RESUMO
BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.
Assuntos
Neoplasias do Colo , Neoplasias Hepáticas , Neoplasias Pulmonares , Camundongos , Animais , Humanos , gama Catenina/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias do Colo/genética , Neoplasias Hepáticas/patologiaRESUMO
BACKGROUND AND AIM: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (â¼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target. METHODS: Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence. RESULTS: KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo, without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture. CONCLUSIONS: RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , Adenocarcinoma/tratamento farmacológico , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Neoplasias Colorretais/tratamento farmacológico , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Transplante de Neoplasias , Fenóis/farmacologia , Fosforilação , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Junções Íntimas , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. METHODS: We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. RESULTS: Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. CONCLUSIONS: Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.
Assuntos
Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Neoplasias do Colo/enzimologia , Células-Tronco Neoplásicas/enzimologia , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Hipóxia Celular , Proliferação de Células , Técnicas de Cocultura , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Comunicação Parácrina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais , Esferoides Celulares , Fator de Células-Tronco/antagonistas & inibidores , Fator de Células-Tronco/genética , Fatores de Tempo , Carga Tumoral , Células Tumorais CultivadasRESUMO
Colorectal cancer metastasizes predominantly to the liver but also to the lungs and the peritoneum. The presence of extra-hepatic metastases limits curative (surgical) treatment options and is associated with very poor survival. The mechanisms governing multi-organ metastasis formation are incompletely understood. Here, we tested the hypothesis that the site of tumor growth influences extra-hepatic metastasis formation. To this end, we implanted murine colon cancer organoids into the primary tumor site (i.e., the caecum) and into the primary metastasis site (i.e., the liver) in immunocompetent mice. The organoid-initiated liver tumors were significantly more efficient in seeding distant metastases compared to tumors of the same origin growing in the caecum (intra-hepatic: 51 vs. 40%, p = 0.001; peritoneal cavity: 51% vs. 33%, p = 0.001; lungs: 30% vs. 7%, p = 0.017). The enhanced metastatic capacity of the liver tumors was associated with the formation of 'hotspots' of vitronectin-positive blood vessels surrounded by macrophages. RNA sequencing analysis of clinical samples showed a high expression of vitronectin in liver metastases, along with signatures reflecting hypoxia, angiogenesis, coagulation, and macrophages. We conclude that 'onward spread' from liver metastases is facilitated by liver-specific microenvironmental signals that cause the formation of macrophage-associated vascular hotspots. The therapeutic targeting of these signals may help to contain the disease within the liver and prevent onward spread.
RESUMO
The liver has a complex and hierarchical segmental organization of arteries, portal veins, hepatic veins and lymphatic vessels. In-depth imaging of liver vasculature and malignancies could improve knowledge on tumor micro-environment, local tumor growth, invasion, as well as metastasis. Non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI) and positron-emission transmission (PET) are routine for clinical imaging, but show inadequate resolution at cellular and subcellular level. In recent years, tissue clearing - a technique rendering tissues optically transparent allowing enhanced microscopy imaging - has made great advances. While mainly used in the neurobiology field, recently more studies have used clearing techniques for imaging other organ systems as well as tumor tissues. In this study, our aim was to develop a reproducible tissue clearing and immunostaining model for visualizing intrahepatic blood microvasculature and tumor cells in murine colorectal liver metastases. CLARITY and 3DISCO/iDISCO+ are two established clearing methods that have been shown to be compatible with immunolabelling, most often in neurobiology research. In this study, CLARITY unfortunately resulted in damaged tissue integrity of the murine liver lobes and no specific immunostaining. Using the 3DISCO/iDISCO+ method, liver samples were successfully rendered optically transparent. After which, successful immunostaining of the intrahepatic microvasculature using panendothelial cell antigen MECA-32 and colorectal cancer cells using epithelial cell adhesion molecule (EpCAM) was established. This approach for tumor micro-environment tissue clearing would be especially valuable for allowing visualization of spatial heterogeneity and complex interactions of tumor cells and their environment in future studies.
