Comprehensive Analysis of CLCA1 Expression, Immune Infiltration and Immune Checkpoints in Colon Adenocarcinoma / Análisis Completo de la Expresión de CLCA1, Infiltración Inmune y Puntos de Control Inmunológico en Colonadenocarcinoma

SUMMARY: Colon adenocarcinoma (COAD) is a prevalent disease worldwide, known for its high mortality and morbidity rates. Despite this, the extent of investigation concerning the correlation between COAD's CLCA1 expression and immune cell infiltration remains insufficient. This study seeks to examine the expression and prognosis of CLCA1 in COAD, along with its relationship to the tumor immune microenvironment. These findings will offer valuable insights for clinical practitioners and contribute to the existing knowledge in the field. In order to evaluate the prognostic significance of CLCA1 in individuals diagnosed with colorectal cancers, we conducted a comprehensive analysis using univariate and multivariate Cox regression models along with receiver operating characteristic curve (ROC) analysis. This study was performed on the patient data of COAD obtained from The Cancer Genome Atlas (TCGA) database. Nomograms were developed to anticipate CLCA1 prognostic influence. Furthermore, the CLCA1 association with tumor immune infiltration, immune checkpoints, immune checkpoint blockade (ICB) response, interaction network, and functional analysis of CLCA1-related genes was analyzed. We found that Colon adenocarcinoma tissues significantly had decreased CLCA1 expression compared to healthy tissues. Furthermore, the study revealed that the group with high expression of CLCA1 demonstrated a significantly higher overall survival rate (OS) as compared to the group with low expression. Multivariate and Univariate Cox regression analysis revealed the potential of CLCA1 as a standalone risk factor for COAD. These results were confirmed using nomograms and ROC curves. In addition, protein-protein interaction (PPI) network analysis and functional gene enrichment showed that CLCA1 may be associated with functional activities such as pancreatic secretion, estrogen signaling and cAMP signaling, as well as with specific immune cell infiltration. Therefor, as a new independent predictor and potential biomarker of COAD, CLCA1 plays a crucial role in the advancement of colon cancer.

El adenocarcinoma de colon (COAD) es una enfermedad prevalente a nivel mundial, conocida por sus altas tasas de mortalidad y morbilidad. Sin embargo, el alcance de la investigación sobre la correlación entre la expresión de CLCA1 de COAD y la infiltración de células inmunes sigue siendo insuficiente. Este estudio busca examinar la expresión y el pronóstico de CLCA1 en COAD, junto con su relación con el microambiente inmunológico del tumor. Estos hallazgos ofrecerán conocimientos valiosos para los profesionales clínicos y contribuirán al conocimiento existente en el campo. Para evaluar la importancia de pronóstico de CLCA1 en personas diagnosticadas con cáncer colorrectal, realizamos un análisis exhaustivo utilizando modelos de regresión de Cox univariados y multivariados junto con un análisis de la curva característica operativa del receptor (ROC). Este estudio se realizó con los datos de pacientes de COAD obtenidos de la base de datos The Cancer Genome Atlas (TCGA). Se desarrollaron nomogramas para anticipar la influencia pronóstica de CLCA1. Además, se analizó la asociación de CLCA1 con la infiltración inmunitaria tumoral, los puntos de control inmunitarios, la respuesta de bloqueo de los puntos de control inmunitarios (ICB), la red de interacción y el análisis funcional de genes relacionados con CLCA1. Descubrimos que los tejidos de adenocarcinoma de colon tenían una expresión significativamente menor de CLCA1 en comparación con los tejidos sanos. Además, el estudio reveló que el grupo con alta expresión de CLCA1 demostró una tasa de supervivencia general (SG) significativamente mayor en comparación con el grupo con baja expresión. El análisis de regresión de Cox multivariado y univariado reveló el potencial de CLCA1 como factor de riesgo independiente de COAD. Estos resultados se confirmaron mediante nomogramas y curvas ROC. Además, el análisis de la red de interacción proteína- proteína (PPI) y el enriquecimiento de genes funcionales mostraron que CLCA1 puede estar asociado con actividades funcionales como la secreción pancreática, la señalización de estrógenos y la señalización de AMPc, así como con la infiltración de células inmunes específicas. Por lo tanto, como nuevo predictor independiente y biomarcador potencial de COAD, CLCA1 desempeña un papel crucial en el avance del cáncer de colon.

