MHC heterogeneity and response of metastases to immunotherapy.

In recent years, immunotherapy has proven to be an effective treatment against cancer. Cytotoxic T lymphocytes perform an important role in this anti-tumor immune response, recognizing cancer cells as foreign, through the presentation of tumor antigens by MHC class I molecules. However, tumors and metastases develop escape mechanisms for evading this immunosurveillance and may lose the expression of these polymorphic molecules to become invisible to cytotoxic T lymphocytes. In other situations, they may maintain MHC class I expression and promote immunosuppression of cytotoxic T lymphocytes. Therefore, the analysis of the expression of MHC class I molecules in tumors and metastases is important to elucidate these escape mechanisms. Moreover, it is necessary to determine the molecular mechanisms involved in these alterations to reverse them and recover the expression of MHC class I molecules on tumor cells. This review discusses the role and regulation of MHC class I expression in tumor progression. We focus on altered MHC class I phenotypes present in tumors and metastases, as well as the molecular mechanisms responsible for MHC-I alterations, emphasizing the mechanisms of recovery of the MHC class I molecules expression on cancer cells. The individualized study of the HLA class I phenotype of the tumor and the metastases of each patient will allow choosing the most appropriate immunotherapy treatment based on a personalized medicine.

Generation of MHC class I diversity in primary tumors and selection of the malignant phenotype.

Intratumor heterogeneity among cancer cells is promoted by reversible or irreversible genetic alterations and by different microenvironmental factors. There is considerable experimental evidence of the presence of a variety of malignant cell clones with a wide diversity of major histocompatibility class I (MHC-I) expression during early stages of tumor development. This variety of MHC-I phenotypes may define the evolution of a particular tumor. Loss of MHC-I molecules frequently results in immune escape of MHC-negative or -deficient tumor cells from the host T cell-mediated immune response. We review here the results obtained by our group and other researchers in animal models and humans, showing how MHC-I intratumor heterogeneity may affect local oncogenicity and metastatic progression. In particular, we summarize the data obtained in an experimental mouse cancer model of a methylcholanthrene-induced fibrosarcoma (GR9), in which isolated clones with different MHC-I expression patterns demonstrated distinct local tumor growth rates and metastatic capacities. The observed "explosion of diversity" of MHC-I phenotypes in primary tumor clones and the molecular mechanism ("hard"/irreversible or "soft"/reversible) responsible for a given MHC-I alteration might determine not only the metastatic capacity of the cells but also their response to immunotherapy. We also illustrate the generation of further MHC heterogeneity during metastatic colonization and discuss different strategies to favor tumor rejection by counteracting MHC-I loss. Finally, we highlight the role of MHC-I genes in tumor dormancy and cell cycle control.

The pro-oxidant buthionine sulfoximine (BSO) reduces tumor growth of implanted Lewis lung carcinoma in mice associated with increased protein carbonyl, tubulin abundance, and aminopeptidase activity.

This study evaluated the effects of the pro-oxidant buthionine sulfoximine (BSO) and of the interaction between BSO and TETRAC, an antagonist of αvß3 integrin, on tumor development and aminopeptidase (AP) activity in a murine model of implanted Lewis's carcinoma. Male CBA-C57 mice were untreated (controls) or treated with BSO (222 mg/100 mL in drinking water), TETRAC (10 mg/kg/day, i.p.), or BSO + TETRAC. BSO for 28 days and TETRAC were given for the last 20 days. Mice were subcutaneously inoculated with 1 × 10(6) Lewis carcinoma 3LL cells into the dorsum. Study variables were tumor weight (TW); Hb, as index of tumor-mediated angiogenesis; vascular endothelial growth factor (VEGF) protein abundance; protein carbonyl content; α-tubulin abundance; and GluAp, AlaAp, and AspAp activities. BSO produced a major decrease in TW (203 ± 18 mg) with respect to controls (365 ± 26) and a reduction in Hb content. The TETRAC group also showed marked reductions in TW (129 ± 15) and Hb concentration associated with a reduced VEGF content. The BSO + TETRAC group showed a major TW reduction (125 ± 13); although, the difference with the TETRAC group was not significant. BSO treatment increased protein carbonyl and tubulin abundance in comparison to controls. The activity of all APs was increased in the three experimental groups and was strongly and negatively correlated with TW. In conclusion, administration of BSO reduced the TW, which inversely correlated with protein carbonyl content, suggesting a loss of microtubule polymerization. The finding of a negative correlation between TW and AP activity opens up new perspectives for the study of APs as tumor growth modulators.

