ABSTRACT
Regenerative stem cell-like memory (TSCM) CD8+ T cells persist longer and produce stronger effector functions. We found that MEK1/2 inhibition (MEKi) induces TSCM that have naive phenotype with self-renewability, enhanced multipotency and proliferative capacity. This is achieved by delaying cell division and enhancing mitochondrial biogenesis and fatty acid oxidation, without affecting T cell receptor-mediated activation. DNA methylation profiling revealed that MEKi-induced TSCM cells exhibited plasticity and loci-specific profiles similar to bona fide TSCM isolated from healthy donors, with intermediate characteristics compared to naive and central memory T cells. Ex vivo, antigenic rechallenge of MEKi-treated CD8+ T cells showed stronger recall responses. This strategy generated T cells with higher efficacy for adoptive cell therapy. Moreover, MEKi treatment of tumor-bearing mice also showed strong immune-mediated antitumor effects. In conclusion, we show that MEKi leads to CD8+ T cell reprogramming into TSCM that acts as a reservoir for effector T cells with potent therapeutic characteristics.
Subject(s)
Antineoplastic Agents/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Immunologic Memory/drug effects , Immunotherapy, Adoptive , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neoplasms/therapy , Stem Cells/cytology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Cycle/drug effects , Humans , Immunologic Memory/immunology , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Receptors, Antigen, T-Cell/physiology , Tumor MicroenvironmentABSTRACT
Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.
Subject(s)
Colorectal Neoplasms/diagnosis , Immunoassay/methods , Pathology, Molecular/methods , T-Lymphocyte Subsets/immunology , Th1 Cells/immunology , Aged , Aged, 80 and over , Cells, Cultured , Colorectal Neoplasms/mortality , Cytotoxicity Tests, Immunologic , DNA Mutational Analysis , Female , Frameshift Mutation/genetics , Humans , Immunologic Memory , Male , Microsatellite Instability , Microsatellite Repeats , Predictive Value of Tests , Prognosis , Survival Analysis , TranscriptomeABSTRACT
The NLRP3 inflammasome is a multiprotein complex that plays a critical role in activating the immune system in response to danger signals. Small molecule agonists of NLRP3 may offer clinical benefits in cancer immunology either as a monotherapy or in combination with checkpoint blockade, where it is hypothesised that their application can help to initiate an antitumor immune response. In this study, we report the discovery of quinazolines and 8-azaquinazolines as NLRP3 agonists and their chemical optimization to afford compounds with oral bioavailability in mice. We confirm that these compounds engage the NLRP3 inflammasome by verifying their dependence upon lipopolysaccharide (LPS) priming for cytokine release and the activation of Caspase-1. We further demonstrate pathway engagement through loss of activity in an NLRP3-knockout THP1 cell line. Based on their pharmacokinetic profile and biological activity, these compounds represent valuable tools to evaluate the therapeutic potential of NLRP3 activation in a pre-clinical setting.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , Mice , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Biological Availability , Quinazolines/pharmacology , Quinazolines/metabolism , Macrophages/metabolism , Caspase 1/metabolism , Lipopolysaccharides/pharmacology , Interleukin-1beta/metabolismABSTRACT
BACKGROUND: Major histocompatibility complex (MHC) class I chain-related protein A (MICA) and MHC class I chain-related protein B (MICB) are polymorphic proteins that are induced upon stress, damage or transformation of cells which act as a 'kill me' signal through the natural-killer group 2, member D receptor expressed on cytotoxic lymphocytes. MICA/B are not thought to be constitutively expressed by healthy normal cells but expression has been reported for most tumour types. However, it is not clear how much of this protein is expressed on the cell surface. METHODS: Using a novel, well-characterised antibody and both standard and confocal microscopy, we systematically profiled MICA/B expression in multiple human tumour and normal tissue. RESULTS: High expression of MICA/B was detected in the majority of tumour tissues from multiple indications. Importantly, MICA/B proteins were predominantly localised intracellularly with only occasional evidence of cell membrane localisation. MICA/B expression was also demonstrated in most normal tissue epithelia and predominantly localised intracellularly. Crucially, we did not observe qualitative differences in cell surface expression between tumour and MICA/B expressing normal epithelia. CONCLUSIONS: This demonstrates for the first time that MICA/B is more broadly expressed in normal tissue and that expression is mainly intracellular with only a small fraction appearing on the cell surface of some epithelia and tumour cells.
