RESUMEN
Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Recurrencia Local de Neoplasia/genética , Macrófagos Asociados a Tumores/metabolismo , Adulto , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Estudios de Cohortes , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Riñón/metabolismo , Neoplasias Renales/patología , Linfocitos Infiltrantes de Tumor/patología , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Pronóstico , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Microambiente Tumoral , Macrófagos Asociados a Tumores/fisiologíaRESUMEN
Activation of the stimulator of interferon genes (STING) pathway by both exogenous and endogenous cytosolic DNA results in the production of interferon beta (IFN-ß) and is required for the generation of cytotoxic T-cell priming against tumor antigens. In the clinical setting, pharmacological stimulation of the STING pathway has the potential to synergize with immunotherapy antibodies by boosting anti-tumor immune responses. We report the discovery of two highly potent cyclic dinucleotide STING agonists, IACS-8803 and IACS-8779, which show robust activation of the STING pathway in vitro and a superior systemic anti-tumor response in the B16 murine model of melanoma when compared to one of the clinical benchmark compounds.
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Antineoplásicos/química , Compuestos Heterocíclicos/química , Interferón beta/metabolismo , Melanoma Experimental/inmunología , Melanoma/inmunología , Nucleótidos Cíclicos/antagonistas & inhibidores , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Línea Celular , Citosol/metabolismo , Compuestos Heterocíclicos/farmacología , Humanos , Inmunidad Innata , Inmunoterapia/métodos , Proteínas de la Membrana/inmunología , Ratones , Fosfatos/metabolismo , Transducción de Señal , Microambiente TumoralRESUMEN
We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12D TP53+/R172H Pdx1-Cre-derived (KPC-derived) model of pancreatic adenocarcinoma to examine the tumor response and adaptive resistance mechanisms involved in response to 2 established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast with prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8+ T cell response. Bulk tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid-derived suppressor cells (G-MDSCs) through CCL9 secretion. Blockade of the CCL9/CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302, anti-VEGFR-2, and ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.
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Adenocarcinoma , Células Supresoras de Origen Mieloide , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patología , Adenocarcinoma/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Hipoxia/metabolismoRESUMEN
Lack of robust activation of Stimulator of Interferon Genes (STING) pathway and subsequent induction of type I IFN responses is considered a barrier to antitumor immunity in acute myeloid leukemia (AML). Using common human AML cell lines as in vitro tools to evaluate the efficacy of novel STING agonists, we found most AML lines to be poor producers of IFNs upon exposure to extremely potent agonists, suggesting cell-intrinsic suppression of STING signaling may occur. We observed unexpected patterns of response that did not correlate with levels of STING pathway components or of known enzymes associated with resistance. To identify a genetic basis for these observations, we cloned and sequenced STING from the cDNA of human AML cell lines and found both frequent mutations and deviations from normal RNA splicing. We identified two novel spliced isoforms of STING in these lines and validated their expression in primary human AML samples. When transduced into reporter cells, these novel STING isoforms exhibited complete insensitivity to agonist stimulation. These observations identify alternative splicing as a mechanism of STING pathway suppression and suggest that most AML silences the STING pathway through direct modification rather than through engagement of external inhibitory factors. SIGNIFICANCE: We find that AML acquires resistance to innate immune activation via the STING pathway through aberrant splicing of the STING transcript including two novel forms described herein that act as dominant negatives. These data broaden understanding of how cancers evolve STING resistance, and suggest that the AML tumor microenvironment, not the cancer cell, should be the target of therapeutic interventions to activate STING.
