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Journal of Cancer Research and Therapeutics, Vol. 6, No. 4, October-December, 2010, pp. 427-431 Review Article From tumor immunology to cancer immunotherapy: Miles to go Manjul Tiwari Departments of Oral Pathology and Microbiology, School of Dental Sciences, Sharda University, Greater Noida Uttar Pradesh, India Correspondence Address: Manjul Tiwari, D-97, Anupam Apartments, B/13, Vasundhara Enclave, Delhi - 110096, India, manjultiw@gmail.com Code Number: cr10111 PMID: 21358075 DOI: 10.4103/0973-1482.77071 Abstract Advances in tumor immunology are supporting the clinical implementation of several immunological approaches to cancer in the clinical setting. However, the alternate success of current immunotherapeutic regimens underscores the fact that the molecular mechanisms underlying immune-mediated tumor rejection are still poorly understood. Given the complexity of the immune system network and the multidimensionality of tumor/host interactions, the comprehension of tumor immunology might greatly benefit from high-throughput microarray analysis, which can portrait the molecular kinetics of immune response on a genome-wide scale, thus accelerating the discovery pace and ultimately catalyzing the development of new hypotheses in cell biology. Although the general principles of microarray-based gene profiling with the application of DNA microarray have rapidly spread in the scientific community, the need for mastering this technique to produce meaningful data and correctly interpret the enormous output of information generated by this technology is critical and represents a tremendous challenge for investigators. To describe the sequence of events conductive to an effective immune recognition and killing of malignant cells, clinicians might use gene profiling as a powerful tool to assess the activation status of the immune system before and during immunotherapy.Keywords: DNA microarray, immune-mediated tumors, microarray, tumor immunology Introduction Recent years have witnessed important breakthroughs in the understanding of tumor immunology. [1] Moreover, a variety of immunotherapeutic strategies have shown that immune manipulation can mediate the regression of established cancer in humans. [2],[3] The identification of the genes encoding tumor associated antigens (TAA) and the development of therapies for immunizing against these antigens have opened new avenues for the development of an effective anticancer immunotherapy. [4] Although several immunotherapeutic approaches have demonstrated that immune cells can be polarized against tumors, in most cases the absence of these findings has halted back on the employment of new modalities, and cancer immunotherapy seems to have reached a plateau of results. However, at present, the mechanism underlying tumor immune rejection is still poorly understood. Only when the molecular matrix governing immune responsiveness of cancer is deciphered, new therapeutic strategies will be designed to fit biologically defined mechanisms of cancer immune rejection. Tumor Immunology and the Post-Genomic ERA Traditional molecular analyses are "reductionist" as they only assess the expression of one or a few genes at a time. Thus, the output of single gene analysis is hardly applicable to biologic models whose outcome is likely to be governed by the combined influence of a global gene network. The development of other molecular methods, such as comparative genomic hybridization (CGH), [5] differential display, [6] serial analysis of gene expression (SAGE), [7] and DNA microarrays, [8] together with the sequencing of the human genome, has provided an opportunity to monitor and investigate the complex cascade of molecular events that regulate tumor-host interactions. The availability of such large amounts of information has shifted the attention of scientists from a hypothesis-driven approach to biological phenomena (the analysis of one event at a time) to a "nonreductionist" approach, where thousands of observations are recorded at once. [9] Global gene expression analysis should be of great use in the field of immunology, [10] as it is clearly understood that the study of a single immunological parameter at one time is not sufficient to generate a general view of how the immune system fights a given pathogen or tumor, maintains self-tolerance or "memorizes" past encounters with antigens. [11] The analysis of complexity in biological systems might start from a simplified representation of static gene networks moving toward an increasingly well-defined and integrated description of biological phenomena, bearing in mind that only dynamic network models will probably explain the reality adequately. [12] High-throughput technologies can be used to follow changing patterns of gene expression over time. Among them, microarrays have become prominent because they are