Effect of thyroid hormone-nitric oxide interaction on tumor growth, angiogenesis, and aminopeptidase activity in mice.

This study evaluated the effects of thyroid hormone-NO interaction on tumor development, vascularization, vascular endothelial growth factor (VEGF), and aminopeptidase (AP) activity in a murine model of implanted Lewis's carcinoma. Experiments were performed in male CBA-C57 mice. Animals were untreated (controls) or treated with: T4, the antithyroid drug methimazole, the NO inhibitor L-NAME, T4+L-NAME, methimazole+NAME, the αvß3 integrin antagonist tetrac, T4+tetrac, the iNOS inhibitor aminoguanidine (AG), and T4 + AG; all treatments were for 6 weeks except for tetrac, administered for the last 11 days. Mice were subcutaneously inoculated with 1 × 10(6) exponentially growing Lewis carcinoma 3LL cells into the dorsum. Study variables 9 days later were tumor weight (TW), Hb content, an index of tumor vascularization, VEGF, and AP activity. T4 produced parallel increases in TW and angiogenesis. L-NAME reduced TW and angiogenesis in control, hyperthyroid, and hypothyroid mice, whereas AG had no effect on these variables. Tetrac arrested TW in normal and T4-treated mice but did not decrease angiogenesis in T4-treated animals. Negative correlations were found between TW and AP activity in tumors from control hyper- and hypothyroid groups and an inverse relationship was observed between TW and AP activities in tetrac-treated mice. T4 enhances TW and angiogenesis, in which NO participates, but requires activation of integrin αvß3 to promote carcinogenesis. NO blockade reduces TW, regardless of the thyroid status. Thyroid hormone negatively modulates AP activity in the tumor. Accordingly, blockade of the membrane TH receptor αvß3 integrin reduces TW associated with an increase in AP activity.

The tumour suppressor Fhit positively regulates MHC class I expression on cancer cells.

MHC class I (MHC-I) molecules are ubiquitously expressed on the cells of an organism. Study of the regulation of these molecules in normal and disease conditions is important. In tumour cells, the expression of MHC-I molecules is very frequently lost, allowing these cells to evade the immune response. Cancers of different histology have shown total loss of MHC-I molecule expression, due to a coordinated transcriptional down-regulation of various antigen-processing machinery (APM) components and/or MHC-I heavy chains. The mechanisms responsible for these alterations remain unclear. We determined the possible genes involved by comparing MHC-I-positive with MHC-I-negative murine metastases derived from the same fibrosarcoma tumour clone. MHC-I-negative metastases showed transcriptional down-regulation of APM and MHC-I heavy chains. The use of microarrays and subtraction cDNA libraries revealed four candidate genes responsible for this alteration, but two of them were ruled out by real-time RT-PCR analyses. The other two genes, AP-2α and Fhit tumour suppressors, were studied by using siRNA to silence their expression in a MHC-I-positive metastatic cell line. AP-2α inhibition did not modify transcriptional expression of APM components or MHC-I heavy chains or surface expression of MHC-I. In contrast, silencing of the Fhit gene produced the transcriptional down-regulation of APM components and MHC-I heavy chains and decreased MHC-I surface expression. Moreover, transfection of Fhit in MHC-I-negative tumour cell lines restored MHC-I cell surface expression. These data indicate that defects in Fhit expression may promote MHC-I down-regulation in cancer cells and allow escape from immunosurveillance(#).

MHC class I molecules act as tumor suppressor genes regulating the cell cycle gene expression, invasion and intrinsic tumorigenicity of melanoma cells.

The alteration of MHC class I (MHC-I) expression is a frequent event during cancer progression, allowing tumor cells to evade the immune system. We report that the loss of one major histocompatibility complex haplotype in human melanoma cells not only allowed them to evade immunosurveillance but also increased their intrinsic oncogenic potential. A second successive defect in MHC-I expression, MHC-I total downregulation, gave rise to melanoma cells that were more oncogenic per se in vivo and showed a higher proliferation rate and greater migratory and invasive potential in vitro. All these processes were reversed by restoring MHC-I expression via human leukocite antigen-A2 gene transfection. MHC-I cell surface expression was inversely correlated with intrinsic oncogenic potential. Modifications in the expression of various cell cycle genes were correlated with changes in MHC-I expression; the most important differences among the melanoma cell lines were in the transcriptional level of AP2-alpha, cyclin A1 and p21WAF1/CIP1. According to these results, altered MHC-I expression in malignant cells can directly increase their intrinsic oncogenic and invasive potential and modulate the expression of cell cycle genes. These findings suggest that human leukocite antigen class I molecules may act directly as tumor suppressor genes in melanoma.

