N-glycosylation Regulates Intrinsic IFN-γ Resistance in Colorectal Cancer: Implications for Immunotherapy.

BACKGROUND & AIMS: Advanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. METHODS: Spontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. RESULTS: The conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ-resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling. CONCLUSIONS: Together, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC.

The impact of body mass index on prognosis in patients with colon carcinoma.

BACKGROUND: The impact of body mass index (BMI) on prognosis in patients with curatively resected stage I-III colon carcinoma was analyzed. METHODS: The prospectively collected data of 694 patients who underwent complete mesocolic excision between 2003 and 2014 were analyzed. BMI was classified into four categories: underweight (BMI < 18.5 kg/m2; n = 13), normal weight (BMI 18.5 to 24.9 kg/m2; n = 221), overweight (BMI 25.0 to 29.9 kg/m2; n = 309), and obese (BMI ≥ 30.0 kg/m2; n = 151). Univariate and multivariate analyses for comparison of prognosis were performed. RESULTS: The 5-year rate of locoregional recurrence in all 694 patients was 2.1%, and no differences were found with respect to BMI (p = 0.759). For distant metastasis, the 5-year rate for all patients was 13.4%, and BMI did not have a significant impact (p = 0.593). The 5-year rate of disease-free survival for all 694 patients was 72.4%. The differences with respect to BMI were not found to be significant in univariate analysis (p = 0.222). In multivariate Cox regression analysis, disease-free survival was significantly better in obese patients (HR 0.7; p = 0.034). Regarding overall survival, the 5-year rate for all patients was 78.1%. In univariate analyses, no significant differences were found for BMI (p = 0.094). In the Cox regression analysis, overweight and obese patients had significantly better survival (overweight: HR 0.7; p = 0.027; obese: HR 0.6; p = 0.019). CONCLUSION: The better survival of overweight and obese patients in multivariate analyses must be interpreted with caution. It is influenced by several factors and seems to correspond to the phenomenon of the obesity paradox.

Donor Site Morbidity of Patients Receiving Vertical Rectus Abdominis Myocutaneous Flap for Perineal, Vaginal or Inguinal Reconstruction.

BACKGROUND: Management of donor site closure after harvesting a vertical rectus abdominis myocutaneous (VRAM) flap is discussed heterogeneously in the literature. We aim to analyze the postoperative complications of the donor site depending on the closure technique. METHODS: During a 12-year period (2003-2015), 192 patients in our department received transpelvic VRAM flap reconstruction. Prospectively collected data were analyzed retrospectively. RESULTS: 182 patients received a VRAM flap reconstruction for malignant, 10 patients for benign disease. The median age of patients was 62 years. 117 patients (61%) received a reconstruction of donor site by Vypro® mesh, 46 patients (24%) by Vicryl® mesh, 23 patients (12%) by direct closure and 6 patients (3%) by combination of different meshes. 32 patients (17%) developed in total 34 postoperative complications at the donor site. 22 complications (11%) were treated conservatively, 12 (6%) surgically. 17 patients (9%) developed incisional hernia during follow-up, with highest incidence in the Vicryl® group (n = 8; 17%) and lowest in the Vypro® group (n = 7; 6%). Postoperative parastomal hernias were found in 30 patients (16%) including three patients with simultaneous hernia around an urostomy and a colostomy. The highest incidence of parastomal hernia was found in patients receiving primary closure of the donor site (n = 6; 26%), the lowest incidence in the Vypro® group (n = 16; 14%). CONCLUSION: The use of Vypro® mesh for donor site closure appears to be associated with a low postoperative incidence of complications and can therefore be recommended as a preferred technique.

Soluble intercellular adhesion molecule-1 is a prognostic marker in colorectal carcinoma.

