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Commonly used intestinal in vitro models are limited in their potential to predict oral drug absorption. They either lack the capability to form a tight cellular monolayer mimicking the intestinal epithelial barrier or the expression of cytochrome P450 3A4 (CYP3A4). The aim of this study was to establish a platform of colorectal cancer patient-derived cell lines for evaluation of human intestinal drug absorption and metabolism. We characterized ten 2D cell lines out of our collection with confluent outgrowth and long-lasting barrier forming potential as well as suitability for high throughput applications with special emphasis on expression and inducibility of CYP3A4. By assessment of the transepithelial electrical resistance (TEER) the cells barrier function capacity can be quantified. Very high TEER levels were detected for HROC60. A high basal CYP3A4 expression and function was found for HROC32. Eight cell lines showed higher CYP3A4 induction by stimulation via the vitamin D receptor compared to Caco-2 cells (5.1- to 16.8-fold change). Stimulation of the pregnane X receptor led to higher CYP3A4 induction in two cell lines. In sum, we identified the two cell lines HROC183 T0 M2 and HROC217 T1 M2 as useful tools for in vitro drug absorption studies. Due to their high TEER values and inducibility by drug receptor ligands, they may be superior to Caco-2 cells to analyze oral drug absorption and intestinal drug-drug interactions. Significance statement: Selecting appropriate candidates is important in preclinical drug development. Therefore, cell models to predict absorption from the human intestine are of the utmost importance. This study revealed that the human cell lines HROC183 T0 M2 and HROC217 T1 M2 may be better suited models and possess higher predictive power of pregnane X receptor- and vitamin D-mediated drug metabolism than Caco-2 cells. Consequently, they represent useful tools for predicting intestinal absorption and simultaneously enable assessment of membrane permeability and first-pass metabolism.
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Citocromo P-450 CYP3A , Intestinos , Células CACO-2 , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Humanos , Absorción Intestinal , Receptor X de Pregnano/metabolismoRESUMEN
BACKGROUND: Recent evidence proves that intravenous human immunoglobulin G (IgG) can impair cancer cell viability. However, no study evaluated whether IgG application benefits cancer patients receiving chemotherapeutics. METHODS: Influence of pharmaceutical-grade human IgG on the viability of a series of patient-derived colon cancer cell lines with and without chemotherapeutic intervention was determined. Cell death was analysed flow cytometrically. In addition, the influence of oxaliplatin and IgG on the ERK1/2-signalling pathway was evaluated by western blots. RESULTS: We evaluated the effects of pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, in combination with chemotherapeutics. We did not observe any significant effects of IgG on tumour cell viability directly; however, human IgG significantly impaired the anti-tumoral effects of oxaliplatin. Primary cancer cell lines express IgG receptors and accumulate human IgG intracellularly. Moreover, while oxaliplatin induced the activation of ERK1/2, the pharmaceutical IgG inhibited ERK1/2 activity. CONCLUSIONS: The present study demonstrates that pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, can impair the anti-carcinoma activity of oxaliplatin. These data strongly suggest that therapeutic IgG as co-medication might have harmful side effects in cancer patients. The clinical significance of these preclinical observations absolutely advises further preclinical, as well as epidemiological and clinical research.
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Neoplasias del Colon/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Inmunoglobulinas Intravenosas/administración & dosificación , Oxaliplatino/farmacología , Anciano , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Interacciones Farmacológicas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulinas Intravenosas/farmacología , Masculino , Persona de Mediana Edad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults, highlighting the need for novel treatment strategies. Patient derived xenografts (PDX) represent a valuable tool to accomplish this task. METHODS: PDX were established by implanting GBM tissue subcutaneously. Engraftment success was compared between NMRI Foxn1nu and NOD/SCID as well as between fresh and cryopreserved tissue. Established PDX were analyzed histologically and molecularly. Five PDX were experimentally treated with different drugs to assess their potential for preclinical drug testing. RESULTS: Establishment of PDX was attempted for 36 consecutive GBM cases with an overall success rate of 22.2% in NMRI Foxn1nu mice. No difference was observed between fresh or cryopreserved (20-1057 days) tissue in direct comparison (n = 10 cases). Additionally, engraftment was better in NOD/SCID mice (38.8%) directly compared to NMRI Foxn1nu mice (27.7%) (n = 18 cases). Molecular data and histology of the PDX compare well to the primary GBM. The experimental treatment revealed individual differences in the sensitivity towards several clinically relevant drugs. CONCLUSIONS: The use of vitally frozen GBM tissue allows a more convenient workflow without efficiency loss. NOD/SCID mice appear to be better suited for initial engraftment of tumor tissue compared to NMRI Foxn1nu mice.
