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The tumour microenvironment is infiltrated by immunosuppressive cells, such as regulatory T cells (Tregs), which contribute to tumour escape and impede immunotherapy outcomes. Soluble fibrinogen-like protein 2 (sFGL2), a Treg effector protein, inhibits immune cell populations, via receptors FcγRIIB and FcγRIII, leading to downregulation of CD86 in antigen presenting cells and limiting T cell activation. Increased FGL2 expression is associated with tumour progression and poor survival in several different cancers, such as glioblastoma multiforme, lung, renal, liver, colorectal, and prostate cancer. Querying scRNA-seq human cancer data shows FGL2 is produced by cells in the tumour microenvironment (TME), particularly monocytes and macrophages as well as T cells and dendritic cells (DCs), while cancer cells have minimal expression of FGL2. We studied the role of FGL2 exclusively produced by cells in the TME, by leveraging Fgl2 knockout mice. We tested two murine models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer and melanoma. We show that absence of FGL2 leads to a more activated TME, including activated DCs (CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 leads to more activated natural killer cells (DNAM-1+, NKG2D+) in the TME. Furthermore, the absence of FGL2 leads to prolonged survival in the B16F10 melanoma model, while the absence of FGL2 synergizes with oncolytic virus to prolong survival in the ID8-p53-/-Brca2-/- ovarian cancer model. In conclusion, targeting FGL2 is a promising cancer treatment strategy alone and in combination immunotherapies.
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Fibrinogênio , Melanoma , Neoplasias Ovarianas , Animais , Feminino , Humanos , Camundongos , Células Apresentadoras de Antígenos , Carcinoma Epitelial do Ovário , Melanoma/genética , Melanoma/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Microambiente TumoralRESUMO
Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer. The disease is often diagnosed after wide-spread dissemination, and the standard treatment combines aggressive surgery with platinum-based chemotherapy; however, most patients experience relapse in the form of peritoneal carcinomatosis, resulting in a 5-year mortality below 45%. There is clearly a need for the development of novel treatments and cancer immunotherapies offering a different approach. Immunotherapies have demonstrated their efficacy in many types of cancers; however, only <15% of EOC patients show any evidence of response. One of the main barriers behind the poor therapeutic outcome is the reduced expression of Major Histocompatibility Complexes class I (MHC I) which occurs in approximately 60% of EOC cases. This review aims to gather and enhance our current understanding of EOC, focusing on its distinct cancer characteristics related to MHC I expression, immunogenicity, antigen presentation, epithelial-to-mesenchymal transition, and various ongoing immunotherapeutic strategies designed to stimulate antitumor immunity.
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Cancer cells often metastasize by undergoing an epithelial-mesenchymal transition (EMT). Although abundance of CD8+ T-cells in the tumor microenvironment correlates with improved survival, mesenchymal cancer cells acquire greater resistance to antitumor immunity in some cancers. We hypothesized the EMT modulates the immune response to ovarian cancer. Here we show that cancer cells from infiltrated/inflamed tumors possess more mesenchymal cells, than excluded and desert tumors. We also noted high expression of LGALS3 is associated with EMT in vivo, a finding validated with in vitro EMT models. Dissecting the cellular communications among populations in the tumor revealed that mesenchymal cancer cells in infiltrated tumors communicate through LGALS3 to LAG3 receptor expressed by CD8+ T cells. We found CD8+ T cells express high levels of LAG3, a marker of T cell exhaustion. The results indicate that EMT in ovarian cancer cells promotes interactions between cancer cells and T cells through the LGALS3 - LAG3 axis, which could increase T cell exhaustion in infiltrated tumors, dampening antitumor immunity.
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Linfócitos T CD8-Positivos , Neoplasias Ovarianas , Humanos , Feminino , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Galectina 3/genética , Galectina 3/metabolismo , Exaustão das Células T , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Microambiente TumoralRESUMO
Ovarian cancers exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate human disease are critical to develop new therapeutic approaches. Syngeneic mouse models allow for the generation of tumours comprising the full repertoire of non-malignant cell types but have expanded in number, varying in the cell type of origin, method for transformation, and ultimately, the properties of the tumours they produce. Here we have performed a comparative analysis of high-grade serous ovarian cancer models based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12. Among cell lines, we identify distinct signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic differences, such as reduced glycolysis-associated expression in several engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Among tumour samples, we observe increased variability and stromal content among intrabursal tumours. Finally, we predict differences in the microenvironment of ID8 models engineered with clinically relevant mutations. We anticipate that this work will serve as a valuable resource, providing new insight to help select models for specific experimental objectives.