RESUMO
BACKGROUND & AIMS: Stem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors. METHODS: Colonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity. RESULTS: Colonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh/ABCB1negative cells generated nontumorigenic ALDHlow/ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice. CONCLUSIONS: The resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh/ABCB1negative cells and their differentiated, drug-expelling, ALDHlow/ABCB1positive daughter cells.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Aldeído Desidrogenase/metabolismo , Animais , Antineoplásicos Fitogênicos/metabolismo , Biomarcadores Tumorais/metabolismo , Western Blotting , Camptotecina/metabolismo , Camptotecina/farmacologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclosporinas/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo/métodos , Humanos , Irinotecano , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares , Fatores de Tempo , Inibidores da Topoisomerase I/metabolismo , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Anastomotic leakage is a feared complication after esophagectomy. The purpose of this study was to investigate whether the use of a fibrin-thrombin coated collagen patch (TachoSil; Nycomed, Zurich, Switzerland), applied as a sealant, would strengthen the esophagogastric anastomosis and stimulate anastomotic healing in a rat model. METHODS: Hand sewn, end-to-side esophagogastric anastomoses were performed in 54 rats. Animals were randomized for an unsealed or sealed anastomosis. Rats were sacrificed on postoperative d 0, 3, 5, and 7. Primary parameter was bursting pressure. Secondary outcomes were complications, weight, and immunohistochemical staining for collagen formation and fibroblast activity. RESULTS: Bursting pressure at d 0 and 3 was significantly increased when a sealant was used (55.1 ± 4.6 mmHg versus 102.4 ± 7.3 mmHg, P < 0.010; and 19.7 ± 3.3 mmHg versus 34.6 ± 4.9 mmHg, P < 0.050 respectively). There was no difference in bursting pressure at d 5 and 7 between unsealed and sealed anastomoses (60.9 ± 18.2 mmHg versus 53.4 ± 6.6 mmHg, P = 0.690; and 118.8 ± 20.2 mmHg versus 97.2 ± 8.3 mmHg, P = 0.374 respectively). Application of sealant independently influenced bursting pressure (P < 0.010). Increased fibroblastic activity was noticed at d 7 in sealed anastomoses (P < 0.050). There were no differences in weight gain between groups. CONCLUSIONS: Additional sealing of the anastomosis increased anastomotic strength during early postoperative recovery when anastomotic strength is at its weakest. The findings indicate that sealing of the anastomosis has the potential to prevent leakage after esophagectomy in humans.
Assuntos
Esofagectomia/efeitos adversos , Adesivo Tecidual de Fibrina/farmacologia , Fibrinogênio/farmacologia , Complicações Pós-Operatórias/prevenção & controle , Trombina/farmacologia , Cicatrização/efeitos dos fármacos , Anastomose Cirúrgica/efeitos adversos , Animais , Peso Corporal/fisiologia , Modelos Animais de Doenças , Combinação de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Masculino , Complicações Pós-Operatórias/fisiopatologia , Pressão , Ratos , Ratos Wistar , Ruptura Espontânea/prevenção & controle , Adesivos Teciduais/farmacologiaRESUMO
Peritoneal metastases in colorectal cancer (CRC) belong to Consensus Molecular Subtype 4 (CMS4) and are associated with poor prognosis. Conventional imaging modalities, such as Computed Tomography (CT) and Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET), perform very poorly in the detection of peritoneal metastases. However, the stroma-rich nature of these lesions provides a basis for developing molecular imaging strategies. In this study, conducted from 2019 to 2021, we aimed to generate a Platelet-Derived Growth Factor Receptor beta (PDGFRB)-binding molecular imaging tracer for the detection of CMS4 CRC, including peritoneal metastases. The expression of PDGFRB mRNA discriminated CMS4 from CMS1-3 (AUROC = 0.86 (95% CI 0.85-0.88)) and was associated with poor relapse-free survival. PDGFRB mRNA and protein levels were very high in all human peritoneal metastases examined (n = 66). Therefore, we generated a PDGFRB-targeting llama nanobody (VHH1E12). Biotin-labelled VHH1E12 bound to immobilized human and mouse PDGFRB with high affinity (EC50 human PDGFRB = 7 nM; EC50 murine PDGFRB = 0.8 nM), and to PDGFRB-expressing HEK293 cells grown in vitro. A pharmacokinetic analysis of IRDye-800CW-conjugated VHH1E12 in mice showed that the plasma half-life was 6 min. IRDye-800CW-conjugated VHH1E12 specifically accumulated in experimentally induced colorectal cancer peritoneal metastases in mice. A tissue analysis subsequently demonstrated co-localization of the nanobody with PDGFRB expression in the tumour stroma. Our results demonstrate the potential value of PDGFRB-targeted molecular imaging as a novel strategy for the non-invasive detection of CMS4 CRC, in particular, peritoneal metastases.
