RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and iNOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors including head and neck cancers. We show for the first time that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti-PD1, and anti-CTLA4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid-T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid-T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.
RESUMO
This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Técnicas de Cocultura , Transição Epitelial-Mesenquimal , Inflamação , Integrina beta1 , Neoplasias Pancreáticas , Análise de Célula Única , Microambiente Tumoral , Humanos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Inflamação/patologia , Inflamação/metabolismo , Integrina beta1/metabolismo , Integrina beta1/genética , Organoides/patologia , Organoides/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Comunicação CelularRESUMO
The immune system defines a complex network of tissues and cell types that orchestrate responses across the body in a dynamic manner. The local and systemic interactions between immune and cancer cells contribute to disease progression. Lymphocytes are activated in lymph nodes, traffic through the periphery, and impact cancer progression through their interactions with tumor cells. As a result, therapeutic response and resistance are mediated across tissues, and a comprehensive understanding of lymphocyte dynamics requires a systems-level approach. In this review, we highlight experimental and computational methods that can leverage the study of leukocyte trafficking through an immunomics lens and reveal how adaptive immunity shapes cancer.
Assuntos
Imunoinformática , Neoplasias , Humanos , Linfócitos , Neoplasias/terapia , Leucócitos , LinfonodosRESUMO
Recent advances in spatial transcriptomics (STs) enable gene expression measurements from a tissue sample while retaining its spatial context. This technology enables unprecedented in situ resolution of the regulatory pathways that underlie the heterogeneity in the tumor as well as the tumor microenvironment (TME). The direct characterization of cellular co-localization with spatial technologies facilities quantification of the molecular changes resulting from direct cell-cell interaction, as it occurs in tumor-immune interactions. We present SpaceMarkers, a bioinformatics algorithm to infer molecular changes from cell-cell interactions from latent space analysis of ST data. We apply this approach to infer the molecular changes from tumor-immune interactions in Visium spatial transcriptomics data of metastasis, invasive and precursor lesions, and immunotherapy treatment. Further transfer learning in matched scRNA-seq data enabled further quantification of the specific cell types in which SpaceMarkers are enriched. Altogether, SpaceMarkers can identify the location and context-specific molecular interactions within the TME from ST data.
Assuntos
Algoritmos , Microambiente Tumoral , Comunicação Celular , Biologia Computacional , Perfilação da Expressão GênicaRESUMO
Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.
RESUMO
Mass cytometry, or cytometry by TOF (CyTOF), provides a robust means of determining protein-level measurements of more than 40 markers simultaneously. While the functional states of immune cells occur along continuous phenotypic transitions, cytometric studies surveying cell phenotypes often rely on static metrics, such as discrete cell-type abundances, based on canonical markers and/or restrictive gating strategies. To overcome this limitation, we applied single-cell trajectory inference and nonnegative matrix factorization methods to CyTOF data to trace the dynamics of T cell states. In the setting of cancer immunotherapy, we showed that patient-specific summaries of continuous phenotypic shifts in T cells could be inferred from peripheral blood-derived CyTOF mass cytometry data. We further illustrated that transfer learning enabled these T cell continuous metrics to be used to estimate patient-specific cell states in new sample cohorts from a reference patient data set. Our work establishes the utility of continuous metrics for CyTOF analysis as tools for translational discovery.
Assuntos
Benchmarking , Linfócitos T , Biomarcadores/análise , Ensaios Clínicos como Assunto , Citometria de Fluxo/métodos , Fatores Imunológicos , Imunoterapia , Monitorização ImunológicaRESUMO
Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.
Assuntos
Neoplasias da Mama , Células Supressoras Mieloides , Animais , Benzamidas/farmacologia , Neoplasias da Mama/metabolismo , Feminino , Humanos , Terapia de Imunossupressão , Camundongos , Piridinas , Microambiente TumoralRESUMO
BACKGROUND: The gut microbiome harbors trillions of bacteria that play a major role in dietary nutrient extraction and host metabolism. Metabolic diseases such as obesity and diabetes are associated with shifts in microbiome composition and have been on the rise in Westernized or highly industrialized countries. At the same time, Westernized diets low in dietary fiber have been shown to cause loss of gut microbial diversity. However, the link between microbiome composition, loss of dietary fiber, and obesity has not been well defined. RESULTS: To study the interactions between gut microbiota, dietary fiber, and weight gain, we transplanted captive and wild douc gut microbiota into germ-free mice and then exposed them to either a high- or low-fiber diet. The group receiving captive douc microbiota gained significantly more weight, regardless of diet, while mice receiving a high-fiber diet and wild douc microbiota remained lean. In the presence of a low-fiber diet, the wild douc microbiota partially prevented weight gain. Using 16S rRNA gene amplicon sequencing we identified key bacterial taxa in each group, specifically a high relative abundance of Bacteroides and Akkermansia in captive douc FMT mice and a higher relative abundance of Lactobacillus and Clostridium in the wild douc FMT mice. CONCLUSIONS: In the context of our germ-free mouse experiment, wild douc microbiota could serve as a reservoir for microbes for cross-species transplants. Our results suggest that wild douc microbiota are tailored to diverse fiber diets and can prevent weight gain when exposed to a native diet.