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Neoplasias Ovarianas , Animais , Camundongos , Humanos , Feminino , Neoplasias Ovarianas/patologia , Perfilação da Expressão Gênica , Transdução de Sinais , Microambiente Tumoral/genéticaRESUMO
Clinical trials are currently exploring combinations of PARP inhibitors and immunotherapies for the treatment of ovarian cancer, but their effects on the ovarian tumour microenvironment (TME) remain unclear. Here, we investigate how olaparib, PD-L1 monoclonal antibodies, and their combination can influence TME composition and survival of tumour-bearing mice. We further explored how BRCA deficiencies can influence the response to therapy. Olaparib and combination therapies similarly improved the median survival of Brca1- and Brca2-deficient tumour-bearing mice. Anti-PD-L1 monotherapy improved the survival of mice with Brca1-null tumours, but not Brca2-null tumours. A detailed analysis of the TME revealed that olaparib monotherapy resulted in a large number of immunosuppressive and immunomodulatory effects in the more inflamed Brca1-deficient TME but not Brca2-deficient tumours. Anti-PD-L1 treatment was mostly immunosuppressive, resulting in a systemic reduction of cytokines and a compensatory increase in PD-L1 expression. The results of the combination therapy generally resembled the effects of one or both of the monotherapies, along with unique changes observed in certain immune populations. In-silico analysis of RNA-seq data also revealed numerous differences between Brca-deficient tumour models, such as the expression of genes involved in inflammation, angiogenesis and PD-L1 expression. In summary, these findings shed light on the influence of novel therapeutics and BRCA mutations on the ovarian TME.
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Antígeno B7-H1 , Neoplasias Ovarianas , Feminino , Humanos , Animais , Camundongos , Antígeno B7-H1/genética , Microambiente Tumoral/genética , Genes BRCA2 , Proteína BRCA1/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteína BRCA2/genéticaRESUMO
Many traditional cancer treatments such as radiation and chemotherapy are known to induce cellular DNA damage as part of their cytotoxic activity. The cGAS-STING signaling axis, a key member of the DNA damage response that acts as a sensor of foreign or aberrant cytosolic DNA, is helping to rationalize the DNA-damaging activity of these treatments and their emerging immunostimulatory capacity. Moreover, cGAS-STING, which is attracting considerable attention for its ability to promote antitumor immune responses, may fundamentally be able to address many of the barriers limiting the success of cancer immunotherapy strategies, including the immunosuppressive tumor microenvironment. Herein, we review the traditional cancer therapies that have been linked with cGAS-STING activation, highlighting their targets with respect to their role and function in the DNA damage response. As part of the review, an emerging "chemoimmunotherapy" concept whereby DNA-damaging agents are used for the indirect activation of STING is discussed as an alternative to the direct molecular agonism strategies that are in development, but have yet to achieve clinical approval. The potential of this approach to address some of the inherent and emerging limitations of cGAS-STING signaling in cancer immunotherapy is also discussed. Ultimately, it is becoming clear that in order to successfully employ the immunotherapeutic potential of the cGAS-STING axis, a balance between its contrasting antitumor and protumor/inflammatory activities will need to be achieved.
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Introduction: Epithelial ovarian cancer (OC) stands as one of the deadliest gynecologic malignancies, urgently necessitating novel therapeutic strategies. Approximately 60% of ovarian tumors exhibit reduced expression of major histocompatibility complex class I (MHC I), intensifying immune evasion mechanisms and rendering immunotherapies ineffective. NOD-like receptor CARD domain containing 5 (NLRC5) transcriptionally regulates MHC I genes and many antigen presentation machinery components. We therefore explored the therapeutic potential of NLRC5 in OC. Methods: We generated OC cells overexpressing NLRC5 to rescue MHC I expression and antigen presentation and then assessed their capability to respond to PD-L1 blockade and an infected cell vaccine. Results: Analysis of microarray datasets revealed a correlation between elevated NLRC5 expression and extended survival in OC patients; however, NLRC5 was scarcely detected in the OC tumor microenvironment. OC cells overexpressing NLRC5 exhibited slower tumor growth and resulted in higher recruitment of leukocytes in the TME with lower CD4/CD8 T-cell ratios and increased activation of T cells. Immune cells from peripheral blood, spleen, and ascites from these mice displayed heightened activation and interferon-gamma production when exposed to autologous tumor-associated antigens. Finally, as a proof of concept, NLRC5 overexpression within an infected cell vaccine platform enhanced responses and prolonged survival in comparison with control groups when challenged with parental tumors. Discussion: These findings provide a compelling rationale for utilizing NLRC5 overexpression in "cold" tumor models to enhance tumor susceptibility to T-cell recognition and elimination by boosting the presentation of endogenous tumor antigens. This approach holds promise for improving antitumoral immune responses in OC.