Inmunoexpresión p53, sangre oculta en heces de pacientes colecistectomizados y colelitiasis con adenomas colónicos / P53 Immunoexpression, Faecal Occult Blood from Cholecystectomized Patients and Cholelithiasis with Colonic Adenomas

Introducción: la secuencia adenoma- adenocarcinoma, es resultado de fallos genéticos en las células intestinales heredados o adquiridos. Objetivo: determinar la posible relación entre la inmunoexpresión de la p53 y la positividad de la sangre oculta en heces en los adenomas de colon con alto grado de displasia diagnosticados en pacientes colecistectomizados o con colelitiasis. Métodos: se realizó un estudio descriptivo, de corte transversal, en el Instituto de Gastroenterología, en el período de mayo de 2013 hasta junio de 2015. Se realizaron pruebas estadísticas descriptiva y de chi Cuadrado y probabilidad exacta de Fisher. Resultados: la proporción de adenomas con alto grado de displasia fue similar en pacientes colecistectomizados y con colelitiasis (50 por ciento) respectivamente. Una alta proporción se diagnosticó en colecistectomizados femeninas (35 por ciento), con 60 y más años de edad (53 por ciento) y 11 y más años de colecistectomizados (60 por ciento), mientras que en las colelitiasis fueron masculinos (30 por ciento). Conclusiones: una alta proporción de adenomas con alto grado de displasia presentan inmunoexpresión de la p53 y sangre en heces positiva en pacientes colecistectomizados y con colelitiasis, que se reporta por vez primera(AU)

Introduction: The adenoma - adenocarcinoma sequence is a result of inherited or acquired genetic failures in the intestinal cells. Objective: To determine the immunohistochemical expression of p53 and the positivity of the fecal occult blood test in colon adenomas with high degree of diagnosed dysplasia in cholecystectomized patients or with cholelithiasis. Methods: Descriptive, cross-sectional study conducted in the Institute of Gastroenterology in the period of May, 2013 to June, 2015. Statistical tests were statistics testing, exact Chi Square and Fisher's probability tests. Results: The proportion of adenomas with high degree of dysplasia was similar in cholecystectomized patientsand with cholelithiasis (50 percent) respectively. A high proportion diagnosed in colecistectomizados women (35 percent), 60 and more years of age (53 percent) and 11 and more years of performed cholecystectomy (60 percent), whereas cholelithiasis prevailed in males (30 percent). Conclusions: High proportion of adenomas with high degree of dysplasia present p 53 immunoexpression and positive fecal occult blood test in cholecystectomized patients and patients with cholelithiasis that is reported for the first time(AU)

Phospholipid Remodeling and Eicosanoid Signaling in Colon Cancer Cells.

Phospholipid remodeling and eicosanoid synthesis are central to lipid-based inflammatory reactions. Studies have revealed that membrane phospholipid remodeling by fatty acids through deacylation/reacylation reactions increases the risk of colorectal cancers (CRC) by allowing the cells to produce excess inflammatory eicosanoids, such as prostaglandins, thromboxanes and leukotrienes. Over the years, efforts have been made to understand the lipid remodeling pathways and to design anti-cancer drugs targeting the enzymes of eicosanoid biosynthesis. Here, we discuss the recent progress in phospholipid remodeling and eicosanoid biosynthesis in CRC.

Enhancement of antitumor effect using dendritic cells activated with natural killer cells in the presence of Toll-like receptor agonist

Dendritic cells (DCs) play a role in natural killer (NK) cell activation, while NK cells are also able to activate and mature DCs. Toll-like receptors (TLRs) on the surface of DCs and NK cells induce the maturation and activation of these cells when engaged with their cognate ligand. We investigated to generate potent DCs by maturation with NK cells in the presence of TLR agonist in vitro and tested the efficacy of these DC vaccinations in mouse colon cancer model. The optimal ratios of DCs versus NK cells were 1:1 to 1:2. Immature DCs were mature with NK cells in the presence of lipopolysaccharide, which is TLR4 agonist, and further addition of IL-2 induced phenotypically and functionally mature bone marrow-derived DCs. These potent DCs exhibited not only high expression of several costimulatory molecules and high production of IL-12p40 and IL-12p70, but also high allogeneic T cells stimulatory capacity, and the induction of the high activities to generate tumor-specific CTLs. Consistently, vaccination with these DCs efficiently inhibited CT-26 tumor growth in mouse colon cancer model when compared to other vaccination strategies. Interestingly, combination therapy of these DC-based vaccines and with low-dose cyclophosphamide showed dramatic inhibition effects of tumor growth. These results suggest that the DCs maturated with NK cells in the presence of TLR agonist are potent inducer of antitumor immune responses in mouse model and may provide a new source of DC-based vaccines for the development of immunotherapy against colon cancer.