Subject(s)
Histocompatibility Antigens Class I/biosynthesis , Neoplasms/genetics , Cell Line, Tumor , Cell Membrane/genetics , Cytoplasm/genetics , Gene Expression Regulation, Neoplastic , Histocompatibility Antigens Class I/genetics , Humans , Killer Cells, Natural/immunology , Neoplasms/classification , Neoplasms/pathology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Introduction: Immuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response. Methods: Using aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs. Results: We found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice. Discussion: These differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.
Subject(s)
Neoplasms , Mice , Animals , ImmunotherapyABSTRACT
Single-cell technologies have elucidated mechanisms responsible for immune checkpoint inhibitor (ICI) response, but are not amenable to a clinical diagnostic setting. In contrast, bulk RNA sequencing (RNA-seq) is now routine for research and clinical applications. Our workflow uses transcription factor (TF)-directed coexpression networks (regulons) inferred from single-cell RNA-seq data to deconvolute immune functional states from bulk RNA-seq data. Regulons preserve the phenotypic variation in CD45+ immune cells from metastatic melanoma samples (n = 19, discovery dataset) treated with ICIs, despite reducing dimensionality by >100-fold. Four cell states, termed exhausted T cells, monocyte lineage cells, memory T cells, and B cells were associated with therapy response, and were characterized by differentially active and cell state-specific regulons. Clustering of bulk RNA-seq melanoma samples from four independent studies (n = 209, validation dataset) according to regulon-inferred scores identified four groups with significantly different response outcomes (P < 0.001). An intercellular link was established between exhausted T cells and monocyte lineage cells, whereby their cell numbers were correlated, and exhausted T cells predicted prognosis as a function of monocyte lineage cell number. The ligand-receptor expression analysis suggested that monocyte lineage cells drive exhausted T cells into terminal exhaustion through programs that regulate antigen presentation, chronic inflammation, and negative costimulation. Together, our results demonstrate how regulon-based characterization of cell states provide robust and functionally informative markers that can deconvolve bulk RNA-seq data to identify ICI responders.
Subject(s)
Gene Regulatory Networks , Melanoma , Humans , Melanoma/drug therapy , Melanoma/genetics , Immunotherapy , Leukocytes , Antigen PresentationABSTRACT
The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.
Subject(s)
Interferon Type I , Neoplasms , DNA , DNA Damage , Dendritic Cells/metabolism , Exodeoxyribonucleases , Indoles , Membrane Proteins/genetics , Membrane Proteins/metabolism , Morpholines , Neoplasms/genetics , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Pyrimidines , SulfonamidesABSTRACT
PARP inhibitors are synthetically lethal with BRCA1/2 mutations, and in this setting, accumulation of DNA damage leads to cell death. Because increased DNA damage and subsequent immune activation can prime an anti-tumor immune response, we studied the impact of olaparib ± immune checkpoint blockade (ICB) on anti-tumor activity and the immune microenvironment. Concurrent combination of olaparib, at clinically relevant exposures, with ICB gave durable and deeper anti-tumor activity in the Brca1m BR5 model vs. monotherapies. Olaparib and combination treatment modulated the immune microenvironment, including increases in CD8+ T cells and NK cells, and upregulation of immune pathways, including type I IFN and STING signaling. Olaparib also induced a dose-dependent upregulation of immune pathways, including JAK/STAT, STING and type I IFN, in the tumor cell compartment of a BRCA1m (HBCx-10) but not a BRCA WT (HBCx-9) breast PDX model. In vitro, olaparib induced BRCAm tumor cell-specific dendritic cell transactivation. Relevance to human disease was assessed using patient samples from the MEDIOLA (NCT02734004) trial, which showed increased type I IFN, STING, and JAK/STAT pathway expression following olaparib treatment, in line with preclinical findings. These data together provide evidence for a mechanism and schedule underpinning potential benefit of ICB combination with olaparib.
Subject(s)
Ovarian Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Clinical Trials as Topic , Female , Humans , Immunity , Janus Kinases/metabolism , Janus Kinases/pharmacology , Janus Kinases/therapeutic use , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , STAT Transcription Factors/metabolism , STAT Transcription Factors/pharmacology , STAT Transcription Factors/therapeutic use , Signal Transduction , Tumor MicroenvironmentABSTRACT
Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.