PURPOSE: Serological tumor markers are routinely used to monitor tumor onset and progression. In colorectal carcinoma (CRC), the carcinoembryonic antigen (CEA) is roughly elevated in 50% of patients at initial diagnosis. Soluble ICAM-1 (sICAM-1) is elevated in different cancers. The aim of this study was to evaluate the prognostic relevance of sICAM-1 combined with CEA in patients with CRC. METHODS: In blood samples of 297 CRC patients, sICAM-1 was determined by ELISA and CEA by microparticle enzyme immunoassay the day before oncologic resection. Separation in patients with sICAM-1high and sICAM-1low was performed by minimum p value approach; separation in CEA normal and elevated was performed according to the established diagnostic cutoff. Clinical data were obtained from the prospective collected data from the Erlangen Registry for Colorectal Carcinomas. RESULTS: Cancer-related 5-year survival rate of patients with sICAM-1low (< 290 ng/ml, n = 208) was significantly increased (83.4%) as compared to that of patients with sICAM-1high (≥ 290 ng/ml, n = 89) (66.2%; p < 0.001). Patients with normal CEA concentrations (n = 199; 90.8%) showed a significantly (p < 0.001) improved cancer-related 5-year survival rate compared to patients with elevated CEA concentrations (n = 98; 52.1%). Moreover, high sICAM-1 was an independent risk factor (hazard ratio 1.6) in multivariate analysis. Of note, increased sICAM-1 levels, either within normal or within elevated CEA, allowed to identify high-risk subgroups, both for overall (p < 0.001) and cancer-related survival (p < 0.001). CONCLUSION: Application of a novel risk score combining CEA/sICAM-1 serum concentrations allows the identification of high-risk groups for poor survival in CRC patients.

The influence of tumour site on prognosis in metastatic colorectal carcinomas with primary tumour resection.

PURPOSE: The aim of our study was to compare the characteristics and prognosis between right- and left-sided metastatic colorectal carcinomas. METHODS: Data from 937 patients with stage IV colorectal carcinomas (synchronous distant metastasis) who had a resection of the primary tumour between 1985 and 2014 were analysed. Carcinomas in the caecum to transverse colon were defined as right-sided (n = 250; 26.7%). They were compared to tumours located from the splenic flexure to the rectum categorised as left-sided (n = 687; 73.3%). RESULTS: In right-sided carcinomas, we observed significantly more female patients (50.8 vs 36.2%; p < 0.001), more unfavourable histological types (24.0 vs 8.6%; p < 0.001), more M1c carcinomas (metastases to the peritoneum ± others; 32.0 vs 14.4%; p < 0.001) and more emergencies (11.6 vs 7.1%; p = 0.029), while multimodal treatment was utilised in fewer patients (51.6 vs 63.8%; p = 0.001) and curative resections were less frequently (24.1 vs 35.4%; p = 0.002). Prognosis was significantly worse in patients with right-sided carcinomas (2-year-survival 27.2 vs 44.6%, p < 0.01). This difference was more pronounced after R2 resection (15.3 vs 29.7%; p < 0.001), than after macroscopic curative resection (2-year-survival 63.9 vs 71.9%; p = 0.106). In multivariate Cox regression analysis, tumour site was found to be an independent prognostic factor for overall survival (HR 1.2; 95% CI 1.0-1.5; p = 0.012). During the three 10-year periods, the prognosis improved equally in patients with right- and left-sided carcinomas, while the differences in survival remained identical. CONCLUSIONS: In a surgical patient cohort undergoing primary tumour resection, significant differences in prognosis were observed between patients with metastatic right- and left-sided colorectal carcinomas.

Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS.

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 µl human serum by isotope dilution mass spectrometry, using (15) N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.