RESUMO
Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFß1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFß signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFß signaling, while KIT deletion sensitized PDOs to TGFß-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFß signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFß expression, and protects the tumor cells from tumor-suppressive TGFß signaling by inhibiting SMAD2 expression.
Assuntos
Neoplasias Colorretais , Transdução de Sinais , Fator de Crescimento Transformador beta , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente TumoralRESUMO
The presence of BRAFV600E in colorectal cancer (CRC) is associated with a higher chance of distant metastasis. Oxidative stress in disseminated tumor cells limits metastatic capacity. To study the relationship between BRAFV600E, sensitivity to oxidative stress, and metastatic capacity in CRC, we use patient-derived organoids (PDOs) and tissue samples. BRAFV600E tumors and PDOs express high levels of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis. Deletion of GCL in BRAFV600E PDOs strongly reduces their capacity to form distant liver and lung metastases but does not affect peritoneal metastasis outgrowth. Vice versa, the glutathione precursor N-acetyl-cysteine promotes organ-site-specific metastasis in the liver and the lungs but not in the peritoneum. BRAFV600E confers resistance to pharmacologically induced oxidative stress in vitro, which is partially overcome by treatment with the BRAF-inhibitor vemurafenib. We conclude that GCL-driven glutathione synthesis protects BRAFV600E-expressing tumors from oxidative stress during distant metastasis to the liver and the lungs.
Assuntos
Neoplasias Colorretais , Neoplasias Pulmonares , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Estresse Oxidativo , Glutamato-Cisteína Ligase/genética , Glutationa , Neoplasias Pulmonares/genética , Neoplasias Colorretais/genéticaRESUMO
BACKGROUND: The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. METHODS: We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. RESULTS: PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. CONCLUSIONS: By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Neoplasias Peritoneais , Camundongos , Animais , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Fator de Crescimento Transformador beta3 , Peritônio/metabolismo , Ascite , Neoplasias Peritoneais/tratamento farmacológico , Citocinas/metabolismo , Neoplasias Colorretais/tratamento farmacológicoRESUMO
BACKGROUND: Ischemia/reperfusion (I/R) injury in the liver is associated with accelerated outgrowth of micrometastases. The aim of the study was to test the role of CD95 signaling in accelerated outgrowth of colorectal liver metastases following I/R. METHODS: Mice underwent vascular clamping 5 days after induction of colorectal liver metastases. Invasion and outgrowth of micrometastases following I/R were analyzed by post-mortem confocal microscopy (36 hr post-I/R) and by morphometric assessment of tumor load (5 days post-I/R), respectively. Tumor cell CD95 was suppressed by lentiviral RNA interference. The contribution of host CD95L was assessed by using gld-mice lacking functional CD95L. RESULTS: CD95-knockdown in tumor cells strongly reduced perinecrotic invasion (tumor diameter from â¼830 to â¼470 µm) and largely prevented outgrowth acceleration of perinecrotic liver metastases following I/R (from â¼8- to â¼4.5-fold). In gld-mice, the relative hepatic area with necrosis was markedly reduced. Perinecrotic tumor cell clusters still displayed an invasive phenotype (tumor diameter of â¼980 µm). However, I/R-induced acceleration of tumor outgrowth was reduced in gld-mice from â¼8- to â¼5-fold. CONCLUSIONS: I/R induces invasion and accelerated outgrowth of preestablished metastases in a CD95-dependent manner. Activation of the CD95 system following I/R not only contributes to liver injury, but may also promote aggressive tumor recurrence.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Traumatismo por Reperfusão/metabolismo , Receptor fas/metabolismo , Animais , Neoplasias Colorretais/patologia , Fígado/irrigação sanguínea , Fígado/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transdução de SinaisRESUMO