Subject(s)
Ataxia Telangiectasia Mutated Proteins , Cancer-Associated Fibroblasts , Immunotherapy , Neoplasms , Humans , Ataxia Telangiectasia Mutated Proteins/metabolism , Cancer-Associated Fibroblasts/metabolism , Cell Differentiation , Myofibroblasts/metabolism , Drug Resistance, NeoplasmABSTRACT
Natural killer (NK) cells are innate lymphocytes that play a pivotal role in the immune surveillance and elimination of transformed or virally infected cells. Using a chemo-genetic approach, we identify BET bromodomain containing proteins BRD2 and BRD4 as central regulators of NK cell functions, including direct cytokine secretion, NK cell contact-dependent inflammatory cytokine secretion from monocytes as well as NK cell cytolytic functions. We show that both BRD2 and BRD4 control inflammatory cytokine production in NK cells isolated from healthy volunteers and from rheumatoid arthritis patients. In contrast, knockdown of BRD4 but not of BRD2 impairs NK cell cytolytic responses, suggesting BRD4 as critical regulator of NK cell mediated tumor cell elimination. This is supported by pharmacological targeting where the first-generation pan-BET bromodomain inhibitor JQ1(+) displays anti-inflammatory effects and inhibit tumor cell eradication, while the novel bivalent BET bromodomain inhibitor AZD5153, which shows differential activity towards BET family members, does not. Given the important role of both cytokine-mediated inflammatory microenvironment and cytolytic NK cell activities in immune-oncology therapies, our findings present a compelling argument for further clinical investigation.
Subject(s)
Inflammation/metabolism , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Transcription Factors/antagonists & inhibitors , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , Azepines/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cytokines , Healthy Volunteers , Heterocyclic Compounds, 2-Ring/pharmacology , High-Throughput Nucleotide Sequencing , Humans , Piperazines/pharmacology , Pyrazoles/pharmacology , Pyridazines/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome , Triazoles/pharmacologyABSTRACT
PURPOSE: Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. EXPERIMENTAL DESIGN: AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. RESULTS: AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. CONCLUSIONS: Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.
Subject(s)
Cell Line, Tumor/radiation effects , DNA-Activated Protein Kinase/antagonists & inhibitors , Interferon Type I/drug effects , Neoplasms/radiotherapy , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrans/pharmacology , Radiation-Sensitizing Agents/pharmacology , Triazoles/pharmacology , Animals , MiceABSTRACT
Monoclonal antibodies are now established as a key therapeutic modality for a range of diseases. Owing to the ability of these agents to selectively target tumour cells, cancer has been a major focus of development programmes for monoclonal antibodies so far. Here, we overview trends in the clinical development and regulatory approval of monoclonal antibodies for cancer since 1980, with the aim of informing future research and development for this class of therapeutics.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Medical Oncology/trends , Neoplasms/drug therapy , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Drug Industry , HumansABSTRACT
We report the first disclosure of IRAK3 degraders in the scientific literature. Taking advantage of an opportune byproduct obtained during our efforts to identify IRAK4 inhibitors, we identified ready-to-use, selective IRAK3 ligands in our compound collection with the required properties for conversion into proteolysis-targeting chimera (PROTAC) degraders. This work culminated with the discovery of PROTAC 23, which we demonstrated to be a potent and selective degrader of IRAK3 after 16 h in THP1 cells. 23 induced proteasome-dependent degradation of IRAK3 and required both CRBN and IRAK3 binding for activity. We conclude that PROTAC 23 constitutes an excellent in vitro tool with which to interrogate the biology of IRAK3.
Subject(s)
Interleukin-1 Receptor-Associated Kinases/metabolism , Phthalimides/pharmacology , Proteolysis/drug effects , Pyrroles/pharmacology , Triazines/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Humans , Ligands , Phthalimides/chemical synthesis , Pyrroles/chemical synthesis , THP-1 Cells , Triazines/chemical synthesis , Ubiquitin-Protein Ligases/metabolismABSTRACT
In vitro assays that evaluate CD8+ T cell-mediated cytotoxicity are important to aid in the development of novel therapeutic approaches to enhance anti-tumor immune responses. Here, we describe a novel cytotoxicity co-culture assay that circumvents the problem of highly variable allogeneic responses and obviates the constraints of HLA-restriction between effector and target cells. We show that this assay can be easily applied to a panel of tumor cell lines to provide additional insights into intrinsic drivers of sensitivity/resistance to T cell-mediated killing, and to evaluate the impact of targeted therapies on both tumor and T cell compartments.