NLRP3 Inhibition Leads to Impaired Mucosal Fibroblast Function in Patients with Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) are characterized by mucosal inflammation and sequential fibrosis formation, but the exact role of the hyperactive NLRP3 inflammasome in these processes is unclear. Thus, we studied the expression and function of the NLRP3 inflammasome in the context of inflammation and fibrosis in IBD. METHODS: We analysed intestinal NLRP3 expression in mucosal immune cells and fibroblasts from IBD patients and NLRP3-associated gene expression via single-cell RNA sequencing and microarray analyses. Furthermore, cytokine secretion of NLRP3 inhibitor treated blood and mucosal cells, as well as proliferation, collagen production, and cell death of NLRP3 inhibitor treated intestinal fibroblasts from IBD patients were studied. RESULTS: We found increased NLRP3 expression in the inflamed mucosa of IBD patients and NLRP3 inhibition led to reduced IL-1ß and IL-18 production in blood cells and diminished the bioactive form of mucosal IL-1ß. Single cell analysis identified overlapping expression patterns of NLRP3 and IL-1ß in classically activated intestinal macrophages and we also detected NLRP3 expression in CD163+ macrophages. In addition, NLRP3 expression was also found in intestinal fibroblasts from IBD patients. Inhibition of NLRP3 led to reduced proliferation of intestinal fibroblasts, which was associated with a marked decrease in production of collagen type I and type VI in IBD patients. Moreover, NLRP3 inhibition in intestinal fibroblasts induced autophagy, a cellular process involved in collagen degradation. CONCLUSIONS: In the presented study, we demonstrate that inhibiting NLRP3 might pave the way for novel therapeutic approaches in IBD, especially to prevent the severe complication of intestinal fibrosis formation.

GBP-1 acts as a tumor suppressor in colorectal cancer cells.

The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.

Analysis of the interferon-γ-induced secretome of intestinal endothelial cells: putative impact on epithelial barrier dysfunction in IBD.

The development of inflammatory bowel diseases (IBD) involves the breakdown of two barriers: the epithelial barrier and the gut-vascular barrier (GVB). The destabilization of each barrier can promote initiation and progression of the disease. Interestingly, first evidence is available that both barriers are communicating through secreted factors that may accordingly serve as targets for therapeutic modulation of barrier functions. Interferon (IFN)-γ is among the major pathogenesis factors in IBD and can severely impair both barriers. In order to identify factors transmitting signals from the GVB to the epithelial cell barrier, we analyzed the secretome of IFN-γ-treated human intestinal endothelial cells (HIEC). To this goal, HIEC were isolated in high purity from normal colon tissues. HIEC were either untreated or stimulated with IFN-γ (10 U/mL). After 48 h, conditioned media (CM) were harvested and subjected to comparative hyper reaction monitoring mass spectrometry (HRM™ MS). In total, 1,084 human proteins were detected in the HIEC-CM. Among these, 43 proteins were present in significantly different concentrations between the CM of IFN-γ- and control-stimulated HIEC. Several of these proteins were also differentially expressed in various murine colitis models as compared to healthy animals supporting the relevance of these proteins secreted by inflammatory activated HIEC in the inter-barrier communication in IBD. The angiocrine pathogenic impact of these differentially secreted HIEC proteins on the epithelial cell barrier and their perspectives as targets to treat IBD by modulation of trans-barrier communication is discussed in detail.

Role of guanylate binding protein-1 in vascular defects associated with chronic inflammatory diseases.

Rheumatic autoimmune disorders are characterized by a sustained pro-inflammatory microenvironment associated with impaired function of endothelial progenitor cells (EPC) and concomitant vascular defects. Guanylate binding protein-1 (GBP-1) is a marker and intracellular regulator of the inhibition of proliferation, migration and invasion of endothelial cells induced by several pro-inflammatory cytokines. In addition, GBP-1 is actively secreted by endothelial cells. In this study, significantly increased levels of GBP-1 were detected in the sera of patients with chronic inflammatory disorders. Accordingly we investigated the function of GBP-1 in EPC. Interestingly, stable expression of GBP-1 in T17b EPC induced premature differentiation of these cells, as indicated by a robust up-regulation of both Flk-1 and von Willebrand factor expression. In addition, GBP-1 inhibited the proliferation and migration of EPC in vitro. We confirmed that GBP-1 inhibited vessel-directed migration of EPC at the tissue level using the rat arterio-venous loop model as a novel quantitative in vivo migration assay. Overall, our findings indicate that GBP-1 contributes to vascular dysfunction in chronic inflammatory diseases by inhibiting EPC angiogenic activity via the induction of premature EPC differentiation.

