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1.
Nature ; 611(7937): 810-817, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36385528

RESUMO

The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types1,2. Intratumoral host-microbiota studies have so far largely relied on bulk tissue analysis1-3, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies4 and single-cell RNA sequencing5 to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host-microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling6, we show that bacterial communities populate microniches that are less vascularized, highly immuno­suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion-adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Colorretais , Interações entre Hospedeiro e Microrganismos , Microbiota , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/microbiologia , Carcinoma de Células Escamosas/patologia , Microbiota/genética , Microbiota/imunologia , Microbiota/fisiologia , Neoplasias Bucais/genética , Neoplasias Bucais/imunologia , Neoplasias Bucais/microbiologia , Neoplasias Bucais/patologia , Células Mieloides/imunologia , Microambiente Tumoral , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Análise de Sequência de RNA , Perfilação da Expressão Gênica , Antígeno Ki-67/metabolismo , Progressão da Doença
2.
JCI Insight ; 9(17)2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39052387

RESUMO

Over 200,000 individuals are diagnosed with lung cancer in the United States every year, with a growing proportion of cases, especially lung adenocarcinoma, occurring in individuals who have never smoked. Women over the age of 50 comprise the largest affected demographic. To understand the genomic drivers of lung adenocarcinoma and therapeutic response in this population, we performed whole genome and/or whole exome sequencing on 73 matched lung tumor/normal pairs from postmenopausal women who participated in the Women's Health Initiative. Somatic copy number alterations showed little variation by smoking status, suggesting that aneuploidy may be a general characteristic of lung cancer regardless of smoke exposure. Similarly, clock-like and APOBEC mutation signatures were prevalent but did not differ in tumors from smokers and never-smokers. However, mutations in both EGFR and KRAS showed unique allelic differences determined by smoking status that are known to alter tumor response to targeted therapy. Mutations in the MYC-network member MGA were more prevalent in tumors from smokers. Fusion events in ALK, RET, and ROS1 were absent, likely due to age-related differences in fusion prevalence. Our work underscores the profound effect of smoking status, age, and sex on the tumor mutational landscape and identifies areas of unmet medical need.


Assuntos
Neoplasias Pulmonares , Mutação , Humanos , Feminino , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/epidemiologia , Idoso , Pessoa de Meia-Idade , Fumar/genética , Fumar/epidemiologia , Fumar/efeitos adversos , Saúde da Mulher , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/epidemiologia , não Fumantes/estatística & dados numéricos , Genômica/métodos , Variações do Número de Cópias de DNA , Estados Unidos/epidemiologia , Receptores ErbB/genética , Sequenciamento do Exoma , Proteínas Proto-Oncogênicas p21(ras)/genética , Fumantes/estatística & dados numéricos
3.
Nat Protoc ; 18(11): 3355-3389, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37789194

RESUMO

Single-cell RNA sequencing (scRNAseq) technologies have been beneficial in revealing and describing cellular heterogeneity within mammalian tissues, including solid tumors. However, many of these techniques apply poly(A) selection of RNA, and thus have primarily focused on determining the gene signatures of eukaryotic cellular components of the tumor microenvironment. Microbiome analysis has revealed the presence of microbial ecosystems, including bacteria and fungi, within human tumor tissues from major cancer types. Imaging data have revealed that intratumoral bacteria may be located within epithelial and immune cell types. However, as bacterial RNA typically lacks a poly(A) tail, standard scRNAseq approaches have limited ability to capture this microbial component of the tumor microenvironment. To overcome this, we describe the invasion-adhesion-directed expression sequencing (INVADEseq) approach, whereby we adapt 10x Genomics 5' scRNAseq protocol by introducing a primer that targets a conserved region of the bacterial 16S ribosomal RNA gene in addition to the standard primer for eukaryotic poly(A) RNA selection. This 'add-on' approach enables the generation of eukaryotic and bacterial DNA libraries at eukaryotic single-cell level resolution, utilizing the 10x barcode to identify single cells with intracellular bacteria. The INVADEseq method takes 30 h to complete, including tissue processing, sequencing and computational analysis. As an output, INVADEseq has shown to be a reliable tool in human cancer cell lines and patient tumor specimens by detecting the proportion of human cells that harbor bacteria and the identities of human cells and intracellular bacteria, along with identifying host transcriptional programs that are modulated on the basis of associated bacteria.