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Glioblastoma/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto , Anciano , Animales , Femenino , Glioblastoma/genética , Humanos , Huésped Inmunocomprometido , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Mutación/genética , Coloración y Etiquetado , Resultado del TratamientoRESUMEN
Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response.T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i.We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naïve peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.
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PURPOSE: Research projects and clinical trials strongly rely on high-quality biospecimens which are provided by biobanks. Since differences in sample processing and storage can strongly affect the outcome of such studies, standardization between biobanks is necessary to guarantee reliable results of large, multicenter studies. The German Cancer Aid Foundation (Deutsche Krebshilfe e.V.) has therefore initiated the priority program "tumor tissue banks" in 2010 by funding four biobank networks focusing on central nervous system tumors, melanomas, breast carcinomas, and colorectal carcinomas. The latter one, the North German Tumor Bank of Colorectal Cancer (ColoNet) is managed by surgeons, pathologists, gastroenterologists, oncologists, scientists, and medical computer scientists. METHODS AND RESULTS: The ColoNet consortium has developed and harmonized standard operating procedures concerning all biobanking aspects. Crucial steps for quality assurance have been implemented and resulted in certification according to DIN EN ISO 9001. A further achievement is the construction of a web-based database for exploring available samples. In addition, common scientific projects have been initiated. Thus, ColoNet's repository will be used for research projects in order to improve early diagnosis, therapy, follow-up, and prognosis of colorectal cancer patients. Apart from the routine sample storage at -170 °C, the tumor banks' unique characteristic is the participation of outpatient clinics and private practices to further expand the sample and clinical data collection. CONCLUSION: The first 2 years of funding by the German Cancer Aid Foundation have already led to a closer scientific connection between the participating institutions and to a substantial collection of biospecimens obtained under highly standardized conditions.
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Neoplasias Colorrectales/patología , Bancos de Tejidos/organización & administración , Investigación Biomédica , Neoplasias Colorrectales/epidemiología , Alemania/epidemiología , HumanosRESUMEN
The most common in vitro model for absorption, distribution, metabolism, and excretion (ADME) purposes is currently the Caco-2 cell line. However, clear differences in gene and protein expression towards the small intestine and an, at best, fair prediction accuracy of intestinal drug absorption restrict the usefulness of a model for intestinal epithelial cells. To overcome these limitations, we evaluated a panel of low-passaged patient-derived colorectal cancer cell lines of the HROC collection concerning similarities to small intestinal epithelial cells and their potential to predict intestinal drug absorption. After initial screening of a larger panel, ten cell lines with confluent outgrowth and long-lasting barrier-forming potential were further characterized in close detail. Tight junctional complexes and microvilli structures were detected in all lines, anda higher degree of differentiation was observed in 5/10 cell lines. All lines expressed multiple transporter molecules, with the expression levels in three lines being close to those of small intestinal epithelial cells. Compared with the Caco-2 model, three HROC lines demonstrated both higher similarity to jejunal epithelial tissue cells and higher regulatory potential of relevant drug transporters. In summary, these lines would be better-suited human small intestinal epithelium models for basic and translational research, especially for ADME studies.
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Células Epiteliales , Intestino Delgado , Humanos , Células CACO-2 , Transporte Biológico , Células Epiteliales/metabolismo , Diferenciación Celular , Intestino Delgado/metabolismo , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Clinical utilization of curcumin in colorectal cancer (CRC) was revived as a result of the development of novel curcumin formulations with improved bioavailability. Additionally, identification of biomarkers for curcumin sensitivity would also promote successful clinical applications. Here, we wanted to identify such biomarkers in order to establish a predictive model for curcumin sensitivity. Thirty-two low-passage CRC cell lines with specified tumor characteristics were included. Curcumin suppressed cell proliferation, yet sensitivity levels were distinct. Most curcumin-sensitive CRC cell lines were microsatellite stable and expressed high levels of IκBα. The predictive capacity of this biomarker combination possessed a statistical significance of 72% probability to distinguish correctly between curcumin-sensitive and -resistant CRC cell lines. Detailed functional analyses were performed with three sensitive and three resistant CRC cell lines. As curcumin's mode of action, inhibition of NF-κB p65 activation via IκBα was identified. In consequence, we hypothesize that novel curcumin formulations-either alone or, more likely, in combination with standard therapeutics-can be expected to prove clinically beneficial for CRC patients with high IκBα expression levels.
