RESUMO
Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.
Assuntos
Reprogramação Celular , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Ensaios de Triagem em Larga Escala/métodos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/transplante , Animais , Diferenciação Celular , Linhagem Celular , DNA Mitocondrial/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , TranscriptomaRESUMO
Growing evidence indicates a role for the gut microbiota in modulating anti-tumor treatment efficacy in human cancer. Here we study mucosa-associated invariant T (MAIT) cells to look for evidence of bacterial antigen recognition in human colon, lung, and kidney carcinomas. Using mass cytometry and single-cell mRNA sequencing, we identify a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 and observe high expression of CD39 on MAIT cells from colorectal cancer (CRC) only, which we show in vitro to be expressed specifically after TCR stimulation. We further reveal that these cells are phenotypically and functionally exhausted. Sequencing data show high bacterial infiltration in CRC tumors and highlight an enriched species, Fusobacteria nucleatum, with capability to activate MAIT cells in a TCR-dependent way. Our results provide evidence of a MAIT cell response to microbial antigens in CRC and could pave the way for manipulating MAIT cells or the microbiome for cancer therapy.
Assuntos
Antígenos de Bactérias/imunologia , Neoplasias Colorretais/imunologia , Microbioma Gastrointestinal/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Antígenos CD/imunologia , Apirase/imunologia , Antígenos CD4/imunologia , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/imunologia , Humanos , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is highly effective in microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC); however, specific predictive biomarkers are lacking. PATIENTS AND METHODS: Data and samples from 85 patients with MSI-H mCRC treated with ICIs were gathered. Tumor infiltrating lymphocytes (TILs) and tumor mutational burden (TMB) were analyzed in an exploratory cohort of "super" responders and "clearly" refractory patients; TILs were then evaluated in the whole cohort of patients. Primary objectives were the correlation between the number of TILs and TMB and their role as biomarkers of ICI efficacy. Main endpoints included response rate (RR), progression-free survival (PFS), and overall survival (OS). RESULTS: In the exploratory cohort, an increasing number of TILs correlated to higher TMB (Pearson's test, p = .0429). In the whole cohort, median number of TILs was 3.6 in responders compared with 1.8 in nonresponders (Mann-Whitney test, p = .0448). RR was 70.6% in patients with high number of TILs (TILs-H) compared with 42.9% in patients with low number of TILs (odds ratio = 3.20, p = .0291). Survival outcomes differed significantly in favor of TILs-H (PFS: hazard ratio [HR] = 0.42, p = .0278; OS: HR = 0.41, p = .0463). CONCLUSION: A significant correlation between higher TMB and increased number of TILs was shown. A significantly higher activity and better PFS and OS with ICI in MSI-H mCRC were reported in cases with high number of TILs, thus supporting further studies of TIL count as predictive biomarker of ICI efficacy. IMPLICATIONS FOR PRACTICE: Microsatellite instability is the result of mismatch repair protein deficiency, caused by germline mutations or somatic modifications in mismatch repair genes. In metastatic colorectal cancer (mCRC), immunotherapy (with immune checkpoint inhibitors [ICIs]) demonstrated remarkable clinical benefit in microsatellite instability-high (MSI-H) patients. ICI primary resistance has been observed in approximately 25% of patients with MSI-H mCRC, underlining the need for predictive biomarkers. In this study, tumor mutational burden (TMB) and tumor infiltrating lymphocyte (TIL) analyses were performed in an exploratory cohort of patients with MSI-H mCRC treated with ICIs, demonstrating a significant correlation between higher TMB and increased number of TILs. Results also demonstrated a significant correlation between high number of TILs and clinical responses and survival benefit in a large data set of patients with MSI-H mCRC treated with ICI. TMB and TILs could represent predictive biomarkers of ICI efficacy in MSI-H mCRC and should be incorporated in future trials testing checkpoint inhibitors in colorectal cancer.
Assuntos
Neoplasias Colorretais , Linfócitos do Interstício Tumoral , Biomarcadores Tumorais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Humanos , Instabilidade de MicrossatélitesRESUMO
BACKGROUND: Sebaceous carcinoma (SeC) is an uncommon malignancy arising from sebaceous glands of the conjunctiva and skin. Recurrent mutations in the ZNF750 were recently identified in ocular SeC. We assessed whether ZNF750 loss is a specific feature of ocular SeC or a general feature of sebaceous tumors. METHODS: Immunostaining for ZNF750 expression was performed in 54 benign and malignant sebocytic proliferations. Staining for ZNF750 was scored on a three-tier scale: positive (>75%), partially positive (5%-74%), and negative (<5%). RESULTS: ZNF750 expression was negative in 4/11 ocular SeC, and partially positive in 4/11 ocular SeC and 6/13 cutaneous SeC. No extraocular tumors were negative. No loss was found in sebaceous adenoma or sebaceous hyperplasia. In nine previously sequenced ocular SeCs, two lacked detectable somatic mutations in ZNF750, but showed complete loss of staining, indicating non-mutational inactivation of ZNF750. CONCLUSION: We show complete loss of the ZNF750 epidermal differentiation regulator in about half of ocular SeC, highlighting the most common genetic defect in this cancer type. Loss of ZNF750 expression is seen even in tumors without truncating mutations and reduced in many of the remaining ocular and cutaneous SeC. In contrast, no ZNF750 loss was detected in benign sebaceous proliferations.