easier to use, do not require large-scale DNA sequencing, and allow the parallel quantification of thousands of genes across multiple samples. [13] Although functional genomics studies have demonstrated a tight correlation between the function of a protein and the expression patterns of its gene, [8] however, translational gene expression regulation and post-translational protein modifications are also of crucial importance in determining cell functions. Therefore, gene microarray technology should be complemented with other recently developed high-throughput assays such as tissue microarray [14] and proteomics. [15] Hopefully, by integrating these powerful analytic tools, investigators will be able to comprehensively describe the molecular portrait of the biological phenomena underlying tumor immune rejection. Escaping of Tumor Immunity Despite the evidence that immune effectors can play a significant role in controlling tumor growth in natural conditions or in response to therapeutic manipulation, it is evident that cancer cells can survive their attack as the disease progresses. Several mechanisms underlying immune escape have been proposed, such as down-regulation of human leukocyte antigen molecules/TAA on tumor cell surface, the production of immunosuppressive cytokines and the expression of lymphotoxic molecules (i.e., FAS ligand) by malignant cells. [16] The molecular events must be hypothesized in order to dissect this phenomenon that is acting as a brake for the development of effective anticancer immunotherapeutic strategies. Gene expression profiling led investigators to hypothesize that a tumor suppressor gene [i.e., retinoic acid receptor P2 (RARP2)] exerts its anticancer activity through the stimulation of the immune system. [17] Searching for genes regulated by this receptor, it is found that several of them code for proteins favoring an effective antitumor immune response, suggesting that down-regulation of these genes in RAR32-deficient tumor cells may contribute to immune system evasion. Several methods to make malignant cells "recognizable" by immune cells have been advocated. [18] One such method implies the exposition of target cells to sublethal doses of radiation. In order to dissect the molecules involved in this phenomenon of sensitization of cancer to the cytotoxic activity of the immune system, microarray analysis of various carcinoma cell lines treated with nonlytic doses of radiation (10-20 Gy) is used. [19] Overall, these findings might help design novel immunotherapeutic strategies able to overcome immune tolerance toward cancer. New Targets for Tumor Immunotherapy The molecular identification of TAA has opened new possibilities for the development of effective immunotherapies for patients with cancer. Although some TAA derive from mutated genes, most of them are products of nonmutated genes encoding intracellular proteins that are commonly expressed by autologous cancer cells. [20] Therefore, interest in antigen-specific cellular immune response has triggered enthusiasm for the development of vaccination regimens with T cell epitopes. Classically, the identification of TAA derived T cell epitopes requires patient-derived T cells and either a gene expression approach [21] or mass spectrometry based sequencing of the recognized peptides. [22] More recently, "reverse immunology" has been proposed as a novel approach to select HLA class I restricted epitopes from a given TAA. [23] Main drawbacks of T cell based strategies are the time-consuming culture techniques and, more importantly, their limitation by the frequency of preexisting epitope-specific T cells. Comparative expression profiling of a tumor and the corresponding autologous normal tissue enabled by gene microarray technology is an excellent method for identifying large numbers of candidate TAA from individual tumor samples. [24],[25],[26],[27],[28] Using this strategy, investigators have found that several genes were overexpressed by transplantable thymomas established from an inbred p53 mouse strain. [29],[30] Therefore, it seems appealing to screen the entire transcriptome of any given tumor to identify genes encoding proteins that encompass possible epitopes for peptide-based tumor-specific vaccines. A potential development of such strategy could be the utilization of microarray technology for designing patient-tailored TAA-based vaccination. [31] Dendritic Cell Biology Although tumor cells express TAA that can be recognized by T cells, advanced tumors are generally not immunogenic, at least in part because they do not express costimulatory molecules. The fate of TAA largely depends on their ability to be phagocytosed and processed by dendritic cells, the most powerful antigen presenting cells. [32] Dendritic cells expressing high levels of HLA class I and II and costimulatory molecules have demonstrated high efficiency and potency in presenting