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Interferón Tipo I , Leucemia Mieloide Aguda , Humanos , Isoformas de Proteínas/genética , Leucemia Mieloide Aguda/genética , Empalme Alternativo/genética , Interferón Tipo I/genética , Línea Celular , Microambiente TumoralRESUMEN
Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios-KLRG1+ subset of tumor-infiltrating regulatory T cells was associated with tumor progression from immune equilibrium to escape and was also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumor-infiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1+ CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker discovery.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Linfocitos T CD4-Positivos , Subgrupos de Linfocitos T , Inmunoterapia , Biomarcadores , Receptores Inmunológicos , Lectinas Tipo CRESUMEN
Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios - KLRG1 + subset of tumor-infiltrating regulatory T cells (Tregs) was associated with tumor progression from immune equilibrium to escape, and were also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumorinfiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1 + CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker and/or target discovery.
RESUMEN
Due to their immunosuppressive role, tumor-infiltrating regulatory T cells (TI-Tregs) represent attractive immuno-oncology targets. Analysis of TI vs. peripheral Tregs (P-Tregs) from 36 patients, across four malignancies, identified 17 candidate master regulators (MRs) as mechanistic determinants of TI-Treg transcriptional state. Pooled CRISPR-Cas9 screening in vivo, using a chimeric hematopoietic stem cell transplant model, confirmed the essentiality of eight MRs in TI-Treg recruitment and/or retention without affecting other T cell subtypes, and targeting one of the most significant MRs (Trps1) by CRISPR KO significantly reduced ectopic tumor growth. Analysis of drugs capable of inverting TI-Treg MR activity identified low-dose gemcitabine as the top prediction. Indeed, gemcitabine treatment inhibited tumor growth in immunocompetent but not immunocompromised allografts, increased anti-PD-1 efficacy, and depleted MR-expressing TI-Tregs in vivo. This study provides key insight into Treg signaling, specifically in the context of cancer, and a generalizable strategy to systematically elucidate and target MR proteins in immunosuppressive subpopulations.
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Neoplasias , Linfocitos T Reguladores , Humanos , Linfocitos T Reguladores/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Proteínas Represoras/metabolismoRESUMEN
When compared to other malignancies, the tumor microenvironment (TME) of primary and castration-resistant prostate cancer (CRPC) is relatively devoid of immune infiltrates. While androgen deprivation therapy (ADT) induces a complex immune infiltrate in localized prostate cancer, the composition of the TME in metastatic castration-sensitive prostate cancer (mCSPC), and the effects of ADT and other treatments in this context are poorly understood. Here, we perform a comprehensive single-cell RNA sequencing (scRNA-seq) profiling of metastatic sites from patients participating in a phase 2 clinical trial (NCT03951831) that evaluated standard-of-care chemo-hormonal therapy combined with anti-PD-1 immunotherapy. We perform a longitudinal, protein activity-based analysis of TME subpopulations, revealing immune subpopulations conserved across multiple metastatic sites. We also observe dynamic changes in these immune subpopulations in response to treatment and a correlation with clinical outcomes. Our study uncovers a therapy-resistant, transcriptionally distinct tumor subpopulation that expands in cell number in treatment-refractory patients.
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Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Antagonistas de Andrógenos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Andrógenos/uso terapéutico , Inmunoterapia , Castración , Microambiente TumoralRESUMEN
We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1-84.5) and 65% (95% CI: 52.5-76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0-80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6-83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Humanos , Anticuerpos Monoclonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Neoadyuvante , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Ensayos Clínicos Fase II como AsuntoRESUMEN
The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues.
