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BACKGROUND: Melanoma, the most lethal form of skin cancer, has undergone a transformative treatment shift with the advent of checkpoint blockade immunotherapy (CBI). Understanding the intricate network of immune cells infiltrating the tumor and orchestrating the control of melanoma cells and the response to CBI is currently of utmost importance. There is evidence underscoring the significance of tissue-resident memory (TRM) CD8 T cells and classic dendritic cell type 1 (cDC1) in cancer protection. Transcriptomic studies also support the existence of a TCF7+ (encoding TCF1) T cell as the most important for immunotherapy response, although uncertainty exists about whether there is a TCF1+TRM T cell due to evidence indicating TCF1 downregulation for tissue residency activation. METHODS: We used multiplexed immunofluorescence and spectral flow cytometry to evaluate TRM CD8 T cells and cDC1 in two melanoma patient cohorts: one immunotherapy-naive and the other receiving immunotherapy. The first cohort was divided between patients free of disease or with metastasis 2 years postdiagnosis while the second between CBI responders and non-responders. RESULTS: Our study identifies two CD8+TRM subsets, TCF1+ and TCF1-, correlating with melanoma protection. TCF1+TRM cells show heightened expression of IFN-γ and Ki67 while TCF1- TRM cells exhibit increased expression of cytotoxic molecules. In metastatic patients, TRM subsets undergo a shift in marker expression, with the TCF1- subset displaying increased expression of exhaustion markers. We observed a close spatial correlation between cDC1s and TRMs, with TCF1+TRM/cDC1 pairs enriched in the stroma and TCF1- TRM/cDC1 pairs in tumor areas. Notably, these TCF1- TRMs express cytotoxic molecules and are associated with apoptotic melanoma cells. Both TCF1+ and TCF1- TRM subsets, alongside cDC1, prove relevant to CBI response. CONCLUSIONS: Our study supports the importance of TRM CD8 T cells and cDC1 in melanoma protection while also highlighting the existence of functionally distinctive TCF1+ and TCF1- TRM subsets, both crucial for melanoma control and CBI response.
Assuntos
Linfócitos T CD8-Positivos , Fator 1-alfa Nuclear de Hepatócito , Imunoterapia , Melanoma , Humanos , Melanoma/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunoterapia/métodos , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Feminino , Masculino , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , IdosoRESUMO
Neutrophils infiltrate several types of cancer; however, whether their presence is associated with disease progression remains controversial. Here, we show that colon tumors overexpress neutrophil chemoattractants compared to healthy tissues, leading to their recruitment to the invasive margin and the central part of colon tumors. Of note, tumor-associated neutrophils expressing tumor necrosis factor α, which usually represents an antitumoral phenotype, were predominantly located in the invasive margin. Tumor-associated neutrophils from the invasive margin displayed an antitumoral phenotype with higher ICAM-1 and CD95 expression than neutrophils from healthy adjacent tissues. A higher neutrophil/lymphocyte ratio was found at later stages compared to the early phases of colon cancer. A neutrophil/lymphocyte ratio ≤3.5 predicted tumor samples had significantly more neutrophils at the invasive margin and the central part. Moreover, tumor-associated neutrophils at the invasive margin of early-stage tumors showed higher ICAM-1 and CD95 expression. Coculture of colon cancer cell lines with primary neutrophils induced ICAM-1 and CD95 expression, confirming our in situ findings. Thus, our data demonstrate that tumor-associated neutrophils with an antitumoral phenotype characterized by high ICAM-1 and CD95 expression infiltrate the invasive margin of early-stage colon tumors, suggesting that these cells can combat the disease at its early courses. The presence of tumor-associated neutrophils with antitumoral phenotype could help predict outcomes of patients with colon cancer.