The liver's cellular functions are sustained by a hierarchical, segmentally-organized vascular system. Additionally, liver lymphatic vessels are thought to drain to perihepatic lymph nodes. Surprisingly, while recent findings highlight the importance of organ-specific lymphatics, the functional anatomy of liver lymphatics has not been mapped out. In literature, no segmental or preferential lymphatic drainage patterns are known to exist. We employ a novel murine model of liver lymphangiography and in vivo microscopy to delineate the lymphatic drainage patterns of individual liver lobes. Our data from blue dye liver lymphangiography show preferential lymphatic drainage patterns: Right lobe mainly to hepatoduodenal ligament lymph node 1 (LN1); left lobe to hepatoduodenal ligament LN1 + LN2 concurrently; median lobe showed a more variable LN1/LN2 drainage pattern with increased (sometimes exclusive) mediastinal thoracic lymph node involvement, indicating that part of the liver can drain directly to the mediastinum. Upon ferritin lymphangiography, we observed no functional communication between the lobar lymphatics. Altogether, these results show the existence of preferential lymphatic drainage patterns in the murine liver. Moreover, this drainage can occur directly to mediastinal lymph nodes and there is no interlobar lymphatic flow. Collectively, these data provide the first direct evidence that liver lymphatic drainage patterns follow segmental anatomy.
Assuntos
Microscopia Intravital , Fígado/anatomia & histologia , Linfonodos/anatomia & histologia , Vasos Linfáticos/anatomia & histologia , Animais , Fígado/metabolismo , Linfonodos/metabolismo , Vasos Linfáticos/metabolismo , Masculino , CamundongosRESUMO
BACKGROUND: Infection of pancreatic necrosis by gut bacteria is a major cause of morbidity and mortality in patients with severe acute pancreatitis. Use of prophylactic antibiotics remains controversial. The aim of this experiment was assess if modification of intestinal flora with specifically designed multispecies probiotics reduces bacterial translocation or improves outcome in a rat model of acute pancreatitis. METHODS: Male Sprague-Dawley rats were allocated into 3 groups: (1) controls (sham-operated, no treatment), (2) pancreatitis and placebo, and (3) pancreatitis and probiotics. Acute pancreatitis was induced by intraductal glycodeoxycholate and intravenous cerulein infusion. Daily probiotics or placebo was administered intragastrically from 5 days prior until 7 days after induction of pancreatitis. Tissue and fluid samples were collected for microbiologic and quantitative real-time PCR analysis of bacterial translocation. RESULTS: Probiotics reduced duodenal bacterial overgrowth of potential pathogens (Log(10) colony-forming units [CFU]/g 5.0 +/- 0.7 [placebo] vs 3.5 +/- 0.3 CFU/g [probiotics], P < .05), resulting in reduced bacterial translocation to extraintestinal sites, including the pancreas (5.38 +/- 1.0 CFU/g [placebo] vs 3.1 +/- 0.5 CFU/g [probiotics], P < .05). Accordingly, health scores were better and late phase mortality was reduced: 27% (4/15, placebo) versus 0% (0/13, probiotics), respectively, P < .05. CONCLUSIONS: This experiment supports the hypothesis that modification of intestinal flora with multispecies probiotics results in reduced bacterial translocation, morbidity, and mortality in the course of experimental acute pancreatitis.