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Tafazzin-an acyltransferase-is involved in cardiolipin (CL) remodeling. CL is associated with mitochondrial function, structure and more recently with cell proliferation. Various tafazzin isoforms exist in humans. The role of these isoforms in cardiolipin remodeling is unknown. Aim of this study was to investigate if specific isoforms like Δ5 can restore the wild type phenotype with respect to CL composition, cellular proliferation and gene expression profile. In addition, we aimed to determine the molecular mechanism by which tafazzin can modulate gene expression by applying promoter analysis and (Ingenuity Pathway Analyis) IPA to genes regulated by TAZ-deficiency. Expression of Δ5 and rat full length TAZ in C6-TAZ- cells could fully restore CL composition and-as proven for Δ5-this is naturally associated with restoration of mitochondrial respiration. A similar restoration of CL-composition could not be observed after re-expression of an enzymatically dead full-length rat TAZ (H69L; TAZMut). Re-expression of only rat full length TAZ could restore proliferation rate. Surprisingly, the Δ5 variant failed to restore wild-type proliferation. Further, as expected, re-expression of the TAZMut variant completely failed to reverse the gene expression changes, whereas re-expression of the TAZ-FL variant largely did so and the Δ5 variant to somewhat less extent. Very likely TAZ-deficiency provokes substantial long-lasting changes in cellular lipid metabolism which contribute to changes in proliferation and gene expression, and are not or only very slowly reversible.
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[This corrects the article DOI: 10.3389/fgene.2022.931017.].
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In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.
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Bancos de Muestras Biológicas , Neoplasias Colorrectales , Xenoinjertos , Neoplasias Pancreáticas , Animales , Línea Celular Tumoral , Criopreservación , Humanos , Ratones , Medicina de PrecisiónRESUMEN
Based on our research group's large biobank of colorectal cancers (CRC), we here describe the ongoing activity of establishing a high quality assured PDX biobank for more than 100 individual CRC cases. This includes sufficient numbers of vitally frozen (n > 30 aliquots) and snap frozen (n > 5) backups, "ready to use". Additionally, PDX tumor pieces were paraffin embedded. At the current time, we have completed 125 cases. This resource allows histopathological examinations, molecular characterizations, and gene expression analysis. Due to its size, different issues of interest can be addressed. Most importantly, the application of low-passage, cryopreserved, and well-characterized PDX for in vivo studies guarantees the reliability of results due to the largely preserved tumor microenvironment. All cases described were molecularly subtyped and genetic identity, in comparison to the original tumor tissue, was confirmed by fingerprint analysis. The latter excludes ambiguity errors between the PDX and the original patient tumor. A cancer hot spot mutation analysis was performed for n = 113 of the 125 cases entities. All relevant CRC molecular subtypes identified so far are represented in the Hansestadt Rostock CRC (HROC)-Xenobank. Notably, all models are available for cooperative research approaches.
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BACKGROUND: Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity. METHODS: Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days). RESULTS: Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation. CONCLUSIONS: Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.
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Adenocarcinoma/patología , Carcinoma de Células Grandes/patología , Carcinoma Neuroendocrino/patología , Neoplasias Colorrectales/patología , Criopreservación , Trasplante Heterólogo , Adenocarcinoma/genética , Animales , Carcinoma de Células Grandes/genética , Carcinoma Neuroendocrino/genética , Neoplasias Colorrectales/genética , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCIDRESUMEN
Despite the importance of tumor infiltrating B cells (TiBc) in immunological circuits, their functional role is scarcely investigated. Here, we analyzed immunoglobulin (Ig) secretion of the subtypes IgA, IgG, and IgM of TiBc from freshly resected primary and secondary colorectal carcinomas (CRC) by FluoroSpot (n = 30 CRC) directly ex vivo. High, intermediate, and low secretion was observed in 33%, 37%, and 30% of the tumors for IgA; in 10%, 27%, and 63% for IgG; and in 21%, 36%, and 50% for IgM, respectively. These ex vivo data validate our previous findings: Most TiBc present in the CRC microenvironment are functional since they produce and actively secrete Ig (IgA > IgG > IgM). Of note, the presence of major histocompatibility complex (MHC) class II expressing cells in the tumor micromilieu only correlated with IgG secretion (p = 0.0004). Supporting recent findings in several other tumor entities, TiBc in CRC thus likely can contribute to tumor control in a dual role of sole antigen-presentation and additionally anti-tumoral Ig-production.