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Antígeno B7-H1 , Melanoma , Ratones , Animales , Antígeno B7-H1/genética , Linfocitos T , Antígenos CD58/química , Antígenos CD58/metabolismo , Melanoma/genética , Melanoma/metabolismo , Activación de LinfocitosRESUMEN
The theory of cancer immunoediting, which describes the dynamic interactions between tumors and host immune cells that shape the character of each compartment, is foundational for understanding cancer immunotherapy. Few models exist that facilitate in-depth study of each of the three canonical phases of immunoediting: elimination, equilibrium, and escape. Here, we utilized NPK-C1, a transplantable prostate tumor model that we found recapitulated the three phases of immunoediting spontaneously in immunocompetent animals. Given that a significant portion of NPK-C1 tumors reliably progressed to the escape phase, we were able to delineate cell types and mechanisms differentially prevalent in equilibrium versus escape phases. Using high-dimensional flow cytometry, we found that activated CD4+ effector T cells were enriched in regressing tumors, highlighting a role for CD4+ T cells in antitumor immunity. CD8+ T cells were also important for NPK-C1 control, specifically, central memory-like cytotoxic CD8+ T cells. Regulatory T cells (Treg), as a whole, were counterintuitively enriched in regressing tumors; however, high-dimensional analysis revealed their significant phenotypic diversity, with a number of Treg subpopulations enriched in progressing tumors. In the myeloid compartment, we found that iNOS+ dendritic cell (DC)-like cells are enriched in regressing tumors, whereas CD103+ DCs were associated with late-stage tumor progression. In total, these analyses of the NPK-C1 model provide novel insights into the roles of lymphoid and myeloid populations throughout the cancer immunoediting process and highlight a role for multidimensional, flow-based analyses to more deeply understand immune cell dynamics in the tumor microenvironment.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Cadenas alfa de Integrinas/inmunología , Neoplasias de la Próstata/inmunología , Escape del Tumor , Microambiente Tumoral/inmunología , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Neoplasias de la Próstata/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Carga Tumoral/inmunologíaRESUMEN
BACKGROUND: Intratumoral injection of cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway engages innate immune activation and priming of adaptive immune effectors to foster local and distal tumor clearance. Despite proven therapeutic efficacy in preclinical models, a thorough understanding of how CDNs reprogram suppressive myeloid stroma in mouse and man is lacking. METHODS: Here, we perform deep transcript-level and protein-level profiling of myeloid-derived suppressor cells and M2 macrophages following stimulation with CDNs of ascending potency. Additionally, we leverage orthotopic Kras+/G12DTP53+/R172HPdx1-Cre (KPC) derived models of pancreatic adenocarcinoma (PDAC) to determine the capacity for locally administered CDNs to sensitize PDAC to immune checkpoint blockade. We use bioluminescent in vivo imaging and 30-parameter flow cytometry to profile growth kinetics and remodeling of the tumor stroma post-therapy. RESULTS: Highly potent synthetic STING agonists repolarize suppressive myeloid populations of human and murine origin in part through inhibition of Myc signaling, metabolic modulation, and antagonism of cell cycle. Surprisingly, high-potency synthetic agonists engage qualitatively unique pathways as compared with natural CDNs. Consistent with our mechanistic observations, we find that intratumoral injection of the highest activity STING agonist, IACS-8803, into orthotopic pancreatic adenocarcinoma lesions unmasks sensitivity to checkpoint blockade immunotherapy. Dimensionality reduction analyses of high parameter flow cytometry data reveals substantial contributions of both myeloid repolarization and T cell activation underlying the in vivo therapeutic benefit of this approach. CONCLUSIONS: This study defines the molecular basis of STING-mediated myeloid reprogramming, revealing previously unappreciated and qualitatively unique pathways engaged by CDNs of ascending potency during functional repolarization. Furthermore, we demonstrate the potential for high potency CDNs to overcome immunotherapy resistance in an orthotopic, multifocal model of PDAC.
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Inmunoterapia/métodos , Proteínas de la Membrana/uso terapéutico , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Humanos , Masculino , Proteínas de la Membrana/farmacología , RatonesRESUMEN
PURPOSE: Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1ß may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types. EXPERIMENTAL DESIGN: Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1ß plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model. RESULTS: Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1ß reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1ß plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs. CONCLUSIONS: Single-cell RNA-seq analyses show that IL1ß blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell-independent mechanisms. IL1ß is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Modelos Animales de Enfermedad , Interleucina-1beta/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Células Supresoras de Origen Mieloide/efectos de los fármacos , Anilidas/administración & dosificación , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Ratones , Ratones Endogámicos BALB C , Células Supresoras de Origen Mieloide/metabolismo , Piridinas/administración & dosificación , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Macrófagos Asociados a Tumores/clasificación , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/metabolismoRESUMEN
Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.