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Neoplasias do Colo , Neutrófilos , Humanos , Neutrófilos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Neoplasias do Colo/patologia , FenótipoRESUMO
Acral melanoma (AM) is the most common melanoma in non-Caucasian populations, yet it remains largely understudied. As AM lacks the UV-radiation mutational signatures that characterize other cutaneous melanomas, it is considered devoid of immunogenicity and is rarely included in clinical trials assessing novel immunotherapeutic regimes aiming to recover the antitumor function of immune cells. We studied a Mexican cohort of melanoma patients from the Mexican Institute of Social Security (IMSS) (n = 38) and found an overrepresentation of AM (73.9%). We developed a multiparametric immunofluorescence technique coupled with a machine learning image analysis to evaluate the presence of conventional type 1 dendritic cells (cDC1) and CD8 T cells in the stroma of melanoma, two of the most relevant immune cell types for antitumor responses. We observed that both cell types infiltrate AM at similar and even higher levels than other cutaneous melanomas. Both melanoma types harbored programmed cell death protein 1 (PD-1+) CD8 T cells and PD-1 ligand (PD-L1+) cDC1s. Despite this, CD8 T cells appeared to preserve their effector function and expanding capacity as they expressed interferon-γ (IFN-γ) and KI-67. The density of cDC1s and CD8 T cells significantly decreased in advanced stage III and IV melanomas, supporting these cells' capacity to control tumor progression. These data also argue that AM could respond to anti-PD-1-PD-L1 immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Células Dendríticas , Linfócitos do Interstício Tumoral , Melanoma , Neoplasias Cutâneas , Pele , Humanos , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Melanoma/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Células Dendríticas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Raios Ultravioleta , Exposição à Radiação , Pele/efeitos da radiação , Melanoma Maligno CutâneoRESUMO
Melanoma is the deadliest form of skin cancer. Due to its high mutation rates, melanoma is a convenient model to study antitumor immune responses. Dendritic cells (DCs) play a key role in activating cytotoxic CD8+ T lymphocytes and directing them to kill tumor cells. Although there is evidence that DCs infiltrate melanomas, information about the profile of these cells, their activity states, and potential antitumor function remains unclear, particularly for conventional DCs type 1 (cDC1). Approaches to profiling tumor-infiltrating DCs are hindered by their diversity and the high number of signals that can affect their state of activation. Multiplexed immunofluorescence (mIF) allows the simultaneous analysis of multiple markers, but image-based analysis is time-consuming and often inconsistent among analysts. In this work, we evaluated several machine learning (ML) algorithms and established a workflow of nine-parameter image analysis that allowed us to study cDC1s in a reproducible and accessible manner. Using this workflow, we compared melanoma samples between disease-free and metastatic patients at diagnosis. We observed that cDC1s are more abundant in the tumor infiltrate of the former. Furthermore, cDC1s in disease-free patients exhibit an expression profile more congruent with an activator function: CD40highPD-L1low CD86+IL-12+. Although disease-free patients were also enriched with CD40-PD-L1+ cDC1s, these cells were also more compatible with an activator phenotype. The opposite was true for metastatic patients at diagnosis who were enriched for cDC1s with a more tolerogenic phenotype (CD40lowPD-L1highCD86-IL-12-IDO+). ML-based workflows like the one developed here can be used to analyze complex phenotypes of other immune cells and can be brought to laboratories with standard expertise and computer capacity.
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Immunotherapy has improved the clinical response in melanoma patients, although a relevant percentage of patients still cannot be salvaged. The search for the immune populations that provide the best tumor control and that can be coaxed by immunotherapy strategies is a hot topic in cancer research nowadays. Tumor-infiltrating TCF-1+ progenitor exhausted CD8+ T cells seem to grant the best melanoma prognosis and also efficiently respond to anti-PD-1 immunotherapy, giving rise to a TIM-3+ terminally exhausted population with heightened effector activity. We tested Porins from Salmonella Typhi as a pathogen associated molecular pattern adjuvant of natural or model antigen in prophylactic and therapeutic immunization approaches against murine melanoma. Porins induced protection against melanomas, even upon re-challenging of tumor-free mice. Porins efficiently expanded IFN-γ-producing CD8+ T cells and induced central and effector memory in lymph nodes and tissue-resident (Trm) T cells in the skin and tumors. Porins induced TCF-1+ PD-1+ CD8+ Trm T cells in the tumor stroma and the presence of this population correlated with melanoma growth protection in mice. Porins immunization also cooperated with anti-PD-1 immunotherapy to hamper melanoma growth. Importantly, the potentially protective Trm populations induced by Porins in the murine model were also observed in melanoma patients in which their presence also correlated with disease control. Our data support the use of cancer vaccination to sculpt the tumor stroma with efficient and lasting Trm T cells with effector activities, highlighting the use of Porins as an adjuvant. Furthermore, our data place CD8+ Trm T cells with a progenitor exhausted phenotype as an important population for melanoma control, either independently or in cooperation with anti-PD-1 immunotherapy.
Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Melanoma/imunologia , Porinas/imunologia , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/farmacologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunização , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Porinas/farmacologia , Salmonella typhiRESUMO
BACKGROUND: Similarities between the pathologic progression of cancer and the physiologic process of placentation have been recognized for many years proposing that both present similar mechanisms and processes. Cervical cancer (CC) is one of the most frequent neoplasia among Mexican women turning it into an important health problem. OBJECTIVE: The aim of this study was to determine the degree of the involvement of pregnancy related genes and in cancer progression by in-silico analysis and validated in CC samples. RESULTS: The data mining analysis resulted in the identification of genes expressed in term placenta, first trimester placenta and normal cervical tissues. Finally, we selected KISS1 for the involvement of pregnancy related gene and also in cancer process. In order to explore KISS1 in CC, we analyzed Copy Number Variation (CNV) and gene expression using microarray experiments. KISS1 showed 20% genomic gain in 1q32.1 on CC samples. Furthermore, microarray analysis showed KISS1 as up-regulated genes. Results were validated showing an overexpression of 85% of KISS1 in CC samples. CONCLUSIONS: Data suggest KISS1 as a great candidate for CC molecular markers or as a therapeutic target for CC. Also, HPV presence does not seem to alter the KISS1 expression in CC.
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Biomarcadores Tumorais/genética , Kisspeptinas/genética , Infecções por Papillomavirus/genética , Neoplasias do Colo do Útero/genética , Adolescente , Adulto , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/genética , Mineração de Dados , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , México , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Transcriptoma/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
Objetivo. Evaluar la presencia de IgG contra C. trachomatis en mujeres entre 18 a 30 años con diagnóstico de artritis de la ciudad de Bogotá D.C. Métodos. Los grupos de estudio están compuestos por un grupo de casos AR (mujeres con diagnóstico de artritis reumatoide y reactiva) y un grupo control. Se obtuvieron muestras de suero y se realizó la determinación cuantitativa de IgG contra C. trachomatis, la determinación PCR (proteína C reactiva) y FR (factor reumatoide). Resultados. Las variables de talla y peso descritas en la población de estudio, muestran un comportamiento homogéneo, por lo cual no interfieren en los resultados obtenidos. Las medias se compararon por los test Mann Whitney y t test no pareado. El porcentaje de resultados positivos en la determinación cualitativa de PCR fueron de: 36,6% para el grupo de casos y 14,5% para el grupo control. En la determinación cualitativa de FR el porcentaje de resultados positivos fueron: 48,8% para el grupo de casos y 5,3% para el grupo control. Finalmente, el porcentaje de resultados positivos en la determinación de anticuerpos IgG específicos contra C trachomatis fue de 2,4% para el grupo de casos y 22,4% para el grupo de controles. Las variables de PCR y FR mostraron mayor frecuencia de resultados positivos en el grupo de casos, en comparación con el grupo control, lo cual se correlaciona con la existencia de procesos inflamatorios propios del desarrollo de artritis. Sin embargo el grupo control presentó mayor frecuencia de anticuerpos IgG específicos contra C. trachomatis en comparación con el grupo de casos. Estos resultados hacen necesario evaluar a futuro, el comportamiento de las pacientes del grupo control con resultados positivos de IgG contra C. trichomatis que permitirían observar una posible aparición de síntomas relacionados con artritis.
Objective. Evaluate the presence of IgG in women infected with C. trachomatis, and diagnosed with arthritis, aged 18-30 years, in Bogota, Colombia. Methods. This project is composed of two study groups: Firstly, female cases diagnosed with rheumatoid reactive-arthritis and secondly, a control group. Quantitative determination of IgG in serum samples from patients with C. trachomatis. Were determined also, PCR determination (C reactive protein) and RF (rheumatoid factor) were performed. The statistical analysis included the comparison of means, using Mann Whitney and unpaired t test. Results. Patient samples that were positive by PCR: 36.6% for the female case group, and 14.5% for the control group. In the qualitative determination of RF positives, results were: 48.8% for the case group and 5.3% for the control group. Finally, the percentages of positive results for IgG antibodies determination against C. trachomatis were: 2.4% for the case group and 22.4% for the control group. PCR and FR variables showed higher frequency of positive results in the female case group compared with the control group, which correlates with the presence of inflammatory processes, where it is typical in the development of arthritis. However, the control group had increased frequency of specific IgG antibodies against C. trachomatis, when it is compared with the case female group. These results need to be evaluated in the future, where, the control group that tested positive for IgG against C. Trachomatis, would allow to observe the possible apparition of symptoms related to arthritis.