Assuntos
Microbiota , Neoplasias , Animais , Humanos , Transcriptoma , Bactérias/genética , Genômica/métodos , Neoplasias/patologia , Microbiota/genética , Mamíferos/genética , Microambiente Tumoral
4.
Res Sq ; 2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37886484

RESUMO

Genetic engineering of human lymphocytes for therapeutic applications is constrained by a lack of transgene transcriptional control, resulting in a compromised therapeutic index. Incomplete understanding of transcriptional logic limits the rational design of contextually responsive genetic modules1. Here, we juxtaposed rationally curated transcriptional response element (TRE) oligonucleotides by random concatemerization to generate a library from which we selected context-specific inducible synthetic promoters (iSynPros). Through functional selection, we screened an iSynPro library for "IF-THEN" logic-gated transcriptional responses in human CD8+ T cells expressing a 4-1BB second generation chimeric antigen receptor (CAR). iSynPros exhibiting stringent off-states in quiescent T cells and CAR activation-dependent transcriptional responsiveness were cloned and subjected to TRE composition and pattern analysis, as well as performance in regulating candidate antitumor potency enhancement modules. These data reveal synthetic TRE grammar can mediate logic-gated transgene transcription in human T cells that, when applied to CAR T cell engineering, enhance potency and improve therapeutic indices.

5.
bioRxiv ; 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36712079

RESUMO

Lung cancer in never-smokers disproportionately affects older women. To understand the mutational landscape of this cohort, we performed detailed genome characterization of 73 lung adenocarcinomas from participants of the Women’s Health Initiative (WHI). We find enrichment of EGFR mutations in never-/light-smokers and KRAS mutations in heavy smokers as expected, but we also show that the specific variants of these genes differ by smoking status, with important therapeutic implications. Mutational signature analysis revealed signatures of clock, APOBEC, and DNA repair deficiency in never-/light-smokers; however, the mutational load of these signatures did not differ significantly from those found in smokers. Last, tumors from both smokers and never-/light-smokers shared copy number subtypes, with no significant differences in aneuploidy. Thus, the genomic landscape of lung cancer in never-/light-smokers and smokers is predominantly differentiated by somatic mutations and not copy number alterations.

6.
Cancer Med ; 12(6): 6583-6593, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36341526

RESUMO

BACKGROUND: The anti-tumor immune response plays a key role in colorectal cancer (CRC) progression and survival. The T cell-inflamed gene expression profile (GEP) is a biomarker predicting response to checkpoint inhibitor immunotherapy across immunogenic cancer types, but the prognostic value in CRC is unknown. We evaluated associations with disease-specific survival, somatic mutations, and examined its differentially expressed genes and pathways among 84 sporadic CRC patients from the Seattle Colon Cancer Family Registry. METHODS: Gene expression profiling was performed using Nanostring's nCounter PanCancer IO 360 panel. Somatic mutations were identified by a targeted DNA sequencing panel. RESULTS: The T cell-inflamed GEP was positively associated with tumor mutation burden and microsatellite instability high (MSI-H). Higher T cell-inflamed GEP had favorable CRC-specific survival (hazard ratio [HR] per standard deviation unit = 0.50, p = 0.004) regardless of hypermutation or MSI status. Analysis of recurrently mutated genes having at least 10 mutation carriers, suggested that the T cell-inflamed GEP is positively associated with RYR1, and negatively associated with APC. However, these associations were attenuated after adjusting for hypermutation or MSI status. We also found that expression of genes RPL23, EPCAM, AREG and ITGA6, and the Wnt signaling pathway was negatively associated with the T cell-inflamed GEP, which might indicate immune-inhibitory mechanisms. CONCLUSIONS: Our results show that the T cell-inflamed GEP is a prognostic biomarker in non-hypermutated microsatellite-stable CRC. This also suggests that patient stratification for immunotherapy within this CRC subgroup should be explored further. Moreover, reported immune-inhibitory gene expression signals may suggest targets for therapeutic combination with immunotherapy.


Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/patologia , Transcriptoma , Instabilidade de Microssatélites , Prognóstico , Repetições de Microssatélites , Mutação
7.
Front Immunol ; 12: 782152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868058

RESUMO

Minor histocompatibility antigens (mHAg) composed of peptides presented by HLA molecules can cause immune responses involved in graft-versus-host disease (GVHD) and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (HCT). The current study was designed to identify individual graft-versus-host genomic mismatches associated with altered risks of acute or chronic GVHD or relapse after HCT between HLA-genotypically identical siblings. Our results demonstrate that in allogeneic HCT between a pair of HLA-identical siblings, a mHAg manifests as a set of peptides originating from annotated proteins and non-annotated open reading frames, which i) are encoded by a group of highly associated recipient genomic mismatches, ii) bind to HLA allotypes in the recipient, and iii) evoke a donor immune response. Attribution of the immune response and consequent clinical outcomes to individual peptide components within this set will likely differ from patient to patient according to their HLA types.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Menor/imunologia , Imunologia de Transplantes , Adolescente , Adulto , Idoso , Alelos , Criança , Pré-Escolar , Suscetibilidade a Doenças/imunologia , Feminino , Predisposição Genética para Doença , Variação Genética , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/etiologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Incidência , Lactente , Recém-Nascido , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/genética , Peptídeos/genética , Peptídeos/imunologia , Transplante Homólogo , Adulto Jovem
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