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1.
Am J Physiol Lung Cell Mol Physiol ; 322(6): L822-L841, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35438006

RESUMO

Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.


Assuntos
Pneumopatias , RNA Longo não Codificante , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Epitélio/metabolismo , Humanos , Pneumopatias/metabolismo , RNA Longo não Codificante/genética , Mucosa Respiratória/metabolismo
2.
STAR Protoc ; 4(4): 102735, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37991921

RESUMO

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1.


Assuntos
Células Epiteliais , Pulmão , Humanos , Células Cultivadas , Organoides
3.
bioRxiv ; 2023 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-37034597

RESUMO

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression towards severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an "early" inflammatory/immune signature preceding a "late" type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

4.
iScience ; 26(8): 107374, 2023 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-37520727

RESUMO

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression toward severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an "early" inflammatory/immune signature preceding a "late" type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

5.
STAR Protoc ; 3(4): 101698, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36149794

RESUMO

We describe a pipeline for optimized and streamlined multiplexed immunofluorescence-guided laser capture microdissection allowing the harvest of individual cells based on their phenotype and tissue localization for transcriptomic analysis with next-generation RNA sequencing. Here, we analyze transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages, and melanoma cells in non-dissociated metastatic melanoma tissue. While this protocol is described for melanoma tissues, we successfully applied it to human tonsil, skin, and breast cancer tissues as well as mouse lung tissues. For complete details on the use and execution of this protocol, please refer to Martinek et al. (2022).


Assuntos
Microdissecção e Captura a Laser , Melanoma , Animais , Humanos , Camundongos , Imunofluorescência , Perfilação da Expressão Gênica/métodos , Microdissecção e Captura a Laser/métodos , Melanoma/genética , Melanoma/cirurgia , Transcriptoma/genética
6.
Sci Adv ; 8(3): eabg6711, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35044822

RESUMO

Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is unknown. We developed a long-read (LR) RNA sequencing and analytical platform that identifies and annotates full-length isoforms and infers tumor-specific splicing events. Application of this platform to breast cancer samples identifies thousands of previously unannotated isoforms; ~30% affect protein coding exons and are predicted to alter protein localization and function. We performed extensive cross-validation with -omics datasets to support transcription and translation of novel isoforms. We identified 3059 breast tumor­specific splicing events, including 35 that are significantly associated with patient survival. Of these, 21 are absent from GENCODE and 10 are enriched in specific breast cancer subtypes. Together, our results demonstrate the complexity, cancer subtype specificity, and clinical relevance of previously unidentified isoforms and splicing events in breast cancer that are only annotatable by LR-seq and provide a rich resource of immuno-oncology therapeutic targets.


Assuntos
Neoplasias da Mama , Processamento Alternativo , Neoplasias da Mama/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma
7.
Cell Rep Med ; 3(5): 100621, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35584631

RESUMO

Modulation of immune function at the tumor site could improve patient outcomes. Here, we analyze patient samples of metastatic melanoma, a tumor responsive to T cell-based therapies, and find that tumor-infiltrating T cells are primarily juxtaposed to CD14+ monocytes/macrophages rather than melanoma cells. Using immunofluorescence-guided laser capture microdissection, we analyze transcriptomes of CD3+ T cells, CD14 + monocytes/macrophages, and melanoma cells in non-dissociated tissue. Stromal CD14+ cells display a specific transcriptional signature distinct from CD14+ cells within tumor nests. This signature contains LY75, a gene linked with antigen capture and regulation of tolerance and immunity in dendritic cells (DCs). When applied to TCGA cohorts, this gene set can distinguish patients with significantly prolonged survival in metastatic cutaneous melanoma and other cancers. Thus, the stromal CD14+ cell signature represents a candidate biomarker and suggests that reprogramming of stromal macrophages to acquire DC function may offer a therapeutic opportunity for metastatic cancers.