The escape of cancer from T lymphocytes: immunoselection of MHC class I loss variants harboring structural-irreversible "hard" lesions.

The discovery of tumor antigens recognized by T lymphocytes has stimulated the development of a variety of cancer treatment protocols aimed at enhancing antitumor-specific T cell responses and tumor rejection. However, immunotherapy-mediated regression of established tumors and clearly positive clinical response to such treatment has not been achieved yet despite the induction of T cells directed against tumor antigens. The failure of the modern immunotherapy protocols can be explained by different tumor escape mechanisms that have been defined in various types of malignancy. The loss or downregulation of MHC class I antigens in tumor cells is one of the best analyzed mechanisms. In this review, we show experimental evidence obtained in our laboratory on human tumors and in a mouse cancer model suggesting that the molecular mechanism responsible for the MHC class I alteration in tumor cells might have a crucial impact on tumor recovery of normal H-2/HLA expression during the natural history of tumor development or after immunotherapy. When the preexisting molecular lesion underlying tumor MHC class I alteration is reversible (regulatory or soft), class I expression can be recovered leading to regression of tumor lesion. In contrast, if the HLA class I alteration is irreversible in nature (structural or hard), the lesion will progress killing the host. This is a new vision of the role of MHC class I alteration in tumors that can explain the failure of immunotherapy in a variety of different clinical protocols.

Alterations of HLA class I expression in human melanoma xenografts in immunodeficient mice occur frequently and are associated with higher tumorigenicity.

Animal models are widely used to study the biological behavior of human tumors in vivo. Murine immunodeficient models are used to test novel human anti-tumor therapies, and humanized mice are employed to study immunotherapeutic protocols. We find that human melanoma cell lines lose HLA class I surface expression after growth in immunodeficient mice and that this phenomenon occurs frequently and is reproducible. This HLA loss is due to a coordinated down-regulation of APM and HLA heavy chain expression at the transcriptional level. It is produced by epigenetic modifications and can be reversed by treatment with histone deacetylase inhibitors or IFN-gamma. These HLA alterations only appear during in vivo growth and not during successive in vitro passages. Interestingly, these new tumor variants with HLA class I loss show higher tumorigenicity per se and may represent a more advanced state of the original tumor. Lack of MHC class I expression on tumor cells represents a frequent escape mechanism from the immune response. Our results indicate that tumor variants with alterations in MHC can also appear in vivo after the immunoescape phase in the absence of anti-tumor immune response. Our findings suggest that any studied parameter, i.e., HLA expression, of malignant cells in xenograft models, has to be evaluated before and after growth in immunodeficient mice, in order to design more appropriate immunotherapy and chemotherapy treatments against tumor cells growing in vivo.

Restoration of MHC-I on Tumor Cells by Fhit Transfection Promotes Immune Rejection and Acts as an Individualized Immunotherapeutic Vaccine.

The capacity of cytotoxic-T lymphocytes to recognize and destroy tumor cells depends on the surface expression by tumor cells of MHC class I molecules loaded with tumor antigen peptides. Loss of MHC-I expression is the most frequent mechanism by which tumor cells evade the immune response. The restoration of MHC-I expression in cancer cells is crucial to enhance their immune destruction, especially in response to cancer immunotherapy. Using mouse models, we recovered MHC-I expression in the MHC-I negative tumor cell lines and analyzed their oncological and immunological profile. Fhit gene transfection induces the restoration of MHC-I expression in highly oncogenic MHC-I-negative murine tumor cell lines and genes of the IFN-γ transduction signal pathway are involved. Fhit-transfected tumor cells proved highly immunogenic, being rejected by a T lymphocyte-mediated immune response. Strikingly, this immune rejection was more frequent in females than in males. The immune response generated protected hosts against the tumor growth of non-transfected cells and against other tumor cells in our murine tumor model. Finally, we also observed a direct correlation between FHIT expression and HLA-I surface expression in human breast tumors. Recovery of Fhit expression on MHC class I negative tumor cells may be a useful immunotherapeutic strategy and may even act as an individualized immunotherapeutic vaccine.

MHC Intratumoral Heterogeneity May Predict Cancer Progression and Response to Immunotherapy.