Results of isolated limb perfusion for metastasized malignant melanoma.

BACKGROUND AND OBJECTIVES: Locoregional metastases are typical biological manifestations of advanced malignant melanomas. Treatment with hyperthermic isolated limb perfusion (HILP) should be considered in affected patients. In the present study, we have analyzed the results of HILPs performed in our department. PATIENTS AND METHODS: Eighty patients with locoregional metastases of the extremities received HILP at the Department of Surgery between January 2007 and December 2016. The mean follow-up was 38 months. RESULTS: The study included 50 men and 30 women (mean age: 63 years). The median time between melanoma diagnosis and HILP was 25 months (range: 1-219 months). HILP was performed in curative (n = 45) and palliative (n = 35) intention. Seventy-five patients received a drug combination of melphalan/dactinomycin and five patients received a drug combination of melphalan/tumor necrosis factor-alpha. Remission rates were determined in 72 of 80 patients (90%) as follows: partial response n = 28, complete response n = 25, no response n = 19. Of the 25 patients with complete response, 13 patients developed a new tumor manifestation during follow-up (locoregional recurrences n = 4; distant metastases n = 3; both n = 6). The median overall survival rate was 33 months. Tumor stage influenced the survival rate significantly (p = 0.001). Patients with complete response showed a significantly better overall survival than patients with partial or no response (p = 0.016). CONCLUSION: HILP is an effective therapeutic option in patients with locoregional metastases. This procedure carries a certain risk of side effects and adverse events but overall results in good response rates. Therefore, HILP should be offered to selected patients based on an individual discussion, considering their health status and oncological prognosis.

Protein tyrosine phosphatase nonreceptor type 2 controls colorectal cancer development.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set out to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC), as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. In particular, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T cells, and increased PTPN2 levels negatively correlated with expression of PD-1, CTLA4, STAT1, and granzyme A. In vivo, T cell- and DC-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44+ effector/memory T cells, as well as CD8+ T cell infiltration and cytotoxicity in the tumor. In direct relevance to CRC treatment, T cell-specific PTPN2 deletion potentiated anti-PD-1 efficacy and induced antitumor memory formation upon tumor rechallenge in vivo. Our data suggest a role for PTPN2 in suppressing antitumor immunity and promoting tumor development in patients with CRC. Our in vivo results identify PTPN2 as a key player in controlling the immunogenicity of CRC, with the strong potential to be exploited for cancer immunotherapy.

Case report: Tension pneumoperitoneum after diagnostic colonoscopy in an 11 y/o boy with Crohns disease.

INTRODUCTION: Endoscopy is an established diagnostic and therapeutic tool in paediatric gastroenterology and a save method with rare complications. PRESENTATION OF CASE: We present the case of an 11-year-old Caucasian boy with a long history of inflammatory bowel disease. Three years prior an ileostomy was created and is still in position. After diagnostic panendoscopy (colonoscopy, gastroscopy, endoscopy of small intestine via ileostomy) the patient showed progressive abdominal distension and pain. After diagnosis of tension pneumoperitoneum by radiological proof of massive intraabdominal air and altered vital signs, we initiated emergency laparotomy. Surgical intervention ruled out a free gastrointestinal perforation as well as peritonitis. There was a gaseous insufflation of the mesenteric tissue of the sigmoid and upper rectum most likely according to microperforations to the mesentery. Due to the pre-existing ileostomy, we took no further surgical action. The abdomen was lavaged and drains inserted. Upon further conservative treatment with intravenous antibiotics, the patient showed quick recovery and was discharged on postoperative day 6. DISCUSSION: With an incidence of 0.01%, perforation after diagnostic colonoscopy in children is very uncommon. The zone most frequently affected is the sigmoid colon due to direct penetration or indirect force due to flexure, or insufflation. Even without macroscopic perforation, the development of a tension pneumoperitoneum seems to be possible. CONCLUSION: Even though Colonoscopy in children is a safe tool, the treating physician must never underestimate the risks of such an intervention. Especially chronically altered intestine as in long-time persisting chronic inflammatory bowel disease demand special care and intensive observation of the patient after intervention.