ABSTRACT
Background: This study assesses how the metastatic immune landscape is impacting the response to treatment and the outcome of colorectal cancer (CRC) patients. Methods: Complete curative resection of metastases (n = 441) was performed for two patient cohorts (n = 153). Immune densities were quantified in the center and invasive margin of all metastases. Immunoscore and T and B cell (TB) score were analyzed in relation to radiological and pathological responses and patient's disease-free (DFS) and overall survival (OS) using multivariable Cox proportional hazards models. All statistical tests were two-sided. Results: The spatial distribution of immune cells within metastases was nonuniform. Patients, as well as metastases of the same patient, had variable immune infiltrates and response to therapy. A beneficial response was statistically significantly associated with increased immune densities. Among all metastases, Immunoscore (I) and TB score evaluated in the least immune-infiltrated metastases were the strongest predictors for DFS and OS (five-year follow-up, Immunoscore: I 3-4: DFS rate = 27.9%, 95% CI = 15.2 to 51.3; vs I 0-1-2: DFS rate = 12.3%, 95% CI = 4.9 to 30.6; HR = 0.45, 95% CI = 0.28 to 0.70, P = .02; I 3-4: OS rate = 64.6%, 95% CI = 46.6 to 89.6; vs I 0-1-2: OS rate = 32.5%, 95% CI = 17.2 to 61.4; HR = 0.32, 95% CI = 0.15 to 0.66, P = .001, C-index = 65.9%; five-year follow-up, TB score: TB 3-4: DFS rate = 25.7%, 95% CI = 14.2 to 46.6; vs TB 0-1-2: DFS rate = 5.0%, 95% CI = 0.8 to 32.4; HR = 0.36, 95% CI = 0.22 to 0.57, P < .001; TB 3-4: OS rate = 63.7%, 95% CI = 46.4 to 87.5; vs TB 0-1-2: OS rate: 21.4%, 95% CI = 9.2 to 49.8; HR = 0.25, 95% CI = 0.12 to 0.51, P < .001, C-index = 67.8%). High TB score and Immunoscore patients had a median survival of 70.5 months, while low patients survived only 25.1 to 38.3 months. Nonresponding patients with high-immune infiltrates had prolonged DFS (HR = 0.28, 95% CI = 0.15 to 0.52, P = .001) and OS (HR = 0.25, 95% CI = 0.1 to 0.62, P = .001). The immune parameters remained the only statistically significant prognostic factor associated with DFS and OS in multivariable analysis (P < .001), while response to treatment was not. Conclusions: Response to treatment and prolonged survival of metastatic CRC patients were statistically significantly associated with high-immune densities quantified into the least immune-infiltrated metastasis.
Subject(s)
B-Lymphocytes , Colorectal Neoplasms/immunology , Liver Neoplasms/immunology , Lung Neoplasms/immunology , Lymphocytes, Tumor-Infiltrating , T-Lymphocytes , Aged , Antigens, CD20/analysis , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B-Lymphocytes/chemistry , CD3 Complex/analysis , CD8-Positive T-Lymphocytes , Chemotherapy, Adjuvant , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Disease-Free Survival , Follow-Up Studies , Forkhead Transcription Factors/analysis , Hepatectomy , Humans , Leukocyte Common Antigens/analysis , Liver Neoplasms/secondary , Liver Neoplasms/therapy , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Lymphocyte Count , Metastasectomy , Middle Aged , Neoplasm Metastasis , Pneumonectomy , Preoperative Period , Response Evaluation Criteria in Solid Tumors , Survival Rate , T-Lymphocytes/chemistry , Tumor Microenvironment/immunologyABSTRACT
Treatment of metastatic colorectal cancer is based upon the assumption that metastases are homogeneous within a patient. We quantified immune cell types of 603 whole-slide metastases and primary colorectal tumors from 222 patients. Primary lesions, and synchronous and metachronous metastases, had a heterogeneous immune infiltrate and mutational diversity. Small metastases had frequently a low Immunoscore and T and B cell score, while a high Immunoscore was associated with a lower number of metastases. Anti-epidermal growth factor receptor treatment modified immune gene expression and significantly increased T cell densities in the metastasis core. The predictive accuracy of the Immunoscore from a single biopsy was superior to the one of programmed cell death ligand 1 (PD-L1). The immune phenotype of the least-infiltrated metastasis had a stronger association with patient outcome than other metastases.
Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , ErbB Receptors/antagonists & inhibitors , Lymphocytes, Tumor-Infiltrating/drug effects , Tumor Microenvironment/drug effects , Adult , Aged , Aged, 80 and over , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Cohort Studies , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , ErbB Receptors/immunology , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/immunology , Gene Ontology , Gene Regulatory Networks , Humans , Kaplan-Meier Estimate , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Male , Middle Aged , Neoplasm Metastasis , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Drug Discovery , Drug Evaluation, Preclinical , Animals , B7-H1 Antigen/antagonists & inhibitors , CTLA-4 Antigen/antagonists & inhibitors , Comparative Genomic Hybridization , DNA Copy Number Variations , Disease Models, Animal , Drug Synergism , Exome , Gene Expression Regulation, Neoplastic/drug effects , Genomics/methods , High-Throughput Nucleotide Sequencing , Immunomodulation/drug effects , Immunomodulation/genetics , Mice , Molecular Targeted Therapy , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/metabolism , Signal Transduction/drug effects , Transcriptome , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Although distant metastases account for most of the deaths in cancer patients, fundamental questions regarding mechanisms that promote or inhibit metastasis remain unanswered. We show the impact of mutations, genomic instability, lymphatic and blood vascularization, and the immune contexture of the tumor microenvironment on synchronous metastases in large cohorts of colorectal cancer patients. We observed large genetic heterogeneity among primary tumors, but no major differences in chromosomal instability or key cancer-associated mutations. Similar patterns of cancer-related gene expression levels were observed between patients. No cancer-associated genes or pathways were associated with M stage. Instead, mutations of FBXW7 were associated with the absence of metastasis and correlated with increased expression of T cell proliferation and antigen presentation functions. Analyzing the tumor microenvironment, we observed two hallmarks of the metastatic process: decreased presence of lymphatic vessels and reduced immune cytotoxicity. These events could be the initiating factors driving both synchronous and metachronous metastases. Our data demonstrate the protective impact of the Immunoscore, a cytotoxic immune signature, and increased marginal lymphatic vessels, against the generation of distant metastases, regardless of genomic instability.
Subject(s)
Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Tumor Microenvironment/immunology , Blood Vessels/pathology , Cell Count , Cell Death , Colorectal Neoplasms/genetics , Disease Progression , Gene Expression Regulation, Neoplastic , Genome, Human , Humans , Lymphatic System/pathology , Lymphocytes/metabolism , Mutation/genetics , Neoplasm Metastasis , Tumor Microenvironment/geneticsABSTRACT
Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.
Subject(s)
Immunotherapy/methods , Neoplasms/therapy , Receptors, Tumor Necrosis Factor, Type II/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Drug Screening Assays, Antitumor/methods , Female , HEK293 Cells , Humans , Jurkat Cells , Mice, Inbred BALB C , NF-kappa B/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/therapy , Phenotype , Receptors, Tumor Necrosis Factor, Type II/agonists , Receptors, Tumor Necrosis Factor, Type II/genetics , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/drug effectsABSTRACT
T and B cell receptor (TCR and BCR, respectively) Vß or immunoglobulin heavy chain complementarity-determining region 3 sequencing allows monitoring of repertoire changes through recognition, clonal expansion, affinity maturation, and T or B cell activation in response to antigen. TCR and BCR repertoire analysis can advance understanding of antitumor immune responses in the tumor microenvironment. TCR and BCR repertoires of sorted CD4+, CD8+ or CD19+ cells in tumor, non-tumoral distant tissue (NT), and peripheral compartments (blood/draining lymph node [P]) from 47 non-small cell lung cancer (NSCLC) patients (agemedian = 68 y) were sequenced. The clonotype spectra were assessed among different tissues and correlated with clinical and immunological parameters. In all tissues, CD4+ and CD8+ TCR repertoires had greater clonality relative to CD19+ BCR. CD4+ T cells exhibited greater clonality in NT compared to tumor (p = 0.002) and P (p < 0.001), concentrated among older patients (age > 68). Younger patients exhibited greater CD4+ T cell diversity in P compared to older patients (p = 0.05), and greater CD4+ T cell clonality in tumor relative to P (p < 0.001), with fewer shared clonotypes between tumor and P than older patients (p = 0.04). More interestingly, greater CD4+ and CD8+ T cell clonality in tumor and P, respectively (both p = 0.05), correlated with high density of tumor-associated tertiary lymphoid structure (TLS) B cells, a biomarker of higher overall survival in NSCLC. Results indicate distinct adaptive immune responses in NSCLC, where peripheral T cell diversity is modulated by age, and tumor T cell clonal expansion is favored by the presence of TLSs in the tumor microenvironment.