An individual tumor can present intratumoral phenotypic heterogeneity, containing tumor cells with different phenotypes that do not present irreversible genetic alterations. We have developed a mouse cancer model, named GR9, derived from a methylcholanthrene-induced fibrosarcoma that was adapted to tissue culture and cloned into different tumor cell lines. The clones showed diverse MHC-I phenotypes, ranging from highly positive to weakly positive MHC-I expression. These MHC-I alterations are due to reversible molecular mechanisms, because surface MHC-I could be recovered by IFN-γ treatment. Cell clones with high MHC-I expression demonstrated low local oncogenicity and high spontaneous metastatic capacity, whereas MHC-I-low clones showed high local oncogenicity and no spontaneous metastatic capacity. Although MHC-I-low clones did not metastasize, they produced MHC-I-positive dormant micrometastases controlled by the host immune system, i.e., in a state of immunodormancy. The metastatic capacity of each clone was directly correlated with the host T-cell subpopulations; thus, a strong decrease in cytotoxic and helper T lymphocytes was observed in mice with numerous metastases derived from MHC-I positive tumor clones but a strong increase was observed in those with dormant micrometastases. Immunotherapy was administered to the hosts after excision of the primary tumor, producing a recovery in their immune status and leading to the complete eradication of overt spontaneous metastases or their decrease. According to these findings, the combination of MHC-I surface expression in primary tumor and metastases with host T-cell subsets may be a decisive indicator of the clinical outcome and response to immunotherapy in metastatic disease, allowing the identification of responders to this approach.

Upregulation of HLA Class I Expression on Tumor Cells by the Anti-EGFR Antibody Nimotuzumab.

Defining how epidermal growth factor receptor (EGFR)-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor - T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR), we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8+ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.

The urgent need to recover MHC class I in cancers for effective immunotherapy.

Immune escape strategies aimed to avoid T-cell recognition, including the loss of tumor MHC class I expression, are commonly found in malignant cells. Tumor immune escape has proven to have a negative effect on the clinical outcome of cancer immunotherapy, including treatment with antibodies blocking immune checkpoint molecules. Hence, there is an urgent need to develop novel approaches to overcome tumor immune evasion. MHC class I antigen presentation is often affected in human cancers and the capacity to induce upregulation of MHC class I cell surface expression is a critical step in the induction of tumor rejection. This review focuses on characterization of rejection, escape, and dormant profiles of tumors and its microenvironment with a special emphasis on the tumor MHC class I expression. We also discuss possible approaches to recover MHC class I expression on tumor cells harboring reversible/'soft' or irreversible/'hard' genetic lesions. Such MHC class I recovery approaches might well synergize with complementary forms of immunotherapy.

Metastases in immune-mediated dormancy: a new opportunity for targeting cancer.

The aim of any anticancer treatment is to avoid, control, or eliminate disseminated tumor cells. Clinical and experimental evidence has revealed that metastases can remain in a latency state, that is, metastasis dormancy. Three mechanisms are thought to be involved in cancer dormancy: cellular dormancy, angiogenic dormancy, and immune-mediated dormancy. Here, we review the mechanisms and cells involved in immune-mediated cancer dormancy and discuss current and future immunotherapeutic strategies. Recent results indicate that the immune system can restrain disseminated cancer cells, promoting their permanent dormancy. CD8(+) T lymphocytes play a relevant role in maintaining immune equilibrium with metastatic dormant cells, and MHC class I surface expression on tumor cells may also be involved. Natural killer (NK) cells have an activator function that triggers a cytotoxic T lymphocyte (CTL) response. Furthermore, immune dormancy promotes cancer cell growth arrest and angiogenic control. Immunotherapeutic interventions in metastatic dormancy may help to control or eradicate cancer disease. Treatments that activate or increase the CTL immune response or reverse cancer cell-induced CTL immunosuppression might be useful to restrain or destroy metastatic cells. These objectives may be achieved by recovering or increasing MHC class I surface expression on cancer cells or even by activating NK cells. Immune-mediated metastasis dormancy provides an opportunity for targeting cancer in novel immune treatments.

A novel preclinical murine model of immune-mediated metastatic dormancy.

The mechanisms underlying cancer dormancy are poorly understood. We have developed a preclinical murine model in which immunosurveillance restrains spontaneous metastases in permanent dormancy. The model faithfully recapitulates human metastatic dormancy and may be useful to decipher the immune mechanisms constraining disease progression, thereby facilitating the development of novel immunotherapeutic approaches to control metastatic disease.

T lymphocytes restrain spontaneous metastases in permanent dormancy.

Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.