Tatjana M.H. Niers

Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals

A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLIf or quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are grafted in mice or rats after transfection with a luciferase gene--usually that of a firefly. To determine tumor load, animals receive the substrate agent luciferin intraperitoneally, which luciferase converts into oxyluciferin in an ATP-dependent manner Light emitted by oxyluciferin in viable cancer cells is captured noninvasively with a highly sensitive charge-coupled device (CCD) camera. Validation studies indicate that BLI is useful to determine tumor load in the course of time, with each animal serving as its own reference. BLI is rapid, easy to perform, and sensitive. It can detect tumor load shortly after inoculation, even when relatively few cancer cells (2500-10,000) are used. BLI is less suited for the determination of absolute tumor mass in an animal because of quenching of bioluminescence by tissue components and the exact location of tumors because its spatial resolution is limited. Nevertheless, BLI is a powerful tool for high-throughput longitudinal monitoring of tumor load in small animals and allows the implementation of more advanced orthotopic tumor models in therapy intervention studies with almost the same simplicity as when measuring traditional ectopic subcutaneous models in combination with calipers.

Endogenous activated protein C limits cancer cell extravasation through sphingosine-1-phosphate receptor 1–mediated vascular endothelial barrier enhancement

Activated protein C (APC) has both anticoagulant activity and direct cell-signaling properties. APC has been reported to promote cancer cell migration/invasion and to inhibit apoptosis and therefore may exacerbate metastasis. Opposing these activities, APC signaling protects the vascular endothelial barrier through sphingosine-1-phosphate receptor-1 (S(1)P(1))activation, which may counteract cancer cell extravasation. Here, we provide evidence that endogenous APC limits cancer cell extravasation, with in vivo use of monoclonal antibodies against APC. The protective effect of endogenous APC depends on its signaling properties. The MAPC1591 antibody that only blocks anticoagulant activity of APC does not affect cancer cell extravasation as opposed to MPC1609 that blocks anticoagulant and signaling properties of APC. Combined administration of anti-APC antibodies and S(1)P(1) agonist (SEW2871) resulted in a similar number of pulmonary foci in mice in presence and absence of APC, indicating that the protective effect of APC depends on the S(1)P(1) pathway. Moreover, endogenous APC prevents cancer cell-induced vascular leakage as assessed by the Evans Blue Dye assay, and SEW2871 treatment reversed MPC1609-dependent vascular leakage. Finally, we show that cancer cells combined with MPC1609 treatment diminished endothelial VE-cadherin expression. In conclusion, endogenous APC limits cancer cell extravasation because of S(1)P(1)-mediated VE-cadherin-dependent vascular barrier enhancement.

Experimental melanoma metastasis in lungs of mice with congenital coagulation disorders

Experimental animal studies as well as clinical trials have shown that interventions targeting the blood coagulation cascade inhibit cancer cell metastasis. These data support the hypothesis that congenital prothrombotic disorders, like factor V Leiden, facilitate metastasis whereas bleeding disorders, like haemophilia impede metastasis. To test this hypothesis, we subjected factor V Leiden and factor VIII deficient mice to a murine model of experimental lung metastasis. In this model, B16F10 murine melanoma cells are injected into the tail vein resulting in multiple lung metastases within 20 days. Both hemi‐ and homozygous factor VIII deficient mice were protected against lung metastasis compared to wild‐type littermate controls. In contrast, homozygous factor V Leiden mice developed more metastases than wild‐type littermates, whereas heterozygous carriers showed an intermediate number of pulmonary foci. Overall, these data show that a congenital susceptibility to either bleeding or thrombosis modifies the metastatic capacity of cancer cells in the bloodstream and suggest that procoagulant phenotypes are a risk factor for tumour metastasis.

Vascular Endothelial Growth Factor in the Circulation in Cancer Patients May Not Be a Relevant Biomarker