Computed Tomography-Guided Wire-Marking for Thoracoscopic Resection of Small Lung Nodules in Children.

Introduction: In pediatric patients, thoracoscopic wedge-resection of pulmonary nodules is an established therapy. However, intraoperative localization of small lesions is still challenging. Purpose of this study was to evaluate the efficacy of preoperative computed tomography (CT)-guided wire-marking of small lung nodules. Materials and Methods: Between 2012 and 2017 a total of six cases receiving thoracoscopic resection of CT-marked lung nodules were analyzed. The nodules were preoperatively tagged by a wire, which was attached to the thoracic wall by sterile dressing. Characteristics of interest were stability of wire, complete resection, and prevention of open thoracotomy. Results: Six procedures were performed on five patients, including four men and one woman. Median age at intervention was 16 years (range 11-19 years). All patients had a history of primary malignancies, including osteosarcoma (n = 4) and synovial sarcoma (n = 1). A total of 10 nodules were visualized in CT of which 9 were marked by wire. The median expected size of nodules was 6 mm (range 2-23 mm). Two patients had bilateral, two left-sided and two right-sided lung lesions. There was no wire slippage. In five procedures wedge resection was possible; one case needed a near total lobe resection. In one case a mini-thoracotomy at port insertion site was performed to extract the specimen. There was no conversion to thoracotomy. Histopathology showed R0 resection in all patients. Malignancy was found in all specimens. In one patient postoperative hemorrhagic anemia necessitated transfusion. Conclusions: Preoperative wire-localization of small lung nodules is a safe and effective tool to enable thoracoscopic resection in children and to avoid thoracotomic interventions.

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Matricellular protein SPARCL1 regulates tumor microenvironment-dependent endothelial cell heterogeneity in colorectal carcinoma.

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

Tumor-associated fibroblasts isolated from colorectal cancer tissues exhibit increased ICAM-1 expression and affinity for monocytes.

Progression of colorectal cancer (CRC) is strongly associated with inflammation and other desmoplastic reactions in the tumor cell-surrounding tissue. We successfully isolated fibroblasts from the desmoplastic stroma of human CRC specimens and uninvolved colon tissue of patients treated surgically for CRC and investigated potential functional capacities. All of the isolated fibroblasts were vimentin-positive and CK-20/CD45-negative confirming the fibroblast phenotype. Differential expression patterns were detected between tumor-associated fibroblasts (TAFs) and normal tissue-associated fibroblasts (NAFs) regarding intercellular adhesion molecule-1 (ICAM-1) expression. In 11 of 12 TAF cultures, basal ICAM-1 expression was increased as compared to corresponding NAF cultures (p=0.001). After stimulation of the cultures with interleukin-1ß, 8 of the 12 TAF cultures presented higher ICAM-1 levels when compared with the level in the corresponding NAF cultures (p=0.001). Moreover, the adhesive capacity of these cultures for U937 was increased in 8 out of 10 unstimulated and in 10 out of 10 stimulated cultures when TAFs and NAFs were compared. In corresponding tumor tissue sections from the same patients, the amount of ICAM-1-positive fibroblasts was significantly higher than that in the corresponding normal colon mucosa, indicating a tumor-specific effect that was maintained in the isolated cultures. These results indicate that fibroblasts from CRC tissue exhibit an increased affinity for monocytic cells. This increased intercellular interaction may contribute to elongated residence times of monocytes in CRC tissue. Therefore, these isolated fibroblasts are a useful tool for further functional investigation of desmoplastic tissue reactions in CRC.

Is coagulation factor VIII a useful marker for colorectal carcinoma?