Assuntos
Melanoma , Segunda Neoplasia Primária , Neoplasias Cutâneas , Humanos , Macrófagos , Melanoma/genética , Fenótipo , Neoplasias Cutâneas/genética , Linfócitos T
8.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857287

RESUMO

Metastasis of melanoma significantly worsens prognosis; thus, therapeutic interventions that prevent metastasis could improve patient outcomes. Here, we show using humanized mice that colonization of distant visceral organs with melanoma is dependent upon a human CD33+CD11b+CD117+ progenitor cell subset comprising <4% of the human CD45+ leukocytes. Metastatic tumor-infiltrating CD33+ cells from patients and humanized (h)NSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNA-seq analysis identified a gene signature of a KIT/CD117-expressing CD33+ subset that correlated with decreased overall survival in a TCGA melanoma cohort. Thus, human CD33+CD11b+CD117+ myeloid cells represent a novel candidate biomarker as well as a therapeutic target for metastatic melanoma.


Assuntos
Melanoma/metabolismo , Melanoma/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos NOD , Prognóstico
9.
Methods Enzymol ; 636: 351-368, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32178826

RESUMO

Mouse models of human cancer have been used extensively to circumvent the complexity in human patients. However, murine models often inadequately recapitulate the human cancer-immune interface partly due to important differences between mouse and human immune systems. Immunodeficient mice when transplanted with CD34+ hematopoietic progenitor cells (HPCs) develop multilineage human immune cells. While there remain limitations, efforts have been made to improve the function of human immune system. Thus, humanized mice, defined as mice with human immune system, have become an emerging model to study human cancers. Humanized mouse models have been used for various areas of cancer research including adoptive transfer of chimeric antigen receptor (CAR)-modified T cells, neoantigen vaccination to increase T cell repertoire and reprograming tumor microenvironment. Here, we describe the essential techniques to generate humanized mouse models for immuno-oncology studies.


Assuntos
Neoplasias , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias/terapia , Linfócitos T , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Int Rev Cell Mol Biol ; 348: 179-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31810553

RESUMO

Dendritic cells (DCs) orchestrate a repertoire of immune responses that bring about resistance to infection and tolerance to self. Cancers can exploit DCs to evade immunity, but DCs also can generate resistance to cancer. Owing to their capacity to capture, process, and present antigens to naïve T cells, thereby launching adaptive immunity, DCs are poised to play a critical role in cancer recognition and rejection. As such, DCs represent a solution for the expansion and infiltration of T cells with tumor-rejecting properties. Indeed, clinical responses to checkpoint blockade, such as anti-PD-1, are linked to the presence of T cell immunity to cancer-specific antigens. However, only a fraction of patients has clinical benefit. Unraveling the molecular pathways controlling DC-cancer interplay will therefore pave the way for identifying new targets for therapy that overcome limitations of current treatments and promote long-term cancer control.


Assuntos
Células Dendríticas/imunologia , Neoplasias/imunologia , Animais , Células Dendríticas/patologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia
11.
Cancer Res ; 78(18): 5243-5258, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30012670

RESUMO

Inflammation affects tumor immune surveillance and resistance to therapy. Here, we show that production of IL1ß in primary breast cancer tumors is linked with advanced disease and originates from tumor-infiltrating CD11c+ myeloid cells. IL1ß production is triggered by cancer cell membrane-derived TGFß. Neutralizing TGFß or IL1 receptor prevents breast cancer progression in humanized mouse model. Patients with metastatic HER2- breast cancer display a transcriptional signature of inflammation in the blood leukocytes, which is attenuated after IL1 blockade. When present in primary breast cancer tumors, this signature discriminates patients with poor clinical outcomes in two independent public datasets (TCGA and METABRIC).Significance: IL1ß orchestrates tumor-promoting inflammation in breast cancer and can be targeted in patients using an IL1 receptor antagonist. Cancer Res; 78(18); 5243-58. ©2018 AACRSee related commentary by Dinarello, p. 5200.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/metabolismo , Transcrição Gênica , Animais , Neoplasias da Mama/tratamento farmacológico , Antígeno CD11c/metabolismo , Capecitabina/administração & dosagem , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Furanos/administração & dosagem , Humanos , Inflamação , Proteína Antagonista do Receptor de Interleucina 1/administração & dosagem , Cetonas/administração & dosagem , Leucócitos Mononucleares/citologia , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Células Mieloides/metabolismo , Metástase Neoplásica , Transplante de Neoplasias , Paclitaxel/administração & dosagem , Projetos Piloto , Fator de Crescimento Transformador beta/metabolismo
12.
J Exp Med ; 214(10): 3123-3144, 2017 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-28904110

