RESUMO
The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.
Assuntos
Linfócitos T CD8-Positivos , Imunoterapia , Linfócitos do Interstício Tumoral , Camundongos Endogâmicos C57BL , Microambiente Tumoral , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Relógios Circadianos , Ritmo Circadiano , Células Endoteliais/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Melanoma/terapia , Melanoma/patologia , Microambiente Tumoral/imunologiaRESUMO
For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.
Assuntos
Linfonodos , Melanoma , Animais , Tolerância Imunológica , Imunoterapia , Metástase Linfática/patologia , Melanoma/patologia , CamundongosRESUMO
Combined LAG-3 and PD-1 blockade is an emerging strategy for the treatment of melanoma. Tawbi et al. and Amaria et al. report in The New England Journal of Medicine and Nature respectively on two clinical trials evaluating relatlimab and nivolumab as front-line treatment for metastatic, and resectable, high-risk, node-positive melanoma.
Assuntos
Melanoma , Nivolumabe , Humanos , Nivolumabe/uso terapêutico , Ipilimumab , Melanoma/tratamento farmacológico , Melanoma/patologia , Anticorpos Monoclonais Humanizados/uso terapêuticoRESUMO
Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.
Assuntos
Melanoma , Nevo Pigmentado , Neoplasias Cutâneas , Animais , Xenoenxertos , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Transplante de Neoplasias , Nevo Pigmentado/congênito , Nevo Pigmentado/tratamento farmacológico , Nevo Pigmentado/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controleRESUMO
Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.
Assuntos
Receptores CXCR6/metabolismo , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral , Animais , Antígeno B7-H1/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Quimiocina CXCL16 , Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Interleucina-15/metabolismo , Ligantes , Linfonodos/metabolismo , Melanoma/imunologia , Melanoma/patologia , Camundongos Endogâmicos C57BLRESUMO
The tumor microenvironment (TME) influences cancer progression and therapy response. Therefore, understanding what regulates the TME immune compartment is vital. Here we show that microbiota signals program mononuclear phagocytes in the TME toward immunostimulatory monocytes and dendritic cells (DCs). Single-cell RNA sequencing revealed that absence of microbiota skews the TME toward pro-tumorigenic macrophages. Mechanistically, we show that microbiota-derived stimulator of interferon genes (STING) agonists induce type I interferon (IFN-I) production by intratumoral monocytes to regulate macrophage polarization and natural killer (NK) cell-DC crosstalk. Microbiota modulation with a high-fiber diet triggered the intratumoral IFN-I-NK cell-DC axis and improved the efficacy of immune checkpoint blockade (ICB). We validated our findings in individuals with melanoma treated with ICB and showed that the predicted intratumoral IFN-I and immune compositional differences between responder and non-responder individuals can be transferred by fecal microbiota transplantation. Our study uncovers a mechanistic link between the microbiota and the innate TME that can be harnessed to improve cancer therapies.
Assuntos
Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Microbiota , Monócitos/metabolismo , Microambiente Tumoral , Akkermansia/efeitos dos fármacos , Akkermansia/fisiologia , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Fibras na Dieta/farmacologia , Fosfatos de Dinucleosídeos/administração & dosagem , Fosfatos de Dinucleosídeos/farmacologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunomodulação/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Melanoma/imunologia , Melanoma/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.
Assuntos
Citotoxicidade Imunológica , Imunoterapia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Serpinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Progressão da Doença , Feminino , Deleção de Genes , Granzimas/metabolismo , Imunidade/efeitos dos fármacos , Melanoma/patologia , Camundongos Endogâmicos C57BL , Neoplasias/prevenção & controle , Bibliotecas de Moléculas Pequenas/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.
Assuntos
Movimento Celular/fisiologia , Miosina Tipo II/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/imunologia , Proteínas do Citoesqueleto , Feminino , Humanos , Interleucina-1alfa/metabolismo , Masculino , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Fosforilação , Proteômica , Receptor Cross-Talk/fisiologia , Transdução de Sinais , Microambiente Tumoral/imunologiaRESUMO
Conventional type 1 dendritic cells (cDC1) are critical for antitumor immunity, and their abundance within tumors is associated with immune-mediated rejection and the success of immunotherapy. Here, we show that cDC1 accumulation in mouse tumors often depends on natural killer (NK) cells that produce the cDC1 chemoattractants CCL5 and XCL1. Similarly, in human cancers, intratumoral CCL5, XCL1, and XCL2 transcripts closely correlate with gene signatures of both NK cells and cDC1 and are associated with increased overall patient survival. Notably, tumor production of prostaglandin E2 (PGE2) leads to evasion of the NK cell-cDC1 axis in part by impairing NK cell viability and chemokine production, as well as by causing downregulation of chemokine receptor expression in cDC1. Our findings reveal a cellular and molecular checkpoint for intratumoral cDC1 recruitment that is targeted by tumor-derived PGE2 for immune evasion and that could be exploited for cancer therapy.