Assuntos
Translocação Bacteriana/efeitos dos fármacos , Bifidobacterium , Lactobacillus , Pancreatite Necrosante Aguda/terapia , Probióticos/uso terapêutico , Animais , Duodeno/microbiologia , Masculino , Pancreatite Necrosante Aguda/microbiologia , Probióticos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms. EXPERIMENTAL DESIGN: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy. RESULTS: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy. CONCLUSIONS: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fosforilação Oxidativa , Sirtuína 1/genética , Fatores de Transcrição/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Metabolismo Energético/efeitos dos fármacos , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/secundário , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Infectious complications often occur in acute pancreatitis, related to impaired intestinal barrier function, with prolonged disease course and even mortality as a result. The bile salt nuclear receptor farnesoid X receptor (FXR), which is expressed in the ileum, liver and other organs including the pancreas, exhibits anti-inflammatory effects by inhibiting NF-κB activation and is implicated in maintaining intestinal barrier integrity and preventing bacterial overgrowth and translocation. Here we explore, with the aid of complementary animal and human experiments, the potential role of FXR in acute pancreatitis. METHODS: Experimental acute pancreatitis was induced using the CCK-analogue cerulein in wild-type and Fxr-/- mice. Severity of acute pancreatitis was assessed using histology and a semi-quantitative scoring system. Ileal permeability was analyzed in vitro by Ussing chambers and an in vivo permeability assay. Gene expression of Fxr and Fxr target genes was studied by quantitative RT-PCR. Serum FGF19 levels were determined by ELISA in acute pancreatitis patients and healthy volunteers. A genetic association study in 387 acute pancreatitis patients and 853 controls was performed using 9 tagging single nucleotide polymorphisms (SNPs) covering the complete FXR gene and two additional functional SNPs. RESULTS: In wild-type mice with acute pancreatitis, ileal transepithelial resistance was reduced and ileal mRNA expression of Fxr target genes Fgf15, SHP, and IBABP was decreased. Nevertheless, Fxr-/- mice did not exhibit a more severe acute pancreatitis than wild-type mice. In patients with acute pancreatitis, FGF19 levels were lower than in controls. However, there were no associations of FXR SNPs or haplotypes with susceptibility to acute pancreatitis, or its course, outcome or etiology. CONCLUSION: We found no evidence for a major role of FXR in acute human or murine pancreatitis. The observed altered Fxr activity during the course of disease may be a secondary phenomenon.
Assuntos
Pancreatite Necrosante Aguda/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Animais , Estudos de Casos e Controles , Impedância Elétrica , Fatores de Crescimento de Fibroblastos/sangue , Técnicas de Inativação de Genes , Humanos , Íleo/metabolismo , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite Necrosante Aguda/genética , Polimorfismo de Nucleotídeo Único , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity.
Assuntos
Neoplasias Colorretais/patologia , Glutationa Peroxidase/fisiologia , Peróxido de Hidrogênio/metabolismo , Animais , Diferenciação Celular , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Metástase Neoplásica , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Tiorredoxina Redutase 1/fisiologiaRESUMO
Drug-resistant cancer stem cells (CSCs) have been implicated in tumor recurrence following chemotherapy. However, the contribution of CSCs to drug-resistance in colorectal cancer is unclear and CSC-intrinsic drug-resistance mechanisms are ill-defined. Here, we address these issues by proteomic analysis of the secretomes of CSCs and isogenic differentiated tumor cells (DTCs) isolated from three distinct metastasized colon tumors. Mass spectrometry-based proteomics identified 1254 unique proteins in the conditioned media of the paired CSC and DTC cultures. Ingenuity Pathway Analysis revealed that proteins governing 'Cell Death' were most significantly enriched in the CSC secretome. The vast majority of these (37/43) promote cell survival. The CSC secretome is also characterized by a pro-survival Nrf2 antioxidant signature. Interestingly, proteome-maintenance networks are highly enriched in the CSC secretome. CSCs also secrete high levels of drug-metabolizing enzymes, including aldehyde dehydrogenase 1 (ALDH1A1) and bleomycin hydrolase (BLMH). We show that these enzymes cause extracellular detoxification of maphosphamide and bleomycin respectively. We conclude that colorectal CSCs are characterized by extensive survival and anti-oxidant networks, which are likely to contribute to CSC-intrinsic drug-resistance. In addition, CSCs may modulate drug responses in nearby tumor cells by detoxifying chemotherapeutic drugs in the extracellular space. BIOLOGICAL SIGNIFICANCE: Cancer stem cells are thought to play an important role in mediating drug resistance and tumor recurrence following chemotherapy. Therefore, it is important to identify the factors that are secreted by them. Our results provide novel insights into the pathways that govern the intrinsic resistance of CSCs to chemotherapy and, furthermore, demonstrate that they can also inactivate chemotherapeutic drugs in the extracellular space. A better understanding of the pathways that govern drug resistance in CSCs may help in developing effective CSC-targeting drugs.