Assuntos
Adenocarcinoma Sebáceo , Neoplasias Oculares , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias das Glândulas Sebáceas , Fatores de Transcrição/biossíntese , Adenocarcinoma Sebáceo/metabolismo , Adenocarcinoma Sebáceo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Oculares/metabolismo , Neoplasias Oculares/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias das Glândulas Sebáceas/metabolismo , Neoplasias das Glândulas Sebáceas/patologia , Proteínas Supressoras de TumorRESUMO
Genetic variation in the NF-κB inhibitors, ABIN1 and A20, increase risk for psoriasis. While critical for hematopoietic immune cell function, these genes are believed to additionally inhibit psoriasis by dampening inflammatory signaling in keratinocytes. We dissected ABIN1 and A20's regulatory role in human keratinocyte inflammation using an RNA sequencing-based comparative genomic approach. Here we show subsets of the IL-17 and tumor necrosis factor-α signaling pathways are robustly restricted by A20 overexpression. In contrast, ABIN1 overexpression inhibits these genes more modestly for IL-17, and weakly for tumor necrosis factor-α. Our genome-scale analysis also indicates that inflammatory program suppression appears to be the major transcriptional influence of A20/ABIN1 overexpression, without obvious influence on keratinocyte viability genes. Our findings thus enable dissection of the differing anti-inflammatory mechanisms of two distinct psoriasis modifiers, which may be directly exploited for therapeutic purposes. Importantly, we report that IL-17-induced targets of A20 show similar aberrant epidermal layer-specific transcriptional upregulation in keratinocytes from diseases as diverse as psoriasis, atopic dermatitis, and erythrokeratodermia variabilis, suggesting a contributory role for epidermal inflammation in a broad spectrum of rashes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Exantema/imunologia , Queratinócitos/imunologia , Transdução de Sinais/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Eritroceratodermia Variável/imunologia , Eritroceratodermia Variável/patologia , Exantema/patologia , Genômica , Humanos , Interleucina-17/imunologia , Interleucina-17/metabolismo , Queratinócitos/patologia , Cultura Primária de Células , Psoríase/imunologia , Psoríase/patologia , RNA-Seq , Análise de Célula Única , Pele/citologia , Pele/imunologia , Pele/patologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, â¼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1ßhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.
Assuntos
Epiderme/metabolismo , Epiderme/patologia , Inflamação/genética , Inflamação/patologia , Análise de Célula Única , Transcrição Gênica , Anfirregulina/farmacologia , Biomarcadores/metabolismo , Agregação Celular/genética , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Prepúcio do Pênis/citologia , Folículo Piloso/metabolismo , Humanos , Inflamação/imunologia , Queratinócitos/metabolismo , Cinética , Masculino , Psoríase/genética , Psoríase/imunologia , Psoríase/patologia , Proteínas S100/metabolismo , Fatores de Tempo , Transcriptoma/genética , Proteínas Wnt/metabolismoRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.
Assuntos
Desaminases APOBEC/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Citosina Desaminase/genética , Epidermólise Bolhosa Distrófica/enzimologia , Epidermólise Bolhosa Distrófica/genética , Mutação/genética , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Variações do Número de Cópias de DNA/genética , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutagênese/genética , Taxa de Mutação , Transcriptoma/genéticaRESUMO
Sebaceous carcinomas (SeC) are cutaneous malignancies that, in rare cases, metastasize and prove fatal. Here we report whole-exome sequencing on 32 SeC, revealing distinct mutational classes that explain both cancer ontogeny and clinical course. A UV-damage signature predominates in 10/32 samples, while nine show microsatellite instability (MSI) profiles. UV-damage SeC exhibited poorly differentiated, infiltrative histopathology compared to MSI signature SeC (p = 0.003), features previously associated with dissemination. Moreover, UV-damage SeC transcriptomes and anatomic distribution closely resemble those of cutaneous squamous cell carcinomas (SCC), implicating sun-exposed keratinocytes as a cell of origin. Like SCC, this UV-damage subclass harbors a high somatic mutation burden with >50 mutations per Mb, predicting immunotherapeutic response. In contrast, ocular SeC acquires far fewer mutations without a dominant signature, but show frequent truncations in the ZNF750 epidermal differentiation regulator. Our data exemplify how different mutational processes convergently drive histopathologically related but clinically distinct cancers.