TAA peptides to enhance cellular immunity both in preclinical models and in humans. [2],[3] Despite the strong preclinical evidence supporting the use of dendritic cells for anticancer vaccination in humans, the results of clinical trials so far carried out do not seem to meet the expectations [2],[3] likely because the physiology of these cells is only partially understood and their therapeutic potential incompletely exploited. Immature dendritic cells capture TAA in the peripheral tissues, process them into peptides bound to HLA molecules, and then migrate to lymphoid organs where they present HLA-peptide complexes to T lymphocytes. Following the interaction with TAA-specific T helper lymphocytes, dendritic cells become activated through the CD40 signaling pathway, up-regulate HLA and costimulatory molecules′ expression on their surface and acquire a mature phenotype, characterized by the expression of new markers such as CD83 and by the secretion of pro-inflammatory and chemotactic cytokines. [33] Gene profiling studies have recently broadened the spectrum of genes distinguishing immature versus mature dendritic cells. [34] Mature dendritic cells prime cytotoxic T lymphocytes, thus polarizing the effector arm of the cell-mediated immune response against the pathogen. By contrast, dendritic cells conditioned by regulatory T suppressor cells are "licensed" to inhibit the initiation of the immune response by inducing T helper lymphocyte energy. [35] Using DNA microarray technology, the molecular portrait characterizing dendritic cells at different stages of maturation [36] is described. In an animal model, these researches could link two different dendritic cell gene patterns with two levels of effectiveness in inducing tumor regression mediated by dendritic cell based vaccine. If confirmed in a human model, these results might explain some vaccination failures observed in the clinical setting and indicate new avenues of research in the design of more effective dendritic cell preparation protocols for antitumor vaccines. Currently, most clinical protocols imply the expansion of dendritic cells with granulocyte/monocyte colony-stimulating factor (GM-CSF) combined with interleukin-4 (IL-4), while their maturation is induced with tumor necrosis factor (TNF). [37] The microarray-based study of the cascade of molecular events leading to a successful expansion/maturation of dendritic cells has already begun, [38] and novel strategies for improving dendritic cell based anticancer immunotherapy are expected in the near future. T Cell Biology As a direct consequence of TAA presentation by antigen presenting cells, native T cells become activated helper and cytotoxic T lymphocytes. Immune polarization can also differentially affect T cells, as demonstrated by the fact that human type 1/type 2 helper lymphocytes and cytotoxic T cell clones express substantially distinct sets of genes. In animal models it has been demonstrated that the activated tumor-specific effector T cells mainly comprise type 1 CD4+ and CD8+ lymphocytes, both of which are important for an effective antitumor immune response. [39] Thus, the cellular and molecular biology of these T cell subsets is of substantial interest in the context of both basic and clinical tumor immunology. Using microarray technology, investigators have started exploring the mRNA steady state of such tumor-specific T cells as compared to naive T cells in mice. [40] Gene expression profiling has been also applied to the study the mechanisms of partial T cell activation, which accounts for different cytotoxic capabilities and might determine the clinical outcome of vaccinated cancer bearing patients. [41],[42] Tumor Microenvironment Until recently, most studies addressing the immunological effects of vaccination in cancer patients have looked at variations in the level of TAA-specific reactivity in circulating lymphocytes. [43] Results from clinical trials have shown that vaccination can be quite effective in inducing tumor-specific T cell responses that can be easily observed among circulating lymphocytes. [2],[3] However, the identification of such immune responses could not be consistently correlated with tumor regression. Moreover, approaching immune follow-up of vaccinated cancer patients at a systemic level presents some intrinsic limitations. For instance, none of the assays currently in use can be considered ideal to reveal the activation status of anticancer immunity. Moreover, the study of circulating T cell responses yields only one type of information, though important, which consists of the documentation of whether a most often locally administered immunogen may induce a systemic effect beyond the draining lymph nodes. To this aim, it was proposed that a dynamic analysis of host-tumor interactions following immunotherapy could be most fruitfully performed by correlating clinical outcome with