Autocrine WNT2 signaling in fibroblasts promotes colorectal cancer progression.

The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/ß-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/ß-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/ß-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/ß-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.

Stromal-derived IGF2 promotes colon cancer progression via paracrine and autocrine mechanisms.

The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-ß (TGFß) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFß/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFß to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFß was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.

Controlled clinical laboratory comparison of two supplemented aerobic and anaerobic media used in automated blood culture systems to detect bloodstream infections.

A 20-ml blood sample was collected from adult patients with suspected bloodstream infections and distributed equally into the four volume-controlled bottles of a blood culture set consisting of aerobic and anaerobic BACTEC Plus/F bottles and aerobic and anaerobic BacT/Alert FAN bottles. All bottles were incubated in their respective instruments for a standard 5-day protocol or until the instruments signalled positivity. Samples in all bottles with negative results by these instruments were terminally subcultured. A total of 8,390 blood culture sets were obtained during the study period, of which 4,402 (52.5%) met the study criteria. Of these, 946 (21.5%) were positive either by instrument signal or by additional terminal subculture of all negative bottles and yielded growth of microorganisms. Five hundred eighty-nine (13.4%) blood culture sets were considered to have recovered 663 clinically significant organisms. When both the BACTEC and the BacT/Alert systems were used, 465 positive sets were detected; BACTEC alone detected 52 positive sets and BacT/Alert alone detected 72 (P = 0.09). No differences were found between the two systems in microbial recovery rate from blood cultures obtained from patients on antibiotic therapy. Significantly more members of the family Enterobacteriaceae (P < 0.01) were detected from patients without antimicrobial therapy by BacT/Alert than by BACTEC. The false-negative rates were 0.20% for BACTEC and 0.32% for BacT/Alert. A significantly higher false-positive rate was found for BACTEC (P < 0.0001). Both systems were comparable for the time to detection of microorganisms. However, gram-positive bacteria were detected faster by BACTEC and Enterobacteriaceae were detected faster on average by BacT/Alert. We concluded that both systems are comparable in their abilities to recover aerobic and anaerobic organisms from blood cultures and a terminal subculture might not be necessary for either of the two systems. The increased positivity rate when using an anaerobic bottle in a two-bottle blood culture set is due to the additional blood volume rather than to the use of an anaerobic medium.

Multicenter clinical comparison of resin-containing bottles with standard aerobic and anaerobic bottles for culture of microorganisms from blood.

In a study comparing the Bactec 9240 (Plus Aerobic/F and Anaerobic/F bottles, containing resins; Becton Dickinson, USA) and the Vital (standard aerobic and anaerobic bottles, with no additives; bioMérieux, France) blood culture systems, 6456 sets of four bottles of 9660 blood cultures submitted were evaluated. There were 531 clinically significant isolates from 795 positive blood cultures. Of the 531 positive blood cultures, 355 were positive in both systems, 141 with the Bactec 9240 alone, and 30 with the Vital alone (p < 0.001); five were not detected by either system. The average time to detection of positive cultures for the matched sets was 10.65 h and 18.41 h by the Bactec 9240 system and the Vital system, respectively. The false-positive rate per bottle was 0.65% in the Bactec 9240 and 0.71% in the Vital. The rate of false-negative pairs (i.e., major errors) was very low (0.12% for the Bactec 9240, 0.19% for the Vital) and not significantly different between the two systems. The striking differences in recovery of microorganisms may be due to the presence of resins in the Bactec medium. However, the observed superiority of the Bactec 9240, even for patients not receiving antibiotics, suggests that resins adsorb other inhibitors present in patients' blood.