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Neoplasias Ovarianas , Vacinas , Humanos , Feminino , Animais , Camundongos , Proteínas NLR , Domínio de Ativação e Recrutamento de Caspases , Microambiente Tumoral , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Classe I , Neoplasias Ovarianas/genética , Antígenos de NeoplasiasRESUMO
Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer with an imperative need for new treatments. Immunotherapy has had marked success in some cancer types; however, clinical trials studying the efficacy of immune checkpoint inhibitors for the treatment of EOC benefited less than 15% of patients. Given that EOC develops from multiple tissues in the reproductive system and metastasizes widely throughout the peritoneal cavity, responses to immunotherapy are likely hindered by heterogeneous tumor microenvironments (TME) containing a variety of immune profiles. To fully characterize and compare syngeneic model systems that may reflect this diversity, we determined the immunogenicity of six ovarian tumor models in vivo, the T and myeloid profile of orthotopic tumors and the immune composition and cytokine profile of ascites, by single-cell RNA sequencing, flow cytometry and IHC. The selected models reflect the different cellular origins of EOC (ovarian and fallopian tube epithelium) and harbor mutations relevant to human disease, including Tp53 mutation, PTEN suppression, and constitutive KRAS activation. ID8-p53-/- and ID8-C3 tumors were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumors were primarily infiltrated by tumor associated macrophages and were unique in MHC class I and II expression. MOE-PTEN/KRAS tumors were capable of forming T cell clusters. This panel of well-defined murine EOC models reflects some of the heterogeneity found in human disease and can serve as a valuable resource for studies that aim to test immunotherapies, explore the mechanisms of immune response to therapy, and guide selection of treatments for patient populations.
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Neoplasias Ovarianas , Proteínas Proto-Oncogênicas p21(ras) , Camundongos , Humanos , Feminino , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Ovarianas/terapia , Carcinoma Epitelial do Ovário/terapia , Imunoterapia , Microambiente TumoralRESUMO
The implications of the epithelial-mesenchymal transition (EMT) mechanisms in the initiation and progression of epithelial ovarian cancer (EOC) remain poorly understood. We have previously shown that suppression of the antigen receptor LY75 directs mesenchymal-epithelial transition (MET) in EOC cell lines with the mesenchymal phenotype, associated with the loss of Wnt/ß-catenin signaling activity. In the present study, we used the LY75-mediated modulation of EMT in EOC cells as a model in order to investigate in vivo the specific role of EOC cells, with an epithelial (E), mesenchymal (M) or mixed epithelial plus mesenchymal (E+M) phenotype, in EOC initiation, dissemination and treatment response, following intra-bursal (IB) injections of SKOV3-M (control), SKOV3-E (Ly75KD) and a mixed population of SKOV3-E+M cells, into severe combined immunodeficiency (SCID) mice. We found that the IB-injected SKOV3-E cells displayed considerably higher metastatic potential and resistance to treatment as compared to the SKOV3-M cells, due to the acquisition of a Ly75KD-mediated hybrid phenotype and stemness characteristics. We also confirmed in vivo that the LY75 depletion directs suppression of the Wnt/ß-catenin pathway in EOC cells, suggestive of a protective role of this pathway in EOC etiology. Moreover, our data raise concerns regarding the use of LY75-targeted vaccines for dendritic-cell EOC immunotherapy, due to the possible occurrence of undesirable side effects.
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Antígenos CD/genética , Carcinogênese/genética , Carcinoma Epitelial do Ovário/genética , Regulação Neoplásica da Expressão Gênica , Lectinas Tipo C/genética , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Ovarianas/genética , Receptores de Superfície Celular/genética , Animais , Antineoplásicos/uso terapêutico , Carboplatina/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Neoplasias Experimentais , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologiaRESUMO
Immunotherapy has emerged as one of the most promising approaches for ovarian cancer treatment. The tumor microenvironment (TME) is a key factor to consider when stimulating antitumoral responses as it consists largely of tumor promoting immunosuppressive cell types that attenuate antitumor immunity. As our understanding of the determinants of the TME composition grows, we have begun to appreciate the need to address both inter- and intra-tumor heterogeneity, mutation/neoantigen burden, immune landscape, and stromal cell contributions. The majority of immunotherapy studies in ovarian cancer have been performed using the well-characterized murine ID8 ovarian carcinoma model. Numerous other animal models of ovarian cancer exist, but have been underutilized because of their narrow initial characterizations in this context. Here, we describe animal models that may be untapped resources for the immunotherapy field because of their shared genomic alterations and histopathology with human ovarian cancer. We also shed light on the strengths and limitations of these models, and the knowledge gaps that need to be addressed to enhance the utility of preclinical models for testing novel immunotherapeutic approaches.