BACKGROUND AND OBJECTIVE: Increased thromboembolic events are well known in patients suffering from malignant diseases. In the following pilot study, we investigated the usefulness of coagulation factor VIII (FVIII) as a possible prognostic marker in patients with colorectal carcinoma (CRC). METHODS: Plasma FVIII levels were measured in 79 patients with CRC, correlated with tumor characteristics, and compared with normal ranges of blood group (BG) 0 and BG A/AB/B and with 19 control patients. RESULTS: In CRC patients mean FVIII levels were elevated compared with controls (BG 0: p=0.283, BG A/AB/B: p=0.001) and normal ranges. Interestingly, mean FVIII levels varied significantly in different blood groups (p=0.002). UICC stage I CRC patients presented with mean FVIII plasma levels within normal ranges, whereas UICC stage II-IV CRC patients presented with elevated FVIII plasma levels. In BG A/AB/B a significantly elevated FVIII level was found in G2 compared with G1 tumors (p< 0.001). Patients with elevated carcinoembryonic antigen also showed significantly elevated FVIII levels (p=0.050). FVIII levels at time of surgery did not correlate with survival within the first 2 years following surgery. CONCLUSION: In this pilot study, we demonstrated that FVIII plasma levels are elevated in patients with CRC and affected by T-stage and differentiation of the tumor. Whether FVIII is a clinical useful marker needs to be tested in a larger cohort.

Isolation of endothelial cells from human tumors.

Antiangiogenic drugs have been used successfully for the treatment of colorectal cancer (CRC) and several other tumor types. Until recently, viable tumor endothelial cells (TEC) and normal endothelial cells of uninvolved colon tissue (NEC) from the same patient have not been available to optimize treatment strategies in vitro. Here, we describe a protocol for the isolation of TEC and NEC. These cells were isolated at a very high purity via magnetic cell sorting of tissue samples obtained from surgical specimens of patients suffering from CRC. Isolated TEC and NEC expressed CD31, CD105, VE-cadherin, VCAM-1, ICAM-1, and E-selectin, formed capillaries in basal membrane extract, and were able to take up acetylated LDL. They were negative for podoplanin, CD45, CD68, and CK-20, indicating blood vessel endothelial lineage. Expression of vWF was more pronounced in NEC cultures, whereas vWF was absent or only slightly expressed in all TEC cultures in vitro. Lower intracellular concentrations of vWF were also detected in TEC as compared to NEC at the tissue level. The latter finding demonstrated that differential features of TEC and NEC in vivo are stably perpetuated in culture. The isolated endothelial cell cultures may provide a useful in vitro model system to elucidate epigenetic effects on angiogenesis in cancer and to optimize antiangiogenic therapy.

The clinical value of von Willebrand factor in colorectal carcinomas.

BACKGROUND: To identify the value of von Willebrand factor (vWF) as a clinical marker in colorectal carcinoma (CRC). METHODS: Plasma levels of vWF were measured in 79 patients with UICC Stage I-IV CRC at time of operation and correlated with TNM categories, levels of the carcinoembryonic antigen (CEA), blood groups (BG) and 19 controls (CO). CO included cancer-free patients without bacterial or viral infections. For tissue analysis paraffin embedded tumour and mucosa sections of operation specimens were stained immunohistochemically for vWF and compared to vWF plasma levels as well as to TNM categories. RESULTS: VWF plasma levels in CRC patients were significantly dependent on blood groups (p=0.012) and elevated compared to the normal ranges as well as to controls (BG 0: p=0.668, BG A/AB/B: p=0.020). CRC-Patients over 60 years of age presented with significantly higher vWF levels than patients below 60 years (BG 0: p=0.005; BG A/AB/B: p=0.035). There was no correlation of vWF plasma levels and UICC stages in CRC. Patients with elevated vWF plasma levels also presented with elevated CEA levels, but significance was missing (p=0.080). VWF concentration within the tumour tissue was independent of concentration within normal mucosa, blood groups, histopathological characteristics and did not correlate with plasma vWF levels. CONCLUSION: VWF plasma levels are elevated in CRC patients, but not in a stage dependent manner. Besides the tumour at least blood groups and age mainly influence plasma vWF levels. In our opinion vWF as a routinely used clinical marker in CRC cannot be recommended.