Assuntos
Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteoma/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular , Biologia Computacional , Meios de Cultivo Condicionados/química , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacologia , Humanos , Células-Tronco Neoplásicas/metabolismo , Estresse Oxidativo , Dobramento de Proteína , Proteômica , RecidivaRESUMO
BACKGROUND: Intestinal barrier failure during acute pancreatitis (AP) is associated with translocation of luminal bacteria, resulting in infectious complications. We examined the effects of multispecies probiotics on the intestinal barrier impairment in a murine model of AP. METHODS: Mice were injected with cerulein to induce AP and were sacrificed 11 (early AP) or 72 hours (late AP) after start of induction. AP and associated systemic effects were confirmed by histology of pancreas and lung. Animals received daily probiotics starting 2 days prior to AP induction (pretreatment) or at the moment of AP induction (treatment). Mucosal barrier function of the distal ileum was assessed in Ussing chambers by measurement of the epithelial electrical resistance and the permeability to Na-fluorescein. RESULTS: Histological analysis revealed pancreatic injury in both phases of AP, and lung damage in the early phase. Epithelial resistance of the ileum was reduced and permeability increased in both phases of AP, indicating impairment of the intestinal barrier. Pretreatment had no effect on resistance or permeability in the early phase of AP. In the late phase of AP, pretreatment but not treatment abolished the AP induced resistance decrease and permeability increase. Administration of probiotics as such (ie, without induction of AP) had no effect on intestinal barrier function. CONCLUSION: Pretreatment with multispecies probiotics for 2 days abolishes intestinal barrier dysfunction in the late phase of AP, while treatment does not. The effectiveness of probiotics in this model depends on the timing of administration. Clinical trials with probiotics should seek conditions where treatment can be started prior to onset of disease or elective surgical intervention.
Assuntos
Translocação Bacteriana , Doenças do Íleo/prevenção & controle , Pancreatite/complicações , Probióticos/administração & dosagem , Animais , Bifidobacterium , Ceruletídeo , Doenças do Íleo/etiologia , Lactobacillus acidophilus , Lacticaseibacillus casei , Lactococcus lactis , Pulmão/patologia , Masculino , Camundongos , Pâncreas/patologia , Pancreatite/patologiaRESUMO
OBJECTIVES: The colon is considered a major source of bacteria causing infection of pancreatic necrosis in acute pancreatitis (AP). Subtotal colectomy before AP in rats reduces mortality, but its role in affecting small bowel flora, bacterial translocation, and infection of pancreatic necrosis is unknown. Our aim was to study these phenomena in rats with AP. METHODS: Fifty rats, allocated in 4 groups, underwent 2 laparotomies: group 1, sham laparotomy and saline biliopancreatic duct infusion; group 2, subtotal colectomy and saline infusion; group 3, sham laparotomy and AP (ductal infusion of glycodeoxycholic acid and intravenous cerulein); group 4, subtotal colectomy and AP. Seventy-two hours later, samples were collected for microbiological analysis. RESULTS: Subtotal colectomy caused small bowel bacterial overgrowth with gram-positive cocci (group 1 versus group 2, duodenum: P = 0.030, ileum: P = 0.029). Bacterial counts of gram-negative rods/anaerobes in the duodenum and ileum and pancreatic bacterial counts of rats with colectomy and AP were significantly higher than in rats with AP only (group 3 versus group 4, duodenum: P = 0.040, ileum: P = 0.029, pancreas: P = 0.017). Duodenal bacterial overgrowth and pancreatic infection correlate significantly (r = 0.45, P = 0.004). CONCLUSIONS: Subtotal colectomy induces small bowel bacterial overgrowth, which is associated with increased bacterial translocation to the pancreas.