RESUMO

Aging is linked to deficiencies in immune responses and increased systemic inflammation. To unravel the regulatory programs behind these changes, we applied systems immunology approaches and profiled chromatin accessibility and the transcriptome in PBMCs and purified monocytes, B cells, and T cells. Analysis of samples from 77 young and elderly donors revealed a novel and robust aging signature in PBMCs, with simultaneous systematic chromatin closing at promoters and enhancers associated with T cell signaling and a potentially stochastic chromatin opening mostly found at quiescent and repressed sites. Combined analyses of chromatin accessibility and the transcriptome uncovered immune molecules activated/inactivated with aging and identified the silencing of the IL7R gene and the IL-7 signaling pathway genes as potential biomarkers. This signature is borne by memory CD8+ T cells, which exhibited an aging-related loss in binding of NF-κB and STAT factors. Thus, our study provides a unique and comprehensive approach to identifying candidate biomarkers and provides mechanistic insights into aging-associated immunodeficiency.


Assuntos
Envelhecimento/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Cromatina/fisiologia , Adulto , Idoso , Envelhecimento/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Epigênese Genética , Feminino , Humanos , Interleucina-7/fisiologia , Subunidade alfa de Receptor de Interleucina-7/fisiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/fisiologia , Masculino , Transdução de Sinais/fisiologia , Adulto Jovem
13.
Cancer Immunol Res ; 2(5): 487-500, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24795361

RESUMO

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2) cells and protumor inflammation. Here, we show that intratumoral delivery of the ß-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated via dectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells, E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvß8 and TGF-ß activation in a dectin-1-dependent fashion. These CD103+ CD8+ mucosal T cells accumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+ CD8+ mucosal T cells elicited by reprogrammed DCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy for cancer rejection.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Mucosa/imunologia , Animais , Neoplasias da Mama/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lectinas Tipo C/metabolismo , Camundongos , Mucosa/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Fator de Crescimento Transformador beta/metabolismo , beta-Glucanas/imunologia , beta-Glucanas/farmacologia
14.
J Exp Med ; 208(3): 479-90, 2011 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-21339324

RESUMO

The human breast tumor microenvironment can display features of T helper type 2 (Th2) inflammation, and Th2 inflammation can promote tumor development. However, the molecular and cellular mechanisms contributing to Th2 inflammation in breast tumors remain unclear. Here, we show that human breast cancer cells produce thymic stromal lymphopoietin (TSLP). Breast tumor supernatants, in a TSLP-dependent manner, induce expression of OX40L on dendritic cells (DCs). OX40L(+) DCs are found in primary breast tumor infiltrates. OX40L(+) DCs drive development of inflammatory Th2 cells producing interleukin-13 and tumor necrosis factor in vitro. Antibodies neutralizing TSLP or OX40L inhibit breast tumor growth and interleukin-13 production in a xenograft model. Thus, breast cancer cell-derived TSLP contributes to the inflammatory Th2 microenvironment conducive to breast tumor development by inducing OX40L expression on DCs.


Assuntos
Neoplasias da Mama/fisiopatologia , Citocinas/fisiologia , Inflamação/fisiopatologia , Células Th2/fisiologia , Animais , Anticorpos Antineoplásicos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/fisiologia , Camundongos , Transplante de Neoplasias , Ligante OX40/fisiologia , Células Th2/imunologia , Linfopoietina do Estroma do Timo
15.
J Virol ; 81(19): 10310-5, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17626076

RESUMO

In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.


Assuntos
Antígenos Virais/imunologia , Infecções por Enterovirus/prevenção & controle , Enterovirus/imunologia , Vacinas Virais , Animais , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Reações Cruzadas , Enterovirus/patogenicidade , Infecções por Enterovirus/imunologia , Feminino , Imunização Passiva , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos ICR , Linfócitos T/imunologia , Vacinação , Carga Viral , Vacinas Virais/imunologia
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