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Humanos , Artrite , Yersiniose , Infecções Sexualmente Transmissíveis , Chlamydia trachomatisRESUMO
Thymopoietin (TMPO) is an inner nuclear membrane protein, the coding gene named equally, can give arise to six isoforms by alternative splicing. This gene has been found up regulated in several types of cancer. At present work, we evaluated the TMPO isoforms generated by alternative splicing as well as the protein signal detection in breast cancer samples. TMPO expression was analyzed by immunohistochemistry in tissue microarray containing 46 breast tissue samples including normal (n = 6), benign lesions (n = 18) (fibroadenomas (n = 6), fibrocystic changes (n = 6), ductal hyperplasias (n = 6)) and breast carcinoma (n = 22). Isoforms -α, -ß and -γ of TMPO were evaluated using RT-PCR; clinical-pathological correlation analysis were done by mean of X(2). Neither the normal nor the benign lesions of the breast showed positive TMPO immunodetection, whilst 45 % of the breast carcinomas were immunopositive (p = 0.000), nine of ten positives carcinomas correspond to the Luminal A subtype. Further, alpha isoform was present in all breast samples analyzed; however, beta and gamma isoforms were only present in ten (p = 0.003) and 17 (p = 0.000), respectively, in the breast cancer samples. According with the present data, we suggest that TMPOß and -γ isoforms could provide a potential reliable diagnostic marker for breast cancer.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Proteínas Nucleares/genética , Timopoietinas/genética , Processamento Alternativo/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Isoformas de Proteínas/genéticaRESUMO
Cervical cancer (CC) as other cancer types, presents molecular deregulations, such as the alterations of transcription factors. Krüppel-like factors (KLF) are a family of transcriptional regulators. They are involved in diverse cellular processes, such as proliferation, apoptosis, and angiogenesis among others. Here, we analyzed the expression of all 17 KLF members at messenger RNA (mRNA) level, and protein expression of the two most commonly altered KLF5 and KLF6 in cervical tissues. Fifty-nine cervical tissues ranging from normal tissue to CC were evaluated for KLF1-17 mRNA expression by end-point RT-PCR and KLF5 by qRT-PCR. For KLF5 and KLF6 protein analysis, a tissue microarray was constructed containing the 59 cases and subjected for immunohistochemistry assay and KLF6 IVS1-27G>A single nucleotide polymorphism by direct DNA sequencing. KLF2-16 expressions were present in normal tissue, whereas all 17 were present in Low-Grade Squamous Intraepithelial Lesion, High-Grade-SIL and CC, unrelated to presence of human papillomavirus (HPV). KLF5 mRNA expression gradually increased throughout the subgroups and overexpressed in CC (p=0.01). KLF5 and KLF6 proteins were immunodetected in all samples. For the KLF6 SNP analysis, 80% of the CC population analyzed presented GG genotype and the remaining 20% presented GA genotype (p=0.491). Our present data show that KLFs expression could not be related to HPV presence, at least at transcriptional level, and KLF5 mRNA overexpression could represent a potential molecular marker for CC; KLF6 SNP has no relation to increased risk of CC.
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Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Estudos de Casos e Controles , Colo do Útero/metabolismo , Colo do Útero/patologia , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Polimorfismo de Nucleotídeo Único/genética , Prognóstico , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Adulto JovemRESUMO
Different lines of evidence support an association between Epstein-Barr virus (EBV) and gastric cancer (GC). The main understood risk factor to develop GC is infection by Helicobacter pylori (H. pylori), which triggers a local inflammatory response critical for progression from gastritis to GC. The role of EBV in early inflammatory gastric lesions has been poorly studied. A recent study proposed a cutoff value of 2000 EBV particles to identify patients with increased chances of infection of the gastric epithelium, which may favor the inflammatory process. To better understand the role of EBV in cancer progression, we analyzed 75 samples of GC, 147 control samples of non-tumor gastric tissue derived from GC patients and 75 biopsies from patients with non-atrophic gastritis (NAG). A first-round PCR was used for EBV detection in tumor and non-tumor controls and a more sensitive nested PCR for gastritis samples; both PCRs had lower detection limits above the proposed cutoff value. With this strategy 10.67% of GC, 1.3% of non-tumor controls and 8% of gastritis samples were found positive. An EBER1 in situ hybridization showed EBV infection of epithelial cells in GC and in a third of NAG samples, while in the other NAGs infection was restricted to the mononuclear cell infiltrate. EBV-positive GCs were enriched in lace and cribriform patterns, while these rare patterns were not observed in EBV negative samples. Our results support a role for EBV in GC and early precursor lesions, either as directly oncogenic infecting epithelial cells or indirectly as an inflammatory trigger.