RESUMO
Pancreatic adenocarcinoma (PDAC) is one the most intractable cancers, in part due to its highly inflammatory microenvironment and paucity of infiltrating dendritic cells (DCs). Here, we find that genetic ablation or antibody blockade of tumor necrosis factor receptor 1 (TNFR1) enhanced intratumor T cell activation and slowed PDAC growth. While anti-PD-1 checkpoint inhibition alone had little effect, it further enhanced intratumor T cell activation in combination with anti-TNFR1. The major cellular alteration in the tumor microenvironment in the absence of TNFR1 signaling was a large increase in DC number and immunostimulatory phenotype. This may reflect a direct effect on DCs, because TNF induced TNFR1-dependent apoptosis of bone-marrow-derived DCs. The therapeutic response to anti-TNFR1 alone was superior to the combination of DC-activating agonistic anti-CD40 and Flt3 ligand (Flt3L). These observations suggest that targeting TNFR1, perhaps in concert with other strategies that promote DC generation and mobilization, may have therapeutic benefits.
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Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.
Assuntos
Antígeno CD47 , Eritropoese , Baço , Trombospondina 1 , Animais , Antígeno CD47/metabolismo , Antígeno CD47/genética , Trombospondina 1/metabolismo , Trombospondina 1/genética , Baço/metabolismo , Camundongos , Camundongos Knockout , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Células Precursoras Eritroides/metabolismoRESUMO
Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.
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Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in thbs1-/- spleens relative to basal levels in wild type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.
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To address CD19 loss from lymphoma after anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we designed a bicistronic construct encoding an anti-CD19 CAR and an anti-CD20 CAR. We detected deletions from the expected bicistronic construct sequence in a minority of transcripts by mRNA sequencing. Loss of bicistronic construct transgene DNA was also detected. Deletions of sequence were present at much higher frequencies in transduced T cell mRNA versus gamma-retroviral vector RNA. We concluded that these deletions were caused by intramolecular template switching of the reverse transcriptase enzyme during reverse transcription of gamma-retroviral vector RNA into transgene DNA of transduced T cells. Intramolecular template switching was driven by repeated regions of highly similar nucleic acid sequence within CAR sequences. We optimized the sequence of the bicistronic CAR construct to reduce repeated regions of highly similar sequences. This optimization nearly eliminated sequence deletions. This work shows that repeated regions of highly similar nucleic acid sequence must be avoided in complex CAR constructs. We further optimized the bicistronic construct by lengthening the linker of the anti-CD20 single-chain variable fragment. This modification increased CD20-specific interleukin-2 release and reduced CD20-specific activation-induced cell death. We selected an optimized anti-CD19/CD20 bicistronic construct for clinical development.
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Thymic epithelial cells (TECs) produce glucocorticoids, which antagonize negative selection of autoreactive thymocytes and promote a competent T cell antigen-specific repertoire. To characterize their source, we generated a knock-in reporter mouse in which endogenous Cyp11b1, the final enzyme in de novo production of active glucocorticoids, was fluorescently tagged with mScarlet. Here, we find that Cyp11b1 is expressed in medullary TECs (mTECs) but not cortical TECs or other cells in the thymus. A distinct characteristic of mTECs is the presence of Aire, a transcription factor that drives expression of tissue-restricted antigens (TRAs) important for establishing immune tolerance. Cyp11b1 expression was highest in Aire+ mTECs, lower in post-Aire mTECs, and absent in mTECs of Aire-deficient mice. Transcriptomic analyses found that multiple enzymatic biosynthetic pathways are expressed specifically in mTECs and are also Aire dependent. In particular, we found that the thymus expresses messenger RNA for enzymes that catalyze production of many bioactive steroids and that glucocorticoids and sex steroids were secreted by cultured thymi. Expression of the transcripts for these genes and production of their final steroid products were markedly reduced in the absence of Aire. Thus, in addition to its well-established role in inducing TRAs that promote negative selection, Aire has an additional and contrary function of inducing glucocorticoids that antagonize negative selection, which together may expand and enhance the TCR repertoire. Furthermore, because Aire drives expression of multiple enzymes responsible for production of other non-gene-encoded bioactive molecules, it might have yet other roles in thymus development and function.
