Andre Verheem

Comparative Proteomics of Colon Cancer Stem Cells and Differentiated Tumor Cells Identifies BIRC6 as a Potential Therapeutic Target

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that “cell death ” regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

Interdigestive small bowel motility and duodenal bacterial overgrowth in experimental acute pancreatitis

Abstract The objective of this study is to investigate the effects of an acute necrotizing pancreatitis (ANP), without biliary obstruction, on the migrating motor complex (MMC), small bowel bacterial overgrowth (SBBO), bacterial translocation (BT) and infection of the pancreas simultaneously. Rats were divided into four groups: mild pancreatitis, control, ANP and sham operated control. Jejunal myoelectrodes were used to measure MMCs. Blood, peritoneal fluid, bile, and abdominal organs were harvested for microbial culturing 72 h after induction of pancreatitis. The splenic portion of the pancreas was taken for histology. During ANP the MMC cycle length was significantly increased from 14.1 ± 0.2 to 22.4 ± 1.9 min (P < 0.05). The duodenum of ANP rats was in contrast with the other groups characterized by Enterobacteriacae (> 3 log 10 CFU g−1 in seven of 12 rats, P < 0.05). A positive correlation (r = 0.78, P < 0.01) existed between duodenal Gram‐negative and anaerobic flora and the MMC cycle. Correlation between MMC cycle length and BT to the pancreas was positive as well (r = 0.70, P < 0.01). A positive correlation (r = 0.85, P < 0.01) was found between the severity of pancreatitis and duodenal bacterial overgrowth. During ANP without biliary obstruction, the jejunal MMC is disturbed and consequently SBBO occurs. The correlation between the severity of pancreatitis, the disturbance of the MMC and SBBO suggests an important pathophysiological role of the proximal small bowel in the infection of pancreatic necrosis.

Differentiated Human Colorectal Cancer Cells Protect Tumor-Initiating Cells From Irinotecan

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.

SIRT1/PGC1α dependent increase in oxidative phosphorylation supports chemotherapy resistance of colon cancer

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. Clin Cancer Res; 21(12); 2870–9. ©2015 AACR.

Adverse effects of the antiangiogenic agent angiostatin on the healing of experimental colonic anastomoses.

BackgroundAntiangiogenic cancer therapy is likely to be administered long term for sustained suppression of tumor outgrowth. Surgeons will encounter more patients undergoing such therapy. Therefore, it is essential to know the effects of antiangiogenic agents on physiological angiogenesis, as occurs during the healing of colonic anastomoses.MethodsAngiostatin was generated from human plasma and administered continuously. In 38 mice, the right colon was anastomosed after transection: group 1 (n=13), anstomotic healing under angiostatin treatment from surgery until death (day 7); group 2 (n=13), phosphate-buffered saline controls. For healing on discontinuation of treatment, group 3 (n=6) received angiostatin treatment preceding surgery during 4 days; group 4 (n=6) included controls. On day 7, all mice were inspected for signs of anastomotic leakage. Bursting pressure measurements were performed to test anastomotic strength. Neovascularization was assessed semiquantitatively by immunohistochemistry.ResultsMice treated with angiostatin postoperatively showed significantly more signs of leakage, more adhesions and peritonitis. One mouse died on day, 5. Five mice had paralytical ileus. The bursting pressure in group 1 was 135±20 mm Hg, versus 175±12 mm Hg in group 2 (mean ±SEM). Significantly fewer new vessels were found surrounding the anastomosis in the treated group (6.6±.9) versus controls (16±1.6). All controls, as well as those animals treated with angiostatin only until surgery (group 3), displayed normal healing and showed no signs of peritonitis or ileus.ConclusionsAngiostatin impairs anastomotic healing in mice. However, on discontinuation of antiangiogenic therapy, normal anastomotic healing is promptly restored.

Pretreatment but not treatment with probiotics abolishes mouse intestinal barrier dysfunction in acute pancreatitis

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.

Disrupted bile flow affects interdigestive small bowel motility in rats

BACKGROUND The role of bile flow in the regulation of small bowel motility and the migrating myoelectric complex (MMC) is unclear. We aimed to study the effects of biliary diversion or obstruction on the MMC in a newly developed rat model. METHODS In rats, myoelectrodes were implanted in the jejunum, and the proximal common bile duct (CBD) was cannulated and exteriorized at the head, enabling us to manipulate biliary flow without influencing pancreatic flow and without the need of anesthesia or additional surgery. Group A were controls without CBD cannulas. Biliary circulation was exteriorized but kept intact in group B; bile was diverted externally in group C; and the CBD was obstructed in group D. MMCs were recorded in unrestrained conditions by jejunal electromyography before and after biliary diversion or obstruction. Spontaneous recanalization of the CBD was monitored by measurement of serum bilirubin and by cholangiography. RESULTS Exteriorization of the CBD without interruption of bile flow did not affect MMC duration (group A, 17.3 +/- 0.3 minutes [mean +/- SEM]; group B, 16.5 +/- 0.6 minutes). MMCs disappeared temporarily after CBD obstruction but not after biliary diversion. MMCs of increased duration were seen after 1 day in rats with biliary diversion or CBD obstruction (group C, 26.1 +/- 4.4 minutes; group D, 36.3 +/- 4.8 minutes [p < 0.05]). MMCs after biliary diversion or obstruction were characterized by an increased duration of phase II-like activity and decreased duration of phase I activity. CONCLUSIONS We conclude that MMCs disappear temporarily early after CBD obstruction, but MMCs of increased duration are seen after 1 day of biliary diversion or obstruction. Thus disrupted bile flow affects interdigestive small bowel motility in rats.

Selective portal clamping to minimize hepatic ischaemia-reperfusion damage and avoid accelerated outgrowth of experimental colorectal liver metastases.

Temporary vascular clamping during local ablation for colorectal liver metastases increases destruction volumes. However, it also causes ischaemia–reperfusion (IR) injury to the liver parenchyma and accelerates the outgrowth of microscopic tumour deposits. The aim of this study was to investigate the effects of selective portal clamping on hepatocellular damage and tumour growth.

Fibrin-thrombin coated sealant increases strength of esophagogastric anastomoses in a rat model

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.

Maintenance of Clonogenic KIT+ Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells

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.