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2.
J Immunother Cancer ; 11(6)2023 06.
Article in English | MEDLINE | ID: mdl-37364933

ABSTRACT

BACKGROUND: PD-1 checkpoint blockade therapy (CBT) has greatly benefited patients with select solid tumors and lymphomas but has limited efficacy against diffuse large B-cell lymphoma (DLBCL). Because numerous inhibitory checkpoint receptors have been implicated in driving tumor-specific T cell dysfunction, we hypothesized that combinatorial CBT would enhance the activity of anti-PD-1-based therapy in DLBCL. T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is a coinhibitory receptor expressed on dysfunctional tumor-infiltrating T cells, and TIGIT blockade has demonstrated encouraging activity in combination with PD-1 blockade in murine tumor models and in clinical studies. However, the degree to which TIGIT mediates T cell dysfunction in DLBCL has not been fully explored. RESULTS: Here, we demonstrate that TIGIT is broadly expressed on lymphoma-infiltrating T cells (LITs) across a variety of human lymphomas and is frequently coexpressed with PD-1. TIGIT expression is particularly common on LITs in DLBCL, where TIGIT+ LITs often form distinct cellular communities and exhibit significant contact with malignant B cells. TIGIT+/PD-1+ LITs from human DLBCL and murine lymphomas exhibit hypofunctional cytokine production on ex vivo restimulation. In mice with established, syngeneic A20 B-cell lymphomas, TIGIT or PD-1 mono-blockade leads to modest delays in tumor outgrowth, whereas PD-1 and TIGIT co-blockade results in complete rejection of A20 lymphomas in most mice and significantly prolongs survival compared with mice treated with monoblockade therapy. CONCLUSIONS: These results provide rationale for clinical investigation of TIGIT and PD-1 blockade in lymphomas, including DLBCL.


Subject(s)
Lymphoma, Large B-Cell, Diffuse , Programmed Cell Death 1 Receptor , Humans , Animals , Mice , Receptors, Immunologic/metabolism , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/pathology
3.
Commun Biol ; 5(1): 1263, 2022 11 18.
Article in English | MEDLINE | ID: mdl-36400937

ABSTRACT

Upcoming technologies enable routine collection of highly multiplexed (20-60 channel), subcellular resolution images of mammalian tissues for research and diagnosis. Extracting single cell data from such images requires accurate image segmentation, a challenging problem commonly tackled with deep learning. In this paper, we report two findings that substantially improve image segmentation of tissues using a range of machine learning architectures. First, we unexpectedly find that the inclusion of intentionally defocused and saturated images in training data substantially improves subsequent image segmentation. Such real augmentation outperforms computational augmentation (Gaussian blurring). In addition, we find that it is practical to image the nuclear envelope in multiple tissues using an antibody cocktail thereby better identifying nuclear outlines and improving segmentation. The two approaches cumulatively and substantially improve segmentation on a wide range of tissue types. We speculate that the use of real augmentations will have applications in image processing outside of microscopy.


Subject(s)
Deep Learning , Humans , Animals , Image Processing, Computer-Assisted/methods , Machine Learning , Cell Nucleus , Mammals
4.
Cell Genom ; 2(8)2022 Aug 10.
Article in English | MEDLINE | ID: mdl-36419822

ABSTRACT

Intra-tumor heterogeneity (ITH) of human tumors is important for tumor progression, treatment response, and drug resistance. However, the spatial distribution of ITH remains incompletely understood. Here, we present spatial analysis of ITH in lung adenocarcinomas from 147 patients using multi-region mass spectrometry of >5,000 regions, single-cell copy number sequencing of ~2,000 single cells, and cyclic immunofluorescence of >10 million cells. We identified two distinct spatial patterns among tumors, termed clustered and random geographic diversification (GD). These patterns were observed in the same samples using both proteomic and genomic data. The random proteomic GD pattern, which is characterized by decreased cell adhesion and lower levels of tumor-interacting endothelial cells, was significantly associated with increased risk of recurrence or death in two independent patient cohorts. Our study presents comprehensive spatial mapping of ITH in lung adenocarcinoma and provides insights into the mechanisms and clinical consequences of GD.

5.
Commun Biol ; 5(1): 1066, 2022 10 07.
Article in English | MEDLINE | ID: mdl-36207580

ABSTRACT

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.


Subject(s)
Epidermal Growth Factor , Proteomics , Epidermal Growth Factor/pharmacology , Extracellular Matrix Proteins , Ligands , Phenotype
6.
Science ; 376(6592): eabi8175, 2022 04 29.
Article in English | MEDLINE | ID: mdl-35482859

ABSTRACT

Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.


Subject(s)
Melanoma , Skin Neoplasms , Humans , Melanocytes/metabolism , Melanoma/pathology , Mutation , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Microenvironment/genetics
7.
Cancer Discov ; 12(6): 1518-1541, 2022 06 02.
Article in English | MEDLINE | ID: mdl-35404441

ABSTRACT

Cutaneous melanoma is a highly immunogenic malignancy that is surgically curable at early stages but life-threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially resolved microregion transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis involving precursor states, melanoma in situ, and invasive tumor. Hallmarks of immunosuppression are already detectable in precursor regions. When tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary. This environment is established by cytokine gradients that promote expression of MHC-II and IDO1, and by PD1-PDL1-mediated cell contacts involving macrophages, dendritic cells, and T cells. A few millimeters away, cytotoxic T cells synapse with melanoma cells in fields of tumor regression. Thus, invasion and immunoediting can coexist within a few millimeters of each other in a single specimen. SIGNIFICANCE: The reorganization of the tumor ecosystem in primary melanoma is an excellent setting in which to study immunoediting and immune evasion. Guided by classic histopathology, spatial profiling of proteins and mRNA reveals recurrent morphologic and molecular features of tumor evolution that involve localized paracrine cytokine signaling and direct cell-cell contact. This article is highlighted in the In This Issue feature, p. 1397.