Assuntos
Glucocorticoides , Esteroide 11-beta-Hidroxilase , Fatores de Transcrição , Animais , Camundongos , Células Epiteliais , Perfilação da Expressão Gênica , Fatores de Transcrição/metabolismo , Timo/metabolismo , Proteína AIRERESUMO
Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by Hsd11b1). Here, we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth, but reduced in vivo tumor progression, which corresponded with increased frequencies of CD8+ tumor-infiltrating lymphocytes (TILs) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of conventional T cell activation in tumor-infiltrating Tregs. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11ß-HSD1 reduced tumor growth to the same degree as gene knockout and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11ß-HSD1 is well tolerated in clinical studies, these data suggest that targeting 11ß-HSD1 may be a beneficial adjunct in cancer therapy.
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Glucocorticoides , Neoplasias , Camundongos , Humanos , Animais , Glucocorticoides/farmacologia , Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Receptores de Glucocorticoides/genética , Técnicas de Inativação de GenesRESUMO
Mucosal delivery of IL-27 has been shown to have a therapeutic benefit in murine models of inflammatory bowel disease (IBD). The IL-27 effect was associated with phosphorylated STAT1 (pSTAT1), a product of IL27 receptor signaling, in bowel tissue. To determine whether IL-27 acted directly on colonic epithelium, murine colonoids and primary intact colonic crypts were shown to be unresponsive to IL-27 in vitro and to lack detectable IL-27 receptors. On the other hand, macrophages, which are present in inflamed colon tissue, were responsive to IL-27 in vitro. IL-27 induced pSTAT1 in macrophages, the transcriptome indicated an IFN-like signature, and supernatants induced pSTAT1 in colonoids. IL-27 induced anti-viral activity in macrophages and MHC Class II induction. We conclude that the effects of mucosal delivery of IL-27 in murine IBD are in part based on the known effects of IL27 inducing immunosuppression of T cells mediated by IL-10. We also conclude that IL-27 has potent effects on macrophages in inflamed colon tissue, generating mediators that in turn act on colonic epithelium.
Assuntos
Doenças Inflamatórias Intestinais , Interleucina-27 , Camundongos , Animais , Interleucina-27/uso terapêutico , Colo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Macrófagos , EpitélioRESUMO
Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.
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Antígeno CD47 , Melanoma , Camundongos , Animais , Antígeno CD47/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Linfócitos T CD8-Positivos , Expressão Gênica , Melanoma/genética , RNA Mensageiro/genética , Trombospondinas/genética , Microambiente TumoralRESUMO
The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.
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Vacinas contra a AIDS , Infecções por HIV , Feminino , Animais , Eficácia de Vacinas , Macaca mulatta , Vacinação , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Anti-HIV , Infecções por HIV/prevenção & controle , Proteína gp120 do Envelope de HIV/genéticaRESUMO
CD47 has established roles in the immune system for regulating macrophage phagocytosis and lymphocyte activation, with growing evidence of its cell-intrinsic regulatory roles in natural killer and CD8+ T cells. CD47 limits antigen-dependent cytotoxic activities of human and murine CD8+ T cells, but its role in T cell activation kinetics remains unclear. Using in vitro and in vivo models, we show here that CD47 differentially regulates CD8+ T cell responses to short- versus long-term activation. Although CD47 was not required for T cell development in mice and early activation in vitro, short-term stimuli elevated pathogen-reactive gene expression and enhanced proliferation and the effector phenotypes of Cd47-deficient relative to Cd47-sufficient CD8+ T cells. In contrast, persistent TCR stimulation limited the effector phenotypes of Cd47 -/- CD8+ T cells and enhanced their apoptosis signature. CD8+ T cell expansion and activation in vivo induced by acute lymphocytic choriomeningitis virus (LCMV) infection did not differ in the absence of CD47. However, the frequency and effector phenotypes of Cd47-/- CD8+ T cells were constrained in chronic LCMV-infected as well as in mice bearing B16 melanoma tumors. Therefore, CD47 regulates CD8+ T cell activation, proliferation, and fitness in a context-dependent manner.
