Angel Miguel Garcia-Lora

During the course of cancer, metastatic cells frequently enter a state of dormancy that can be controlled by the immune system. In our laboratory, we developed a preclinical mouse model of metastatic immunodormancy. Dormant spontaneous metastases are controlled by the immune system of wild‐type mice. Depletion of the host immune system causes these metastases to awaken and progress. Dormant metastases are compared with nude metastases and overt metastases that have never been in dormancy. The findings of the study indicate that the dormant metastases exhibit a unique and differentiated phenotype. This is evidenced by their varied response to nutrient‐restrictive conditions, chemotherapeutic agents, and cytokines in vitro. Furthermore, dormant metastases exhibit a distinctive transcriptional pattern of gene expression, which is predominantly promoted by the Ch25h gene. Additionally, the analysis revealed differential expression of microRNAs, with elevated levels of mir‐142‐3p being expressed de novo. The microenvironment of dormant metastases shows an increase in T lymphocytes (cytotoxic and helper T lymphocytes and γδ T cells) and neutrophils. Immune‐controlled dormant metastases exhibit a unique phenotype that can be exploited to discover new biomarkers, as well as to develop therapies to eradicate them or control overt metastases.

During the progression of cancer, metastatic cells frequently enter a dormant phase. The immune system plays a crucial role by restricting the growth of dormant metastases, although it cannot eradicate them. In our laboratory, we developed a preclinical mouse model of metastatic dormancy. Dormant spontaneous metastases are controlled by the immune system of wild-type mice. Depletion of the host immune system causes these metastases to awaken and progress. The elucidation of the phenotype, genes, miRNAs and immune cells involved in the phenomenon of metastatic dormancy may prove in the development of effective strategies for combating metastatic disease. Dormant Metastases are compared with Nude Metastases and Overt Metastases that have never been in dormancy. The findings of the study indicate that the dormant metastases exhibit a unique and differentiated phenotype. This is evidenced by their varied response to nutrient-restrictive conditions, chemotherapeutic agents, and cytokines in vitro . Additionally, dormant metastases display a unique pattern of gene and miRNA expression. The microenvironment of dormant metastases shows an increase in certain immune cell subpopulations. Immune-controlled dormant metastases exhibit a unique phenotype that can be exploited to discover new biomarkers, as well as to develop therapies to eradicate them or control overt metastases.

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.

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.

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.

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 their combination with various immunotherapeutic agents and cancer vaccines.

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 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. Cancer Res; 74(23); 6750–7. ©2014 AACR.