the gene expression kinetics of a given tumor lesion that could be easily accessed by repeated fine needle aspirates, such as in transit melanoma metastases. [44] The application of such a strategy combined with microarray-based gene profiling led to the finding that melanoma metastases undergoing complete regression in response to peptide/interleukin-2 (IL-2) based vaccination were characterized by a different transcript signature as compared to those progressing. [45] Interestingly, among Tumor Immune Antigen TIA-1 and interleukin-10 (IL-10), [46] many genes overexpressed in responding melanoma metastases were immune related. However, several preclinical models have shown that IL-10 can also mediate tumor regression by stimulating natural killer (NK) cells activity. [46] Furthermore, using cDNA microarray it was observed that in vitro IL-10 induced NK cell (but not cytotoxic T lymphocytes) expression of cytotoxicity related genes, including TIA-1. [47] These observations led to hypothesize that in the presence of high levels of IL-10 in the tumor microenvironment, NK cells might be stimulated to lyze cancer cells, thus increasing TAA availability and "danger signals" delivery required by dendritic cells to be activated and effectively prime cytotoxic T cells against TAA [Figure - 1]. Thus, NK cells may play in the early phase of adaptive immunity engagement against a noxious agent (e.g., infectious agent, tumor cells), thus providing a key link between innate and adaptive immunity. [48] If this theory were proved to be correct, future anticancer immunotherapy strategies should address the challenging task of stimulating both innate and adaptive immunity in a timely fashion. [49] As systemic IL-2 administration significantly increases the frequency of tumor regression induced by peptide-based vaccination of melanoma patients, [50] some authors investigated the role of this cytokine in facilitating an effective immune response. In a study, early changes in transcriptional profiles of circulating mononuclear cells were compared with those occurring within the microenvironment of melanoma metastases following systemic IL-2 administration. [51] The results of this microarray-based work suggested that IL-2 administration induces three predominant effects: (a) activation of antigen-presenting monocytes; (b) a massive production of chemoattractants that may recruit other immune cells to the tumor site, among which are the chemokines MIG (monokine induced by Gamma interferon) specific for T cells; and (c) the activation of lytic mechanisms ascribable to monocytes (calgranulin, grancalcin) and NK cells (e.g., NKG5, NK4). These findings suggest that systemic IL-2 administration may facilitate T cell effector function in the target organ not by sustaining their proliferation, as generally believed, but rather by promoting their migration and by providing a milieu conducive to their activation in situ through activation of antigen presenting cells. If this hypothesis were correct, then adoptive transfer of effector T cells should follow, rather than precede, administration of systemic IL-2. Conclusion Although we are only beginning to exploit the enormous potential of high-throughput technologies for dissecting the molecular events governing tumor immune rejection, preliminary results prompt investigators to pursue the genomic approach to tumor immunology. In fact, the high complexity of the immune network makes it difficult to understand the finely orchestrated molecular and cellular phenomena underlying tumor-host immune system interactions, looking at the expression of one or few molecules at a time, as the traditional research approach has so far sustained. Hopefully, a global view of the expression profiles of the several key players involved in tumor immune surveillance/tolerance will enable investigators to describe the sequence of events conducive to an effective immune recognition and killing of malignant cells, thus giving the opportunity to reproduce them in a larger series of patients. Besides shedding new light on the mechanisms of cancer immune rejection, microarray technology is expected to provide investigators with other information. For instance, clinicians might use gene profiling as a powerful tool to assess the activation status of the immune system (both in the peripheral blood and in the tumor microenvironment) of each patient before and during immunotherapy, thus opening the avenue to the personalization of the treatment. Only the broad implementation of microarray-generated data in the clinical protocols of anticancer immunotherapy will allow one to test the theoretically invaluable potential of such an approach. References
Copyright 2010 - Journal of Cancer Research and Therapeutics The following images related to this document are available:Photo images[cr10111f1.jpg] |
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