Assuntos
Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Microambiente Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Quimiocina CCL5/metabolismo , Quimiocinas C/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação/genética , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Análise de SobrevidaRESUMO
Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasia Residual/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor X Retinoide gama/antagonistas & inibidores , Animais , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos SCID , Mutação , Neoplasia Residual/metabolismo , Neoplasia Residual/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune-checkpoint blockade is able to achieve durable responses in a subset of patients; however, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4- and anti-PD-1-induced tumor rejection. To address these issues, we utilized mass cytometry to comprehensively profile the effects of checkpoint blockade on tumor immune infiltrates in human melanoma and murine tumor models. These analyses reveal a spectrum of tumor-infiltrating T cell populations that are highly similar between tumor models and indicate that checkpoint blockade targets only specific subsets of tumor-infiltrating T cell populations. Anti-PD-1 predominantly induces the expansion of specific tumor-infiltrating exhausted-like CD8 T cell subsets. In contrast, anti-CTLA-4 induces the expansion of an ICOS+ Th1-like CD4 effector population in addition to engaging specific subsets of exhausted-like CD8 T cells. Thus, our findings indicate that anti-CTLA-4 and anti-PD-1 checkpoint-blockade-induced immune responses are driven by distinct cellular mechanisms.
Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Melanoma/imunologia , Melanoma/terapia , Metástase Neoplásica/imunologia , Metástase Neoplásica/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imunoterapia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/patologia , Análise de Célula Única , Transcrição GênicaRESUMO
Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.
Assuntos
Imunoterapia/métodos , Melanoma/imunologia , Melanoma/patologia , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismoRESUMO
Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.
Assuntos
Interferon gama/imunologia , Melanoma/imunologia , Monócitos/imunologia , Metástase Neoplásica/patologia , Neoplasias Cutâneas/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Microambiente Tumoral , Animais , Diferenciação Celular , Células Dendríticas/imunologia , Homeostase , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Monócitos/patologia , Análise de Sequência de RNA , Análise de Célula Única , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , TranscriptomaRESUMO
Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas and showed that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration and thus promote cancer metastasis.
Assuntos
Núcleo Celular/metabolismo , Adesões Focais , Neoplasias Pulmonares/secundário , Melanoma/patologia , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Forminas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido NervosoRESUMO
How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.
Assuntos
Antígenos/imunologia , Proteínas de Checkpoint Imunológico/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Regulação da Expressão Gênica , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas de Checkpoint Imunológico/genética , Ativação Linfocitária , Melanoma/tratamento farmacológico , Melanoma/etiologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Linfócitos T/efeitos dos fármacosRESUMO
Integrated microwave photonics (MWP) is an intriguing technology for the generation, transmission and manipulation of microwave signals in chip-scale optical systems1,2. In particular, ultrafast processing of analogue signals in the optical domain with high fidelity and low latency could enable a variety of applications such as MWP filters3-5, microwave signal processing6-9 and image recognition10,11. An ideal integrated MWP processing platform should have both an efficient and high-speed electro-optic modulation block to faithfully perform microwave-optic conversion at low power and also a low-loss functional photonic network to implement various signal-processing tasks. Moreover, large-scale, low-cost manufacturability is required to monolithically integrate the two building blocks on the same chip. Here we demonstrate such an integrated MWP processing engine based on a 4 inch wafer-scale thin-film lithium niobate platform. It can perform multipurpose tasks with processing bandwidths of up to 67 GHz at complementary metal-oxide-semiconductor (CMOS)-compatible voltages. We achieve ultrafast analogue computation, namely temporal integration and differentiation, at sampling rates of up to 256 giga samples per second, and deploy these functions to showcase three proof-of-concept applications: solving ordinary differential equations, generating ultra-wideband signals and detecting edges in images. We further leverage the image edge detector to realize a photonic-assisted image segmentation model that can effectively outline the boundaries of melanoma lesion in medical diagnostic images. Our ultrafast lithium niobate MWP engine could provide compact, low-latency and cost-effective solutions for future wireless communications, high-resolution radar and photonic artificial intelligence.
