RESUMO
BACKGROUND AND AIMS: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS: We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Linfócitos T Reguladores , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Granzimas/metabolismo , Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1/metabolismo , Ligantes , Microambiente Tumoral , Terapia de Imunossupressão , Hipóxia/metabolismo , Células Dendríticas/metabolismo , Antígenos HLARESUMO
BACKGROUND & AIMS: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. METHODS: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. RESULTS: Single-cell analyses identified CXCR3+CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. CONCLUSIONS: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. CLINICAL TRIAL NUMBER: NCT03695952. LAY SUMMARY: Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/tratamento farmacológico , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Fatores Imunológicos/uso terapêutico , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Neoplasias Hepáticas/tratamento farmacológico , Receptor de Morte Celular Programada 1RESUMO
Hepatocellular carcinoma (HCC), is the most common type of liver cancer which is derived mostly from the background of chronic inflammation. Chronic hepatitis viral infection remains one of the most common etiologies implicated in chronic liver inflammation, cirrhosis, and HCC. With such background inflammation, immunotherapy-particularly the checkpoint inhibitors-have been tested in HCC patients with unprecedented success. However, despite the initial enthusiasm, the response rate to immunotherapy remains modest in most clinical trials (approximately 20%), with mixed reports on response rates in hepatitis viral-related HCC as compared with nonviral HCC. Given such complexity in response to immunotherapy, it is increasingly appreciated that deeper understanding of the tumor molecular features and tumor microenvironment of hepatitis viral-related HCC is crucial for the design of more effective immunotherapeutics. We discuss herein the current knowledge in tumor genomic mutational and immune landscapes as well as the ongoing immunotherapy trials in HCC with the unique focus on their viral etiologies. Based on this understanding, we also outline perspectives and rationale on the design of potential immunotherapeutic strategies in HCC patients according to their viral etiologies.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/terapia , Imunoterapia , Hepatite Crônica , Inflamação , Microambiente TumoralRESUMO
The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8+ T cells (TRMs), resident natural killer cells (NKRs), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3+CD4+ and PD-1+CD8+ T cells. Interestingly, Tregs and TRMs isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and TRMs isolated from the NTME. We found PD-1+ TRMs were the predominant T-cell subset responsive to anti-PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8+ T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NKRs via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed.
Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Microambiente Tumoral/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Carcinoma Hepatocelular/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica/imunologia , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/imunologia , Leucócitos/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Neoplasias Hepáticas/genética , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Proteômica , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
OBJECTIVES: Yttrium-90 (Y90)-radioembolisation (RE) significantly regresses locally advanced hepatocellular carcinoma and delays disease progression. The current study is designed to deeply interrogate the immunological impact of Y90-RE, which elicits a sustained therapeutic response. DESIGN: Time-of-flight mass cytometry and next-generation sequencing (NGS) were used to analyse the immune landscapes of tumour-infiltrating leucocytes (TILs), tumour tissues and peripheral blood mononuclear cells (PBMCs) at different time points before and after Y90-RE. RESULTS: TILs isolated after Y90-RE exhibited signs of local immune activation: higher expression of granzyme B (GB) and infiltration of CD8+ T cells, CD56+ NK cells and CD8+ CD56+ NKT cells. NGS confirmed the upregulation of genes involved in innate and adaptive immune activation in Y90-RE-treated tumours. Chemotactic pathways involving CCL5 and CXCL16 correlated with the recruitment of activated GB+CD8+ T cells to the Y90-RE-treated tumours. When comparing PBMCs before and after Y90-RE, we observed an increase in tumour necrosis factor-α on both the CD8+ and CD4+ T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE, implying a systemic immune activation. Interestingly, a high percentage of PD-1+/Tim-3+CD8+ T cells coexpressing the homing receptors CCR5 and CXCR6 denoted Y90-RE responders. A prediction model was also built to identify sustained responders to Y90-RE based on the immune profiles from pretreatment PBMCs. CONCLUSION: High-dimensional analysis of tumour and systemic immune landscapes identified local and systemic immune activation that corresponded to the sustained response to Y90-RE. Potential biomarkers associated with a positive clinical response were identified and a prediction model was built to identify sustained responders prior to treatment.
Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/radioterapia , Leucócitos Mononucleares/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/radioterapia , Radioisótopos de Ítrio , Células Apresentadoras de Antígenos/imunologia , Biomarcadores Tumorais/imunologia , Quimiocina CCL5/imunologia , Quimiocina CXCL16/imunologia , Progressão da Doença , Feminino , Citometria de Fluxo/métodos , Granzimas/imunologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Singapura , Resultado do Tratamento , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND AND AIMS: Chronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics. METHODS: We interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays. RESULTS: In-depth interrogation of the immune landscapes showed that regulatory T cells (TREG) and CD8+ resident memory T cells (TRM) were enriched in HBV-related HCC, whereas Tim-3+CD8+ T cells and CD244+ natural killer cells were enriched in non-viral-related HCC. NGS of isolated TREG and TRM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. TREG and TRM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1+ TREG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that TREG and TRM contributed to overall patient survival: TREG were associated with a poor prognosis and TRM were associated with a good prognosis in HCC. CONCLUSION: We have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B , Hepatite B Crônica/complicações , Hepatite B Crônica/imunologia , Neoplasias Hepáticas/virologia , Carcinoma Hepatocelular/patologia , Hepatite B Crônica/patologia , Humanos , Neoplasias Hepáticas/patologia , Técnicas de Cultura de Tecidos , Microambiente TumoralRESUMO
The Hippo signaling pathway controls cell growth, proliferation, and apoptosis by regulating the expression of target genes that execute these processes. Acting downstream from this pathway is the YAP transcriptional coactivator, whose biological function is mediated by the conserved TEAD family transcription factors. The interaction of YAP with TEADs is critical to regulate Hippo pathway-responsive genes. Here, we describe the crystal structure of the YAP-interacting C-terminal domain of TEAD4 in complex with the TEAD-interacting N-terminal domain of YAP. The structure reveals that the N-terminal region of YAP is folded into two short helices with an extended loop containing the PXXPhiP motif in between, while the C-terminal domain of TEAD4 has an immunoglobulin-like fold. YAP interacts with TEAD4 mainly through the two short helices. Point mutations of TEAD4 indicate that the residues important for YAP interaction are required for its transforming activity. Mutagenesis reveals that the PXXPhiP motif of YAP, although making few contacts with TEAD4, is important for TEAD4 interaction as well as for the transforming activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ligação a DNA/química , Proteínas Musculares/química , Fosfoproteínas/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Musculares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Sinalização YAPRESUMO
OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.
Assuntos
Antígenos CD/análise , Linfócitos B/imunologia , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral , Linfócitos T/imunologia , ADP-Ribosil Ciclase 1/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD19/genética , Antígenos CD20/análise , Linfócitos B/química , Linfócitos B/patologia , Complexo CD3/análise , Antígenos CD40/análise , Antígenos CD8/análise , Antígenos CD8/genética , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patologia , Caspase 3/análise , Progressão da Doença , Feminino , Expressão Gênica , Granzimas/análise , Humanos , Interferon gama/genética , Antígeno Ki-67/análise , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Taxa de Sobrevida , Linfócitos T/química , Linfócitos T/patologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Adulto JovemRESUMO
Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Actinas/genética , Substituição de Aminoácidos , Angiomotinas , Animais , Proteínas de Transporte/genética , Cães , Células HEK293 , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Madin Darby de Rim Canino , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Mutação de Sentido Incorreto , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Introduction: Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC. Methods: HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses. Results: Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status. Conclusion: Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Microambiente Tumoral , Linfócitos T CD8-Positivos , Imunoterapia/métodosRESUMO
Introduction: Despite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+ T cells and monocytes. However, its specific role in the HCC TME remains unclear. Methods: In this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings. Results: From CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+ T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+ TRM in TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+ TRM from HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+ T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+ CD8+ T cells and CD38+PD-1+ TRM were significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease. Conclusion: Taken together, the concurrent expression of CD38 with exhaustion markers on CD8+ TRM underpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Linfócitos T CD8-Positivos , Leucócitos Mononucleares/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Antígeno CTLA-4/metabolismo , Células T de Memória , Complexo CD3/metabolismo , Microambiente TumoralRESUMO
IL-17-producing CD8 (Tc17) T cells have been shown to play an important role in infection and chronic inflammation, however their implications in hepatocellular carcinoma (HCC) remain elusive. In this study, we performed cytometry by time-of-flight (CyTOF) and revealed the distinctive immunological phenotypes of two IFNγ+ and IFNγ- Tc17 subsets that were preferentially enriched in human HCC. Single-cell RNA-sequencing analysis further revealed regulatory circuits governing the different phenotypes of these Tc17 subsets. In particular, we discovered that IFNγ- Tc17 subset demonstrated pro-tumoral characteristics and expressed higher levels of CCL20. This corresponded to increased tumor infiltration of T regulatory cells (Treg) validated by immunohistochemistry in another independent HCC cohort, demonstrating the immunosuppressive functions of IFNγ- Tc17 subset. Most importantly, higher intra-tumoral proportions of IFNγ- Tc17 were associated with poorer prognosis in patients with HCC and this was further validated in The Cancer Genome Atlas (TCGA) HCC cohort. Taken together, this compendium of transcriptomic and proteomic data of Tc17 subsets sheds light on the immunosuppressive phenotypes of IFNγ- Tc17 and its implications in HCC progression.
Assuntos
Carcinoma Hepatocelular , Tolerância Imunológica , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linfócitos T CD8-Positivos , Interferon gama , Interleucina-17/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , ProteômicaRESUMO
The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity of TAZ and YAP through physical interaction with Amot and AmotL1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiomotinas , Divisão Celular/fisiologia , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Citoplasma/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Fosfoproteínas/química , Fosfoproteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Terciária de Proteína , Transativadores , Fatores de Transcrição , Transcrição Gênica/fisiologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected half a billion people, including vulnerable populations such as cancer patients. While increasing evidence supports the persistence of SARS-CoV-2 months after a negative nasopharyngeal swab test, the effects on long-term immune memory and cancer treatment are unclear. In this report, we examined post-COVID-19 tissue-localized immune responses in a hepatocellular carcinoma (HCC) patient and a colorectal cancer (CRC) patient. Using spatial whole-transcriptomic analysis, we demonstrated spatial profiles consistent with a lymphocyte-associated SARS-CoV-2 response (based on two public COVID-19 gene sets) in the tumors and adjacent normal tissues, despite intra-tumor heterogeneity. The use of RNAscope and multiplex immunohistochemistry revealed that the spatial localization of B cells was significantly associated with lymphocyte-associated SARS-CoV-2 responses within the spatial transcriptomic (ST) niches showing the highest levels of virus. Furthermore, single-cell RNA sequencing data obtained from previous (CRC) or new (HCC) ex vivo stimulation experiments showed that patient-specific SARS-CoV-2 memory B cells were the main contributors to this positive association. Finally, we evaluated the spatial associations between SARS-CoV-2-induced immunological effects and immunotherapy-related anti-tumor immune responses. Immuno-predictive scores (IMPRES) revealed consistent positive spatial correlations between T cells/cytotoxic lymphocytes and the predicted immune checkpoint blockade (ICB) response, particularly in the HCC tissues. However, the positive spatial correlation between B cells and IMPRES score was restricted to the high-virus ST niche. In addition, tumor immune dysfunction and exclusion (TIDE) analysis revealed marked T cell dysfunction and inflammation, alongside low T cell exclusion and M2 tumor-associated macrophage infiltration. Our results provide in situ evidence of SARS-CoV-2-generated persistent immunological memory, which could not only provide tissue protection against reinfection but may also modulate the tumor microenvironment, favoring ICB responsiveness. As the number of cancer patients with COVID-19 comorbidity continues to rise, improved understanding of the long-term immune response induced by SARS-CoV-2 and its impact on cancer treatment is much needed.
Assuntos
COVID-19 , Carcinoma Hepatocelular , Neoplasias Hepáticas , Comorbidade , Humanos , Inibidores de Checkpoint Imunológico , Memória Imunológica , Morbidade , SARS-CoV-2 , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Linfócitos T CD8-Positivos , Carcinoma Hepatocelular/patologia , Humanos , Evasão da Resposta Imune , Neoplasias Hepáticas/patologia , Camundongos , TranscriptomaRESUMO
The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ protein stability is controlled by a phosphodegron recognized by the F-box protein ß-TrCP and ubiquitylated by the SCF/CRL1(ß-TrCP) E3 ligase. The interaction between TAZ and ß-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in TAZ by LATS primes it for further phosphorylation by CK1ε and subsequent binding by ß-TrCP. Therefore, the Hippo pathway negatively regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated TAZ degradation plays an important role in negatively regulating TAZ biological functions.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Animais , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Células NIH 3T3 , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Proteínas Ligases SKP Culina F-Box/genética , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
The Hippo pathway is an evolutionally conserved protein kinase cascade involved in regulating organ size in vivo and cell contact inhibition in vitro by governing cell proliferation and apoptosis. Deregulation of the Hippo pathway is linked to cancer development. Its first core kinase Warts was identified in Drosophila more than 15 years ago, but it gained much attention when other core components of the pathway were identified 8 years later. Major discoveries of the pathway were made during past several years. The core kinase components Hippo, Salvador, Warts, and Mats in the fly and Mst1/2, WW45, Lats1/2, and Mob1 in mammals phosphorylate and inactivate downstream transcriptional co-activators Yorkie in the fly, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in mammals, respectively. Phosphorylated Yorkie, YAP, and TAZ are sequestered in the cytoplasm by interaction with 14-3-3 proteins. Here we review recent progresses of this pathway by focusing on how these proteins communicate with each other and how loss of regulation results in cancers.