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Over the time period from 2006 to 2017, consecutive patients operated on at the University Medical Center Rostock participated in the comprehensive biobanking and tumor-modelling approach known as the HROC collection. Samples were collected using strict standard operating procedures including blood (serum and lymphocytes), tumor tissue (vital and snap frozen), and adjacent normal epithelium. Patient and tumor data including classification, molecular type, clinical outcome, and results of the model establishment are the essential pillars. Overall, 149 patient-derived xenografts with 34 primary and 35 secondary cell lines were successfully established and encompass all colorectal carcinoma anatomic sites, grading and staging types, and molecular classes. The HROC collection represents one of the largest model assortments from consecutive clinical colorectal carcinoma (CRC) cases worldwide. Statistical analysis identified a variety of clinicopathological and molecular factors associated with model success in univariate analysis. Several of them not identified before include localization, mutational status of K-Ras and B-Raf, MSI-status, and grading and staging parameters. In a multivariate analysis model, success solely correlated positively with the nodal status N1 and mutations in the genes K-Ras and B-Raf. These results imply that generating CRC tumor models on the individual patient level is worth considering especially for advanced tumor cases with a dismal prognosis.
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Human tumor in vivo cancer models raised in immunodeficient mice, the so-called patient-derived xenografts, are increasingly in use in preclinical development and evaluation of novel drug candidates including HDAC inhibitors. Here, we describe the techniques needed to generate novel patient-derived xenografts. The focus lies on vitally frozen tumor biopsies as starting material. First, the preparative steps on the animals, followed by the engraftment procedure itself, the tumor growth surveillance, the explantation procedure, and finally the handling of obtained xenograft tissues are described step by step. This technical description is completed by numerous tips and alternatives designed to allow for an easy adaptation and transfer to other laboratories.
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Antineoplásicos/farmacología , Criopreservación/métodos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Trasplante de Neoplasias/métodos , Animales , Biopsia/métodos , Expresión Génica , Histona Desacetilasas/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante HeterólogoRESUMEN
Tumour budding and podia formation are well-appreciated in surgical pathology as an aggressive invasion phenotype of colorectal carcinoma cells that is attained in the microenvironment of the invasive margin. In this study, we addressed how tumour budding and podia formation feature in xenografts. Primary colorectal carcinomas (N = 44) of various molecular types (sporadic standard type, high-degree microsatellite-unstable, CpG island methylator phenotype) were transplanted subcutaneously into T and B cell-deficient NSG mice, making possible immunohistochemistry with routine surgical pathology antibodies. Tumor budding and podia formation were both appreciably present in the xenografts. Quantitative evaluations of cytokeratin immunostains of primaries and their corresponding xenografts showed a reduction of tumour buds in the xenografts. Furthermore, in xenografts tumour cells were completely negative by pSTAT3 immunohistochemistry, indicating absence of cytokine/chemokine signalling, but nuclear ß-catenin and SMAD4 immunostainings as read-out of wnt and BMP pathway activation, respectively, were maintained. Carcinoma cells in most xenografts retained immunostaining of at least some nuclei by immunohistochemistry with antibodies against pERK1/2. K-ras/B-raf mutational status did not correlate with tumour budding or podia formation in the xenografts. Our results indicate that tumour budding and podia formation can be modelled by xenografting, and in NSG mice it can be studied with the same immunohistochemical methods as used for primaries in surgical pathology. Dysregulation of wnt and BMP signalling appears to be transferred into the xenograft microenvironment, but not cytokine/chemokine signalling.