Assuntos
Micro-Ondas , Nióbio , Óptica e Fotônica , Óxidos , Fótons , Inteligência Artificial , Diagnóstico por Imagem/instrumentação , Diagnóstico por Imagem/métodos , Melanoma/diagnóstico por imagem , Melanoma/patologia , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos , Radar , Tecnologia sem Fio , HumanosRESUMO
Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.
Assuntos
Código de Barras de DNA Taxonômico , Genômica , Animais , Humanos , Camundongos , Encéfalo/citologia , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Código de Barras de DNA Taxonômico/métodos , Epigênese Genética , Perfilação da Expressão Gênica , Genômica/métodos , Melanoma/genética , Melanoma/patologia , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Receptores de Antígenos de Linfócitos T/genética , RNA/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Microambiente Tumoral , Hipocampo/citologia , Hipocampo/metabolismo , Análise da Expressão Gênica de Célula Única , Especificidade de Órgãos , Ligantes , Elementos de Resposta/genética , Fatores de Transcrição/metabolismoRESUMO
Emerging data have shown that previously defined noncoding genomes might encode peptides that bind human leukocyte antigen (HLA) as cryptic antigens to stimulate adaptive immunity1,2. However, the significance and mechanisms of action of cryptic antigens in anti-tumour immunity remain unclear. Here mass spectrometry of the HLA class I (HLA-I) peptidome coupled with ribosome sequencing of human breast cancer samples identified HLA-I-binding cryptic antigenic peptides that were noncanonically translated by a tumour-specific circular RNA (circRNA): circFAM53B. The cryptic peptides efficiently primed naive CD4+ and CD8+ T cells in an antigen-specific manner and induced anti-tumour immunity. Clinically, the expression of circFAM53B and its encoded peptides was associated with substantial infiltration of antigen-specific CD8+ T cells and better survival in patients with breast cancer and patients with melanoma. Mechanistically, circFAM53B-encoded peptides had strong binding affinity to both HLA-I and HLA-II molecules. In vivo, administration of vaccines consisting of tumour-specific circRNA or its encoded peptides in mice bearing breast cancer tumours or melanoma induced enhanced infiltration of tumour-antigen-specific cytotoxic T cells, which led to effective tumour control. Overall, our findings reveal that noncanonical translation of circRNAs can drive efficient anti-tumour immunity, which suggests that vaccination exploiting tumour-specific circRNAs may serve as an immunotherapeutic strategy against malignant tumours.
Assuntos
Neoplasias da Mama , Melanoma , Peptídeos , Biossíntese de Proteínas , RNA Circular , Animais , Feminino , Humanos , Camundongos , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Espectrometria de Massas , Melanoma/genética , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/patologia , Peptídeos/genética , Peptídeos/imunologia , Perfil de Ribossomos , RNA Circular/genética , RNA Circular/metabolismo , Análise de SobrevidaRESUMO
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Assuntos
Sistemas CRISPR-Cas , Hepatócitos , Neoplasias Hepáticas , Fígado , Metástase Neoplásica , Proteínas do Tecido Nervoso , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Elementos de DNA Transponíveis , Fluorescência , Hepatócitos/metabolismo , Hepatócitos/citologia , Hepatócitos/patologia , Fator 4 Semelhante a Kruppel/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/secundário , Melanoma/metabolismo , Melanoma/patologia , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Semaforinas/antagonistas & inibidores , Semaforinas/metabolismoRESUMO
Melanoma metastasis is a devastating outcome lacking an effective preventative therapeutic. We provide pharmacologic, molecular, and genetic evidence establishing the liver-X nuclear hormone receptor (LXR) as a therapeutic target in melanoma. Oral administration of multiple LXR agonists suppressed melanoma invasion, angiogenesis, tumor progression, and metastasis. Molecular and genetic experiments revealed these effects to be mediated by LXRß, which elicits these outcomes through transcriptional induction of tumoral and stromal apolipoprotein-E (ApoE). LXRß agonism robustly suppressed tumor growth and metastasis across a diverse mutational spectrum of melanoma lines. LXRß targeting significantly prolonged animal survival, suppressed the progression of established metastases, and inhibited brain metastatic colonization. Importantly, LXRß activation displayed melanoma-suppressive cooperativity with the frontline regimens dacarbazine, B-Raf inhibition, and the anti-CTLA-4 antibody and robustly inhibited melanomas that had acquired resistance to B-Raf inhibition or dacarbazine. We present a promising therapeutic approach that uniquely acts by transcriptionally activating a metastasis suppressor gene.