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Ativação Linfocitária , Coriomeningite Linfocítica , Animais , Antígeno CD47/genética , Linfócitos T CD8-Positivos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-ß (TGF-ß) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-ß activation signatures and collagen-rich environments have both been associated with T cell exclusion and lack of responses to immunotherapy. Here, we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-ß and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations, resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.
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Antígeno B7-H1 , Neoplasias , Receptores Imunológicos , Microambiente Tumoral , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Imunoterapia/métodos , Ligantes , Camundongos , Neoplasias/terapia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
INTRODUCTION: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time. METHODS: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership). RESULTS: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies. CONCLUSION: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes.
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The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.
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Benchmarking , Neoplasias da Mama/genética , Análise Mutacional de DNA/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Sequenciamento Completo do Genoma/normas , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Células Germinativas , Humanos , Mutação , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Poorly inflamed carcinomas do not respond well to immune checkpoint blockade. Converting the tumour microenvironment into a functionally inflamed immune hub would extend the clinical benefit of immune therapy to a larger proportion of cancer patients. Here we show, by using comprehensive single-cell transcriptome, proteome, and immune cell analysis, that Entinostat, a class I histone deacetylase inhibitor, facilitates accumulation of the necrosis-targeted recombinant murine immune-cytokine, NHS-rmIL12, in experimental mouse colon carcinomas and poorly immunogenic breast tumours. This combination therapy reprograms the tumour innate and adaptive immune milieu to an inflamed landscape, where the concerted action of highly functional CD8+ T cells and activated neutrophils drive macrophage M1-like polarization, leading to complete tumour eradication in 41.7%-100% of cases. Biomarker signature of favourable overall survival in multiple human tumor types shows close resemblance to the immune pattern generated by Entinostat/NHS-rmIL12 combination therapy. Collectively, these findings provide a rationale for combining NHS-IL12 with Entinostat in the clinical setting.
Assuntos
Benzamidas/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Imunoglobulina G/administração & dosagem , Interleucina-12/administração & dosagem , Piridinas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Animais , Neoplasias da Mama/mortalidade , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/mortalidade , Quimioterapia Combinada , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microambiente Tumoral/efeitos dos fármacosRESUMO
The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Epiteliais/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timo/enzimologia , Animais , Apoptose/imunologia , Sequência de Bases , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Epiteliais/imunologia , Epitélio/enzimologia , Epitélio/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Sequência de RNA/métodos , Timócitos/imunologia , Timo/imunologia , Éxons VDJRESUMO
Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.
Assuntos
Neoplasias da Mama/patologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Neoplasias Pulmonares/secundário , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral/transplante , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Interleucina-6/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Receptor de Morte Celular Programada 1/imunologia , Estudo de Prova de Conceito , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , DNA Metiltransferase 3BRESUMO
PURPOSE: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH). EXPERIMENTAL DESIGN: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting. RESULTS: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay. CONCLUSIONS: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.
Assuntos
Heterogeneidade Genética , Neoplasias Retais/etiologia , Neoplasias Retais/patologia , Análise de Célula Única , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Terapia Combinada , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Neoplasias Retais/metabolismo , Neoplasias Retais/terapia , Transdução de Sinais , Resultado do Tratamento , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPα. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro Cd47 -/- NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-γ levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47 -/- than in wild-type mice. Melanoma-bearing Cd47 -/- mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47-/- NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas.
Assuntos
Antígeno CD47/genética , Expressão Gênica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Animais , Apoptose , Antígeno CD47/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Melanoma Experimental , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/mortalidade , Prognóstico , RNA Mensageiro , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Trombospondina 1/farmacologiaRESUMO
Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor-product relationships that link the subsets identified. This analysis identified two successive stages of ILC development within T cell factor 1-positive (TCF-1+) early innate lymphoid progenitors (EILPs), which we named 'specified EILPs' and 'committed EILPs'. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate.