correlative biomarkers to define mechanisms of response or resistance to these agents. Predictive biomarkers will enrich for patients most likely to respond in future clinical trials. Additional clinical studies will combine novel targeted therapies with radiation, chemotherapies and immunotherapies.     

Enhancement of tumor uptake and therapeutic efficacy of EGFR‐targeted antibody cetuximab and antibody–drug conjugates by cholesterol sequestration

Cetuximab, a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), has been intensively investigated as a promising cancer treatment strategy. The specific mechanism of cetuximab endocytosis and its influence on cetuximab uptake, biodistribution and efficacy still remain elusive. Recently, statins have been reported to synergize with EGFR‐targeting agents. Our prior work established that nystatin, a cholesterol‐sequestering antifungal drug, facilitates endocytosis via the clathrin‐dependent pathway. This study aimed to investigate whether nystatin regulates the uptake and efficacy of cetuximab and cetuximab‐based antibody–drug conjugates (cetuximab‐ADCs). In vitro and in vivo efficacies of nystatin on the uptake and activity of cetuximab/cetuximab‐ADCs were studied in multiple human carcinoma cell lines and xenograft models, respectively. We identified that cholesterol sequestration by nystatin enhanced cetuximab internalization in EGFR‐positive carcinoma cells by regulating EGFR trafficking/turnover and facilitating a switch from lipid rafts to clathrin‐mediated endocytosis. Combination treatment with cetuximab and nystatin selectively increased cetuximab uptake by tumor tissues, translating into potentiated antitumor efficacy of cetuximab in vivo (A431 and A549 tumors). Nystatin‐enhanced internalization of cetuximab further improved the uptake and potency of cetuximab‐doxorubicin and cetuximab‐methotrexate conjugates in EGFR‐positive cetuximab‐resistant tumors. Combination therapy with nystatin plus either cetuximab or cetuximab‐ADC further prolonged animal survival and significantly suppressed tumor growth, as compared with single‐agent cetuximab or cetuximab‐ADC. In summary, our results identify a novel mechanism whereby cholesterol sequestration enhances the uptake of EGFR‐targeting mAb and ADCs, therefore providing preclinical proof‐of‐concept that combination with nystatin can potentiate the delivery and efficacy of these EGFR‐targeted agents.     

Future developments in the selectivity of anticancer agents: Drug delivery and molecular target strategies

In the past, our limited understanding of the processes involved in the initiation and growth of cancer hindered our ability to effectively treat most human malignancies and therapies were often associated with significant toxic side effects as well as re-emergence of disease. The development of drug delivery systems such as liposomes has improved the specificity of various conventional anticancer agents by enhancing drug accumulation in tumors while often decreasing exposure to susceptible healthy tissues. More recently, the identification of a wide range of genes and corresponding protein products that are altered in various human cancers has revealed new molecular targets for cancer therapy that may provide improved selectivity for tumor cells over traditional cytotoxic agents. This review discusses how advances in the sophistication of liposomal delivery systems may open new opportunities for combining novel molecular targeting strategies with pharmacological targeting via liposomes to optimize the therapy of many human malignancies.     

Intra-arterial infusion chemotherapy: clinical applications and current status of therapeutic effects on various malignant tumors

Intra-arterial infusion chemotherapy for various malignant tumors in order to improve the antitumor effects and to diminish the side effects has been performed in general since the 1950's. Numerous reports have shown favourable therapeutic effects followed by the development of the new anticancer agents. Although in recent years application of intra-arterial administration of anticancer agents alone has been limited to such target tumors as liver cancer because of application of mechanical arterial embolization using gelatin sponge cubes, attempts have been made to enhance the antitumor effect. In order to improve targeting and stagnancy of anticancer agents in the tumor area, drug delivery systems involving arrangement of the hemodynamics of the tumor area (balloon-occluded arterial infusion therapy, administration with vasoconstrictive agents such as noradrenaline or angiotensin II and/or as administration with various drug carriers (microcapsules, lipiodol, albumin microspheres, Degradable Starch Microspheres, liposomes, etc.) have been prepared and made available for clinical use with various tumors. Furthermore, development of totally implantable equipment of intra-arterial use for not only continuous infusion but one-shot injection of anticancer agents contributes to the treatment of patients longer and more frequently with less trouble. In the future intra-arterial infusion chemotherapy will have an important role for treatment of various malignant tumors, especially as one part of multimodal treatments, although the pharmacokinetics should be more fully-studied.