Assuntos
Neoplasias/enzimologia , Neoplasias/patologia , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Transcrição GênicaRESUMO
The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune-ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune-ITH is associated with tumour transcriptomic-ITH, mutational burden and distinct immune microenvironments. Tumours with low immune-ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks contributing to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression.
Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Progressão da Doença , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , DNA/genética , Edição de Genes , Redes Reguladoras de Genes , Humanos , Leucócitos Mononucleares/metabolismo , Filogenia , Prognóstico , RNA/genética , Análise de Sobrevida , Transcriptoma/genética , Resultado do Tratamento , Microambiente Tumoral/imunologiaRESUMO
Intravesical Bacillus Calmette-Guerin (BCG) is an effective immunotherapy for non-muscle invasive bladder cancer (NMIBC). However, recurrence and progression remain frequent warranting deeper insights into its mechanism. We herein comprehensively profiled blood and tissues obtained from NMIBC patients before, during and after BCG treatment using cytometry by time-of-flight (CyTOF) and RNA sequencing to identify the key immune subsets crucial for anti-tumor activity. We observed the temporal changes of peripheral immune subsets including NKT cells, central memory CD4+ T cells, CD8+ T cells and regulatory T cells (Treg) during the course of BCG. Gene expression analysis revealed enriched immune pathways involving in T cell activation and chemotaxis, as well as a more diversified T cell receptor repertoire in post-BCG tissues. Moreover, tissue multiplexed-immunofluorescence (mIF) showed baseline densities of non-Treg and CD8+PD-1+ T cells were predictive of response and better recurrence-free survival after BCG. Remarkably, post-BCG tissues from responders were found to be infiltrated with more active CD8+PD-1- T cells and non-Treg CD4+FOXP3- T cells; but increased exhausted CD8+PD-1+ T cells were found in non-responders. Taken together, we identified predictive biomarkers for response and uncovered the post-treatment expansion of exhausted PD-1+CD8+ T cells as key to BCG resistance, which could potentially be restored by combining with anti-PD-1 immunotherapy.
Assuntos
Vacina BCG/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Imunoterapia Ativa , Subpopulações de Linfócitos/imunologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Carcinoma de Células de Transição/imunologia , Carcinoma de Células de Transição/terapia , Quimiotaxia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Citometria por Imagem/instrumentação , Citometria por Imagem/métodos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Ativação Linfocitária , Contagem de Linfócitos , Subpopulações de Linfócitos/efeitos dos fármacos , Receptor de Morte Celular Programada 1/análise , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/análise , Análise de Célula Única , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Fatores de Tempo , Transcriptoma , Evasão Tumoral , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
Background: CD38 is involved in the adenosine pathway, which represents one of the immunosuppressive mechanisms in cancer. CD38 is broadly expressed across immune cell subsets, including human macrophages differentiated in vitro from monocytes, but expression by tissue-resident macrophages remains to be demonstrated. Methods: Tissue samples were obtained from 66 patients with hepatocellular carcinoma (HCC) from Singapore and analyzed using immunohistochemistry. Tumor-infiltrating leukocytes (TILs) were further examined using DEPArray™, and the phenotype of freshly isolated TILs was determined using flow cytometry. Results: CD38 was frequently co-expressed with the macrophage-specific marker CD68. CD38+CD68+ macrophage density was associated with improved prognosis after surgery, while total CD68+ macrophage density was associated with poor prognosis. DEPArray™ analysis revealed the presence of large (>10 µm), irregularly shaped CD45+CD14+ cells that resembled macrophages, with concurrent CD38+ expression. Flow cytometry also revealed that majority of CD14+HLA-DR+ cells expressed CD38. Conclusion: CD38 expression was clearly demonstrated on human macrophages in an in vivo setting. The positive association identified between CD38+ macrophage density and prognosis may have implications for routine diagnostic work.