Imunogenicidade do vírus vacina recombinante modificado com o gene do antígeno cárcino-embrionário (CEA) humano em modelo experimental de adenocarcinoma de cólon / Immunogenicity of recombinant vaccinia virus modified with human carcinoembryonic antigen gene in an experimental model of colon adenocarcinoma

Introdução: Os adenocarcinomas do trato gastrointestinal desenvolvem inicialmente a partir do epitélio superficial, e em mais de 90% dos casos, expressam o CEA. Existe compartimentalização e alguma independência entre o sistema imunológico mucoso e sistêmico com tráfego assimétrico de células entre eles. Um melhor conhecimento da imunologia dos tumores tem gerado o desenvolvimento de várias abordagens de vacinas direcionadas contra antígenos tumorais específicos. O vírus vaccinia esta entre os mais potentes para a indução de resposta protetora em animais contra infecções virais e câncer. Este tem sido mostrado como imunogênico em humanos. Imunização com o vírus vaccinia recombinante com o antígeno carcinoembrionário (CEA) pode gerar proteção humoral e celular contra tumores CEA +, dependo da dose e via de imunização... Objetivos: Comparar a imunização intra-retal com a venosa, e a resposta ao nível sistêmico e local apos uma dose e dois reforços por estas duas vias. Desenvolvimento de um animal híbrido que desenvolva tumores espontâneos do trato gastrointestinal com evolução previsível:... Material e Métodos: Os animais foram imunizados intra-retal ou intravenoso com 10 milhões de partículas do vírus Vaccinia recombinante com o CEA a cada 15 dias totalizando 3 doses. O grupo controle recebeu a mesma imunização com o mesmo vetor porem, não transduzido com o CEA... Os tumores foram medidos a cada 2 dias. Os animais Apc foram gerados por manipulação genética através da desativação do gene da Polipose colônica adenomatosa (APC) com mutação induzida na posição do aminoácido 1648, gerando proteína instável e não funcional... O camundongo Apc/CEA é um relevante modelo animal para o estudo do desenvolvimento espontâneo do câncer de colón da fase de displasia ao adenocarcinoma invasivo...(AU)

Introduction: The adenocarcinomas of the gastrointestinal tract (TGI) develop from superficial epithelium and in more than 90% of cases express CEA. There is compartmentalization with some independence among mucosal versus systemic immune systems and the asymmetric trafficking of cells between them. An improved understanding of tumor immunology has led to the development of several vaccine approaches targeted against specific human tumors antigens. Recombinant vaccinia virus vaccines are among the most potent for inducing protective immunity in animais against many viral infections and cancer. They have been shown to be immunogenic in human. Immunization with recombinant carcinoembryonic antigen (CEA) vaccinia virus can generate humoral and cellular protection against tumors CEA+, depending on the dose and route of administration. One of the major obstacles in vaccine development has been the lack of a relevant animal model for appropriately evaluating vaccine agents and immunization protocols. These results could be more accurate using an animal model that mimics the human situation with development of spontaneous tumors from the gastrointestinal tract with high expression of a specific antigen. Objectives: Comparing intra-rectal and venous immunization, and to evaluate systemic and local immune response after one dose and 2 booster after these routes. Development of a hybrid mouse model that generates spontaneous TGI' s tumors with expected evolution: hyperplasia ~ dysplasia ~ adenoma ~ superficial adenocarcinoma ~ invasive adenocarcinoma. The animal model should have high CEA expression in tumors and low at normal tissues as observed in humans. Material e Methods: Animais had intra-rectal (IR) or endovenous immunization with 10 million units of recombinant vaccinia-CEA virus each 15 days in a total of 3 doses. Control had the same immunization routes with vaccinia virus without CEA expression. Half of the animais were sacrificed 7 days after the last immunization to evaluate the induction of an immune response against CEA at the spleen (systemic response) and at the Payer's patches (mucosa! response). Subcutaneous colon tumors, with or without the expression of CEA, were implanted in the other animais. Tumors were measured every 2 days. Animais Apc were generated by genetic manipulation with partia! knock out of the APC (Adenomatous Polyposis coli) gene at the amino acid position 1648. The Apc pro te in was unstable and not functional. The CEA mo use recei ved the complete human CEA DNA. Those 2 transgenic and knock out mice models were crossed generating a hybrid transgenic animal model with spontaneous development of TGI' s tumors with high expression of CEA. Results: The animais immunized with rVac-CEA presented humoral and cellular specific CEA response and some tumor protection for CEA + tumors.The contrai group immunized without CEA had no immune response or protection when challenged with CEA tumor implants. Intra-rectal immunization showed to be similar to intravenous immunization, with systemic and mucosa! responses but low-grade tumor protection. Some immunized animais were tumor free and ali were able to generate cytolytic activity against CEA+ cells. Among those that developed tumor, these were smaller and had decreased growing rate. Immunization did not show protection when animais received CEAtumors indicating specificity. The double transgenic animais presented at 2 months hyperplasic polyps and occult blood in the feces. At 4 months old they showed gastrointestinal polyps that progressed to invasive adenocarcinomas with a similar pattem of dysplasia and CEA expression as observed in human colorectal cancer. Tumoral tissues had high expression of human CEA and tissues like colon, intestine and stomach also expressed CEA but at low levei. These animais exhibited incomplete or partial tolerance to CEA. Conclusions: Intra-rectal immunization with rVac-CEA showed an immune response bigger than 40%. CTL against CEA after intra-rectal and systemic immunization was present at the spleen and Peyer' s patch. Both route of immunizations induce CEA + tumors protection but the EV route was more effective for subcutaneous tumor. The Apc/CEA mouse is an important animal model for the studying of spontaneous cancer colon development from the dysplasia to adenocarcinoma. The human CEA is over expressed in tumors of these animais. This model represents a system to evaluate different treatment options and induction of tolerance and tumor immunity (AU)