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Neoplasias Colorrectales/patología , Invasividad Neoplásica/genética , Células del Estroma/patología , Microambiente Tumoral/genética , Animales , Neoplasias Colorrectales/genética , Islas de CpG/genética , Metilación de ADN/genética , Humanos , Ratones , Mutación , Invasividad Neoplásica/patología , Proteína Smad4/genética , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genéticaRESUMEN
Background. For development of individualized treatment on a routine basis, transfer of patients' tumor tissue in a xenograft model (i.e., generation of patient-derived xenografts (PDX)) is desirable for molecular, biochemical, or functional analyses. Drawbacks are dissatisfactory tumor take rates, the necessity of fast tumor tissue processing, and extensive logistics demanding teamwork of surgeons, pathologists, and laboratory researchers. Methods. The take rates of ten colorectal cancer (CRC) tissue samples in immunodeficient mice were compared after direct cryopreservation and after a 24 h cooling period at 4°C prior to cryopreservation. Additionally, the effect of simultaneous Matrigel application on the take rates was investigated. Beside take rates, tumor growth characteristics and cell culture success were analyzed. Results. Tumor takes of CRC tissue samples were significantly improved after Matrigel application (8 versus 15 takes, p = 0.04). As expected, they diminished furthermore after 24 h cooling. Application of Matrigel could counteract this decrease significantly (2 versus 7 takes, p = 0.03). Cumulative take rate after cryopreservation was satisfactory (70%). Conclusion. Matrigel application after 24 h delay in tissue processing facilitates CRC PDX model development. These data help developing strategies for individualized tumor therapies in the context of multicenter clinical studies and for basic research on primary patient tumors.
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Neoplasias Colorrectales , Criopreservación , Trasplante de Neoplasias , Neoplasias Experimentales , Animales , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/fisiopatología , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/fisiopatología , Factores de TiempoRESUMEN
Colitis-associated colorectal cancer (CAC) seems to be a rather unique entity and differs in its genetic alterations, tumour formation capacities, and clinical features from sporadic colorectal carcinoma. Most descriptions about tumour biology of CAC refer to ulcerative colitis; data about Crohn´s colitis related carcinomas are scarce. The majority of patients with Crohn´s disease are under immunosuppression which generates a different environment for tumour growth. We first describe the clinical case of a fast growing CAC in a long-term immunosuppressed patient with Crohn´s disease and successful establishment and characterization of carcinoma cell lines along with their corresponding patient-derived xenograft. Subsequently, these tumor models were molecularly and functionally analysed. Beside numerous chromosomal alterations, mutations in TP53, APC, PTEN and SMAD3 were identified. The cell lines express numerous cancer testis antigens, surface molecules involved in immune evasion but low levels of HLA class I molecules. They show strong invasive but in comparison weak migratory activity. The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient. They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.
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Carcinoma/patología , Técnicas de Cultivo de Célula/métodos , Neoplasias del Colon/patología , Enfermedad de Crohn/patología , Xenoinjertos/patología , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma/etiología , Carcinoma/genética , Células Cultivadas , Aberraciones Cromosómicas , Neoplasias del Colon/etiología , Neoplasias del Colon/genética , Enfermedad de Crohn/complicaciones , Genes MHC Clase I , Células HCT116 , Xenoinjertos/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
INTRODUCTION: A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). RESULTS: The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. CONCLUSION: Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.
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Anticuerpos Monoclonales/inmunología , Colon/inmunología , Colon/virología , Retrovirus Endógenos/inmunología , Productos del Gen gag/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/virología , Citoplasma/inmunología , Citoplasma/virología , Femenino , Genoma Humano/genética , Células HEK293 , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunologíaRESUMEN
The phosphatase and tensin homologue (PTEN) gene is considered to be a tumour-suppressor gene in various types of cancer, colorectal carcinoma among them. According to the 'two-hit' tumour-suppressor gene concept, inactivation occurs by any combination of the following three pathogenetic processes: mutation, loss of one allele [i.e. loss of heterozygosity (LOH)] or promoter methylation. To determine the frequencies of PTEN tumour-suppressor gene features in colorectal carcinoma, we used DNA from colorectal carcinoma xenografts/primary tumour cell lines (N=22) or neoplastic glands isolated by laser-capture microdissection (N=20). Sequencing exons 1-9 of the gene revealed a total of 8 somatic mutations in 5 tumours (3 with high-degree microsatellite instability). In 1 tumour, a truncating mutation of one allele was combined with two missense mutations of the other allele. Polymorphic microsatellite marker analyses (D10S5412, D10S579 and D10S1765) showed complete loss of one allele (i.e. LOH sensu stricto) in 3 tumours, but combined LOH and mutation was found only once. Promoter methylation, tested by MethyLight technology, was found in only 1 of the tumours, not combined with mutation or LOH. In contrast, by immunohistochemistry (mAb 6H2.1), reduction or even loss of PTEN expression was found in 18 tumours. Taken together, PTEN downregulation is a fairly frequent event in colorectal carcinoma, but this apparently is not usually caused by two hits on the gene.