RESUMO
Radiation therapy is a mainstay of cancer treatment but does not always lead to complete tumor regression. Here we combine radiotherapy with blockade of the 'don't-eat-me' cell-surface molecule CD47 in small cell lung cancer (SCLC), a highly metastatic form of lung cancer. CD47 blockade potently enhances the local antitumor effects of radiotherapy in preclinical models of SCLC. Notably, CD47 blockade also stimulates off-target 'abscopal' effects inhibiting non-irradiated SCLC tumors in mice receiving radiation. These abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by radiation and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal antitumor effects were observed in other cancer models treated with radiation and CD47 blockade. The systemic activation of antitumor macrophages following radiotherapy and CD47 blockade may be particularly important in patients with cancer who suffer from metastatic disease.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Camundongos , Animais , Antígeno CD47 , Macrófagos , Fagocitose , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/genética , Sistemas CRISPR-Cas , Citofagocitose/genética , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Antígeno CD47/antagonistas & inibidores , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Humanos , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antígeno CD24/antagonistas & inibidores , Imunoterapia/métodos , Lectinas/metabolismo , Macrófagos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CD24/deficiência , Antígeno CD24/genética , Antígeno CD24/imunologia , Linhagem Celular Tumoral , Humanos , Lectinas/antagonistas & inibidores , Lectinas/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Fagocitose/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologiaRESUMO
Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-ß aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Magnetismo/métodos , Fagocitose/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação da Expressão Gênica , Estudos de Associação Genética/métodos , Genoma Humano , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos , Células RAW 264.7 , Transdução de Sinais/genética , Células U937RESUMO
Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component ß2-microglobulin (ß2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo, which defines the MHC class I-LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/imunologia , Macrófagos/imunologia , Neoplasias/imunologia , Fagocitose/imunologia , Animais , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoterapia/métodos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapiaRESUMO
Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.
Assuntos
Cromatina , Mesoderma/fisiologia , Células-Tronco Pluripotentes , Transcrição Gênica , Biomarcadores , Diferenciação Celular , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologiaRESUMO
Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.
Assuntos
Mesoderma/citologia , Transdução de Sinais , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Coração/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Mesoderma/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/metabolismo , Análise de Célula Única , Somitos/metabolismo , Células-Tronco , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismoRESUMO
Enhancers and promoters commonly occur in accessible chromatin characterized by depleted nucleosome contact; however, it is unclear how chromatin accessibility is governed. We show that log-additive cis-acting DNA sequence features can predict chromatin accessibility at high spatial resolution. We develop a new type of high-dimensional machine learning model, the Synergistic Chromatin Model (SCM), which when trained with DNase-seq data for a cell type is capable of predicting expected read counts of genome-wide chromatin accessibility at every base from DNA sequence alone, with the highest accuracy at hypersensitive sites shared across cell types. We confirm that a SCM accurately predicts chromatin accessibility for thousands of synthetic DNA sequences using a novel CRISPR-based method of highly efficient site-specific DNA library integration. SCMs are directly interpretable and reveal that a logic based on local, nonspecific synergistic effects, largely among pioneer TFs, is sufficient to predict a large fraction of cellular chromatin accessibility in a wide variety of cell types.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Modelos Genéticos , Animais , Cromatina/metabolismo , Genoma Humano , Humanos , Aprendizado de MáquinaRESUMO
Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Cromatina/metabolismo , Receptores do Ácido Retinoico/metabolismo , Ativação Transcricional , Animais , Proteínas Associadas a CRISPR/genética , Linhagem Celular , Cromatina/genética , Loci Gênicos , Genoma , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , MutaçãoRESUMO
We describe protein interaction quantitation (PIQ), a computational method for modeling the magnitude and shape of genome-wide DNase I hypersensitivity profiles to identify transcription factor (TF) binding sites. Through the use of machine-learning techniques, PIQ identified binding sites for >700 TFs from one DNase I hypersensitivity analysis followed by sequencing (DNase-seq) experiment with accuracy comparable to that of chromatin immunoprecipitation followed by sequencing (ChIP-seq). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into prepancreatic and intestinal endoderm. We identified 120 and experimentally validated eight 'pioneer' TF families that dynamically open chromatin. Four pioneer TF families only opened chromatin in one direction from their motifs. Furthermore, we identified 'settler' TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and nondirectional pioneer activity shapes the chromatin landscape for population by settler TFs.