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Antígenos CD58/inmunología , Resistencia a Antineoplásicos/inmunología , Melanoma/patología , Análisis de la Célula Individual/métodos , Escape del Tumor , Antígenos CD58/genética , Antígenos CD58/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cocultivo , Biología Computacional/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Epítopos/genética , Técnicas de Inactivación de Genes , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Análisis de Secuencia de ARN , Escape del Tumor/genéticaRESUMEN
Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a "hypermetabolic" phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.
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Inmunoterapia/métodos , Melanoma Experimental/genética , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Melanoma Experimental/metabolismo , Ratones , Fosforilación Oxidativa , FenotipoRESUMEN
Tumors that lack pre-existing immune infiltration respond poorly to T cell checkpoint blockade immunotherapy. These cancers often surround themselves with high densities of suppressive myeloid stroma while excluding immunostimulatory dendritic cells. Tumor-resident myeloid cells and selected lymphocyte populations retain expression of Toll-like Receptors (TLR) that sense common features of pathogens and activate innate immunity in response. We explored whether agonists of TLR9 could augment innate immunity to promote tumor regression alone or in combination with T cell checkpoint blockade. In the setting of the immunogenic B16-Ova (Ovalbumin) expressing melanoma model, local injection of the CpG oligonucleotide TLR9 agonist ODN1826 combined with systemic CTLA-4 blockade cured 45% of mice of both their treated and an untreated tumor on the opposite flank demonstrating the synergistic potential of this combination. Next, in the non-immunogenic B16-F10 melanoma model, we showed that only intra-tumoral, but not systemic TLR9 activation augments the therapeutic potential of checkpoint blockade. In this setting, intra-tumoral TLR9 activation cooperated equally with either CTLA-4 or PD-1 blockade co-administered locally or given systemically; however, the uninjected tumor rarely regressed. Anti-CTLA-4 combinations were associated with improved intra-tumoral CD8 to regulatory T cell ratios, while anti-PD-1 combinations elicited improved ratios of CD8 T cells relative to suppressive myeloid stroma. Using both a TLR9 agonist (MGN1703) and a CTLA-4 antibody (9D9-IgG2a) of increased potency cured 50% of bi-lateral B16-F10 melanoma. These findings suggest that intra-tumoral TLR9 agonists can improve sensitivity of poorly immunogenic tumors to T cell checkpoint blockade, and that newer, higher potency TLR agonists and checkpoint antibodies can raise the therapeutic ceiling for this combination therapy.
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Melanoma/inmunología , Melanoma/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor Toll-Like 9/agonistas , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor , Antígeno CTLA-4/antagonistas & inhibidores , Citotoxicidad Inmunológica , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Melanoma Experimental , Ratones , Oligodesoxirribonucleótidos/farmacología , Pronóstico , Linfocitos T/efectos de los fármacos , Resultado del TratamientoRESUMEN
Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist-driven tumor immunity from hepatotoxicity.Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines.Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist-induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma.Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27-producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138-51. ©2018 AACR.
Asunto(s)
Antineoplásicos Inmunológicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Interleucinas/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Línea Celular Tumoral/trasplante , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Evaluación Preclínica de Medicamentos , Humanos , Interleucinas/inmunología , Hígado/citología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , Masculino , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/metabolismo , Receptores CCR2/antagonistas & inhibidores , Receptores CCR2/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.
Asunto(s)
Adenocarcinoma/terapia , Inmunoterapia , Neoplasias Experimentales/terapia , Nitroimidazoles/farmacología , Mostazas de Fosforamida/farmacología , Neoplasias de la Próstata/terapia , Linfocitos T/inmunología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Hipoxia de la Célula/genética , Hipoxia de la Célula/inmunología , Línea Celular Tumoral , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Linfocitos T/patologíaRESUMEN
Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment-including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs-can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint-modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676-84. ©2017 AACR.