Imunogenicidade do vírus vaccinia recombinante modificado com o gene do Antígeno Cárcino-Embrionário (CEA) humano em modelo experimental de adenocarcinoma de cólon / Immunogenicity of recombinant modified vaccinia virus with the gene-Carcino Embryonic Antigen (CEA) in an experimental model of human colon adenocarcinoma

Os adenocarcinomas do trato gastrointestinal desenvolvem inicialmente a partir do epitélio superficial, e em mais de 90% dos casos expressam o CEA. Existe compartimentalização e alguma independência entre o sistema imunológico mucoso e sistêmico com tráfego assimétrico de células entre eles. Um melhor conhecimento da imunologia dos tumores tem gerado o desenvolvimento de várias abordagens de vacinas direcionadas contra antígenos tumorais específicos. O vírus vaccinia está entre os mais potentes para a indução de resposta protetora em animais contra infecções virais e câncer. Os animais foram imunizados intra-retal ou intravenoso com 10 milhões de partículas do vírus Vaccinia recombinante com o CEA a cada 15 dias totalizando 3 doses. O grupo controle recebeu a mesma imunização com o mesmo vetor porém, não transduzido com o CEA. Metade dos animais em cada grupo foi sacrificada 7 dias após a última imunização para se avaliar a indução de resposta imunológica contra o CEA no baço (sistêmica) e nas placas de Peyer intestinais (mucosa). A imunização intra-retal com o rVac-CEA se mostrou factível com índice de resposta maior que 50%. A citotoxicidade contra o CEA após imunização intra-retal bem como mucosa após imunização sistêmica foi medida no baço e placas de Peyer. Ambas as vias de imunização induziram proteção contra tumores CEA+mas a via EV foi mais eficiente para tumores implantados no subcutâneo. O camundongo Apc/CEA é um relevante modelo animal para o estudo do desenvolvimento espontâneo do câncer de cólon da fase de displasia ao adenocarcinoma invasivo. O CEA humano é superexpressado nos tumores. Este modelo representa um sistema para se avaliar diversas opções de tratamentos contra o câncer e para estudar a tolerância induzida e imunidade tumoral.

Funcion Citotoxica Natural y Subpoblaciones Linfocitarias en Pacientes con Cancer de Colon / Natural cytotoxic function and lymphocitic subpopulations in patients with colon cancer

Se analizo la accion de las celulas citotoxicas en un grupo de pacientes con neoplasias de colon frente a las lineas celulares Sw-948 y K-562. Se obtuvieron valores de dos tiempos de incubaciones distintos ( 4 y 18 horas) y se estudiaron ademas los marcadores fenotipicos de membrana CD3, CD4, CD6, CD8 y HLA-DR, en celulas linfoides de sangre periferica. Los resultados se compararon con los de un grupo control; solo el grupo de pacientes mostro un incremento significativo de la actividad citotoxica al pasar de 4 a 18 horas de incubacion para ambas lineas celulares, a su vez este grupo expreso niveles bajos de linfocitos CD3 + CD6. Se encontro una correlacion positiva entre estos dos marcadores y la actividad citotoxica