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Immunotherapy as a treatment for cancer is a growing field of endeavor but reports of success have been limited for epithelial ovarian cancer. Overcoming the challenges to developing more effective therapeutic approaches lies in a better understanding of the factors in cancer cells and the surrounding tumor microenvironment that limit response to immunotherapies. This article provides an overview of some ovarian cancer cell features such as tumor-associated antigens, ovarian cancer-derived exosomes, tumor mutational burden and overexpression of immunoinhibitory molecules. Moreover, we describe relevant cell types found in epithelial ovarian tumors including immune cells (T and B lymphocytes, Tregs, NK cells, TAMs, MDSCs) and other components found in the tumor microenvironment including fibroblasts and the adipocytes in the omentum. We focus on how those components may influence responses to standard treatments or immunotherapies.
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Cancers can escape immunesurveillance by diminishing the expression of MHC class-I molecules (MHC-I) and components of the antigen-processing machinery (APM). Developing new approaches to reverse these defects could boost the efforts to restore antitumor immunity. Recent studies have shown that the expression of MHC-I and antigen-processing molecules is transcriptionally regulated by NOD-like receptor CARD domain containing 5 (NLRC5). To investigate whether NLRC5 could be used to improve tumor immunogenicity, we established stable lines of B16-F10 melanoma cells expressing NLRC5 (B16-5), the T cell co-stimulatory molecule CD80 (B16-CD80) or both (B16-5/80). Cells harboring NLRC5 constitutively expressed MHC-I and LMP2, LMP7 and TAP1 genes of the APM. The B16-5 cells efficiently presented the melanoma antigenic peptide gp10025-33 to Pmel-1 TCR transgenic CD8(+) T cells and induced their proliferation. In the presence of CD80, B16-5 cells stimulated Pmel-1 cells even without the addition of gp100 peptide, indicating that NLRC5 facilitated the processing and presentation of endogenous tumor antigen. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that the NLRC5-expressing tumor cells elicited antitumor immunity. Following intravenous injection, B16-5 and B16-5/80 cells formed fewer lung tumor foci compared to control cells. In mice depleted of CD8(+) T cells, B16-5 cells formed large subcutaneous and lung tumors. Finally, immunization with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Collectively, our findings indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity.
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Suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of T lymphocyte homeostasis. SOCS1-deficient mice accumulate CD8(+) T cells, which display a memory-like phenotype and proliferate strongly to IL-15. Socs1(-/-) mice develop inflammatory skin lesions, however, the underlying mechanisms are not well understood. In order to investigate the role of SOCS1 in regulating CD8(+) T cells potentially reactive to tissue antigens (Ags) of the skin, we generated Socs1(-/-) mice expressing MHC-I-restricted Pmel-1 transgenic TCR specific to the melanoma-derived gp100 Ag, which is also expressed by normal melanocytes. Socs1(-/-) Pmel-1 cells express increased levels of memory markers CD44, Ly6C, CD122, and CD62L, and show downregulation of TCR and upregulation of CD5, suggesting in vivo TCR stimulation. However, stimulation of Socs1(-/-)Pmel-1 cells with gp100-derived peptide induced only marginal proliferation in vitro despite eliciting strong effector functions, which was associated with elevated Blimp-1 induction. Following adoptive transfer to Rag1(-/-) mice, Socs1(-/-)Pmel-1 cells underwent lymphopenia-induced proliferation and caused severe skin pathology characterized by inflammatory lesions in ears, muzzle, extremities, and eyes. These findings underscore the importance of SOCS1 in regulating potentially skin-reactive cytotoxic T lymphocytes, which could get activated under conditions that promote Ag-nonspecific, cytokine-driven proliferation.
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Dermatite/imunologia , Memória Imunológica/imunologia , Linfopenia/imunologia , Proteínas Supressoras da Sinalização de Citocina/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Ly/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Dermatite/genética , Dermatite/patologia , Proteínas de Homeodomínio/genética , Receptores de Hialuronatos/imunologia , Subunidade beta de Receptor de Interleucina-2/imunologia , Selectina L/imunologia , Ativação Linfocitária/imunologia , Linfopenia/genética , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T Citotóxicos/patologia , Fatores de Transcrição/imunologia , Antígeno gp100 de Melanoma/imunologiaRESUMO
Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHC expression and revealed NLRC5 as an important regulator of basal MHC class I expression.