Subject(s)
Melanoma , Skin Neoplasms , Cytokines , Ecosystem , Humans , Melanoma/pathology , Skin Neoplasms/genetics , Melanoma, Cutaneous Malignant
8.
Nat Methods ; 19(3): 311-315, 2022 03.
Article in English | MEDLINE | ID: mdl-34824477

ABSTRACT

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.


Subject(s)
Image Processing, Computer-Assisted , Neoplasms , Diagnostic Imaging , Humans , Image Processing, Computer-Assisted/methods , Neoplasms/diagnostic imaging , Neoplasms/pathology , Software
9.
Cancer Cell ; 39(12): 1623-1642.e20, 2021 12 13.
Article in English | MEDLINE | ID: mdl-34739845

ABSTRACT

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.


Subject(s)
Antigen-Presenting Cells/metabolism , CD28 Antigens/metabolism , Immune Checkpoint Inhibitors/therapeutic use , Myeloid Cells/metabolism , Neoplasms/drug therapy , Stem Cell Niche/genetics , Humans , Immune Checkpoint Inhibitors/pharmacology , Neoplasms/immunology
10.
Cell ; 183(7): 1848-1866.e26, 2020 12 23.
Article in English | MEDLINE | ID: mdl-33301708

ABSTRACT

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.


Subject(s)
Immunity , Neoplasms/immunology , Neoplasms/metabolism , Obesity/metabolism , Tumor Microenvironment , Adiposity , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Cell Proliferation , Diet, High-Fat , Fatty Acids/metabolism , HEK293 Cells , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Kinetics , Lymphocytes, Tumor-Infiltrating , Mice, Inbred C57BL , Mice, Knockout , Oxidation-Reduction , Principal Component Analysis , Procollagen-Proline Dioxygenase/metabolism , Proteomics
11.
Cell ; 183(5): 1354-1366.e13, 2020 11 25.
Article in English | MEDLINE | ID: mdl-33065030

ABSTRACT

The COVID-19 pandemic has led to extensive morbidity and mortality throughout the world. Clinical features that drive SARS-CoV-2 pathogenesis in humans include inflammation and thrombosis, but the mechanistic details underlying these processes remain to be determined. In this study, we demonstrate endothelial disruption and vascular thrombosis in histopathologic sections of lungs from both humans and rhesus macaques infected with SARS-CoV-2. To define key molecular pathways associated with SARS-CoV-2 pathogenesis in macaques, we performed transcriptomic analyses of bronchoalveolar lavage and peripheral blood and proteomic analyses of serum. We observed macrophage infiltrates in lung and upregulation of macrophage, complement, platelet activation, thrombosis, and proinflammatory markers, including C-reactive protein, MX1, IL-6, IL-1, IL-8, TNFα, and NF-κB. These results suggest a model in which critical interactions between inflammatory and thrombosis pathways lead to SARS-CoV-2-induced vascular disease. Our findings suggest potential therapeutic targets for COVID-19.


Subject(s)
COVID-19/complications , COVID-19/immunology , SARS-CoV-2/genetics , Thrombosis/complications , Vascular Diseases/complications , Aged, 80 and over , Animals , Bronchoalveolar Lavage , C-Reactive Protein/analysis , COVID-19/blood , COVID-19/pathology , Complement Activation , Cytokines/blood , Female , Humans , Inflammation/blood , Inflammation/immunology , Inflammation/virology , Lung/pathology , Macaca mulatta , Macrophages/immunology , Male , Platelet Activation , Thrombosis/blood , Thrombosis/pathology , Transcriptome , Vascular Diseases/blood , Vascular Diseases/pathology
13.
Nat Commun ; 11(1): 1459, 2020 03 19.
Article in English | MEDLINE | ID: mdl-32193378

ABSTRACT

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B7-H1 Antigen/genetics , Neoplasm Recurrence, Local/drug therapy , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , DNA Mutational Analysis , Drug Monitoring/methods , Female , Gene Amplification , Humans , Indazoles/pharmacology , Indazoles/therapeutic use , Interferons/immunology , Interferons/metabolism , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/immunology , Neoplasm Recurrence, Local/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Ovary/pathology , Piperidines/pharmacology , Piperidines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Programmed Cell Death 1 Ligand 2 Protein/genetics , Programmed Cell Death 1 Ligand 2 Protein/metabolism , Recombinational DNA Repair/genetics , Single-Cell Analysis , Treatment Outcome , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
14.
Cell Syst ; 8(5): 412-426.e7, 2019 05 22.
Article in English | MEDLINE | ID: mdl-31078528

ABSTRACT

Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance.


Subject(s)
Induced Pluripotent Stem Cells/drug effects , Myocytes, Cardiac/metabolism , Protein Kinase Inhibitors/pharmacology , Acclimatization , Antineoplastic Agents/pharmacology , Cardiotoxicity/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Erlotinib Hydrochloride/pharmacology , Gene Expression Profiling/methods , Humans , Induced Pluripotent Stem Cells/metabolism , Lapatinib/pharmacology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Sorafenib/pharmacology , Sunitinib/pharmacology
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