Reduction of interleukin 1 and interleukin 2 production by patients with advanced cancer

The concentrations of interleukin 1 (IL-1) and interleukin 2 (IL-2) produced in the supernatants of peripheral blood mononuclear cell cultures from patients with advanced cancer were measured to identify some of the causes of the immunological impairment characteristic of malignant disease. Mononuclear cells obtained from 19 cancer patients were stimulated to produce IL-1 and IL-2 and compared with those of healthy controls. A severe reduction of both IL-1 and IL-2 activity was observed. There was no correlation between the lower number of OKT4+ cells observed in these patients and the levels of IL-2 production. The removal of monocytes did not bring IL-2 levels to normal. Impaired IL-2 production could not be restored to normal by addition of IL-1. These results suggest that exogenous IL-01 and IL-2 may be useful in cancer immunotherapy

Respuesta inmune en conejos a extractos de tejidos tumoral de colón humano / Immune response in rabbits to tumoral tissue extracts of human colon

Se aisló y purificó antígeno carcinoembriónico (CEA) a partir de tumores de colon humano; se obtuvieron antisueros al extracto perclórico de glicoproteínas de colon normal y de colon tumoral, y al CEA puro en conejos. Se comprobó por electroforesis en gel de poliacrilamida que, al inmunizar en forma prolongada con el extracto tumoral, hubo aumento de proteínas de movilidad alfa2. Los antisueros obtenidos rinden por imnunodifusión bajos títulos al enfrentarlos a los distintos antígenos examinados. En general se obtienen los mayores títulos con la fracción obtenida por focalización isoeléctrica del extracto perclórico de glico proteínas de colon tumoral, y este fenómeno se repite al titular los antisueros por radioinmunoensayo (RIE). Al efectuar el análisis estadístico de los resultados obtenidos al medir la concentración de CEA en muestras de sueros de pacientes, no se obtuvieron diferencias significativas al usar sueros provenientes de distintas sangrías. Se concluye que para cubrir un amplio rango de concentraciones en la medición de CEA por RIE es suficiente purificar al antígeno que se usará, como "standard" y trazador hasta la etapa de focalización, y utilizar de anti-sueros obtenidos de animales inyectados con extracto perclórico de glicoproteínas de colon tumoral con esquemas de inmunización largos o con CEA puro con un esquema corto

Estudios in vitro sobre reconocimiento autólogo y alogénico en pacientes con adenocarcinoma gástrico y colorectal / In vitro studies of autologus and allogeneic recognition in patients with gastric and colorectal adenocarcinoma

La reactividad de linfocitos de sangre periférica en cultivo mixto autólogo (AMLC) y en cultivo mixto alogénico (MLC), fue explorada en 10 pacientes con adenocarcinoma gástrico y 10 pacientes con adenocarcinoma colorectal, comparada con 30 controles sanos. Ambos ensayos, fueron realizados en presencia de suero humano normal (SHN) y suero de cada paciente (suero autólogo = SA). El 95% de las células T aisladas por rosetas con glóbulos rojos de carnero se marcaron con el anticuerpo monoclonal OKT3. Nuestros resultados en AMLC tanto en pacientes como en controles, demostraron tres tipos de respondedores: bajos (alfa cpm < 1.000); intermedios (alfa cpm 1.000 - 10.000) y altos (alfa cpm > 10.000), con diferencias no significativas entre los grupos cultivados tanto en presencia de SHN como de SA. En contraste, en MLC, el 30% (6/20) de los pacientes demostraron hiporespuesta comparados con los (Indice Relativo de Proliferación < 0.66). El SA indujo inhibición significativa de la respuesta en MLC: 11 de 20 pacientes (55%) demostraron un IRP < 0.66 en SA. La acción inhibitoria del SA fue también observada en el MLC praticado con células de los controles. El 25% de los pacientes (5/20) presentaron niveles elevados de complejos inmunológicos circulantes, detectados por la microtécnica de C1q fase sólida. Nuestros hallazgos sugieren cue los pacientes portadores de adenocarcinoma gástrico o colorectal mientras expresan alteración del reconocimiento aloantigénico influenciado por SA, preservan su habilidad de reconocer autoantígenos no alterada por SA