Background Levels of circulating vascular endothelial growth factor (VEGF) have widely been used as biomarker for angiogenic activity in cancer. For this purpose, non-standardized measurements in plasma and serum were used, without correction for artificial VEGF release by platelets activated ex vivo. We hypothesize that “true” circulating (c)VEGF levels in most cancer patients are low and unrelated to cancer load or tumour angiogenesis. Methodology We determined VEGF levels in PECT, a medium that contains platelet activation inhibitors, in citrate plasma, and in isolated platelets in 16 healthy subjects, 18 patients with metastatic non-renal cancer (non-RCC) and 12 patients with renal cell carcinoma (RCC). In non-RCC patients, circulating plasma VEGF levels were low and similar to VEGF levels in controls if platelet activation was minimized during the harvest procedure by PECT medium. In citrate plasma, VEGF levels were elevated in non-RCC patients, but this could be explained by a combination of increased platelet activation during blood harvesting, and by a two-fold increase in VEGF content of individual platelets (controls: 3.4 IU/106, non-RCC: 6.2 IU/106 platelets, p = 0.001). In contrast, cVEGF levels in RCC patients were elevated (PECT plasma: 64 pg/ml vs. 21 pg/ml, RCC vs. non-RCC, p<0.0001), and not related to platelet VEGF concentration. Conclusions Our findings suggest that “true” freely cVEGF levels are not elevated in the majority of cancer patients. Previously reported elevated plasma VEGF levels in cancer appear to be due to artificial release from activated platelets, which in cancer have an increased VEGF content, during the blood harvest procedure. Only in patients with RCC, which is characterized by excessive VEGF production due to a specific genetic defect, were cVEGF levels elevated. This observation may be related to limited and selective success of anti-VEGF agents, such as bevacizumab and sorafenib, as monotherapy in RCC compared to other forms of cancer.

Differential effects of anticoagulants on tumor development of mouse cancer cell lines B16, K1735 and CT26 in lung

Abstract Cancer progression is facilitated by blood coagulation. Anticoagulants, such as Hirudin and low molecular weight heparins (LMWHs), reduce metastasis mainly by inhibition of thrombin formation and L- and P-selectin-mediated cell-cell adhesion. It is unknown whether the effects are dependent on cancer cell type. The effects of anticoagulants on tumor development of K1735 and B16 melanoma cells and CT26 colon cancer cells were investigated in mouse lung. Tumor load was determined noninvasively each week up to day 21 in all experiments using bioluminescence imaging. Effects of anticoagulants on tumor development of the three cell lines were correlated with the fibrin/fibrinogen content in the tumors, expression of tissue factor (TF), protease activated receptor (PAR)-1 and -4 and CD24, a ligand of L- and P-selectins. Hirudin inhibited tumor development of B16 cells in lungs completely but did not affect tumor growth of K1735 and CT26 cells. Low molecular weight heparin did not have an effect on K1735 melanoma tumor growth either. TF and PAR-4 expression was similar in the three cell lines. PAR-1 and CD24 were hardly expressed by K1735, whereas CT26 cells expressed low levels and B16 high levels of PAR-1 and CD24. Fibrin content of the tumors was not affected by LMWH. It is concluded that effects of anticoagulants are dependent on cancer cell type and are correlated with their CD24 and PAR-1 expression.

Validation of Serum Amyloid α as an Independent Biomarker for Progression-Free and Overall Survival in Metastatic Renal Cell Cancer Patients

BACKGROUND We recently identified apolipoprotein A2 (ApoA2) and serum amyloid α (SAA) as independent prognosticators in metastatic renal cell carcinoma (mRCC) patients, thereby improving the accuracy of the Memorial-Sloan Kettering Cancer Center (MSKCC) model. OBJECTIVE Validate these results prospectively in a separate cohort of mRCC patients treated with tyrosine kinase inhibitors (TKIs). DESIGN, SETTING, AND PARTICIPANTS For training we used 114 interferon-treated mRCC patients (inclusion 2001-2006). For validation we studied 151 TKI-treated mRCC patients (inclusion 2003-2009). MEASUREMENTS Using Cox proportional hazards regression analysis, SAA and ApoA2 were associated with progression-free survival (PFS) and overall survival (OS). In 72 TKI-treated patients, SAA levels were analyzed longitudinally as a potential early marker for treatment effect. RESULTS AND LIMITATIONS Baseline ApoA2 and SAA levels significantly predicted PFS and OS in the training and validation cohorts. Multivariate analysis identified SAA in both separate patient sets as a robust and independent prognosticator for PFS and OS. In contrast to our previous findings, ApoA2 interacted with SAA in the validation cohort and did not contribute to a better predictive accuracy than SAA alone and was therefore excluded from further analysis. According to the tertiles of SAA levels, patients were categorized in three risk groups, demonstrating accurate risk prognostication. SAA as a single biomarker showed equal prognostic accuracy when compared with the multifactorial MSKCC risk mode. Using receiver operating characteristic analysis, SAA levels >71 ng/ml were designated as the optimal cut-off value in the training cohort, which was confirmed for its significant sensitivity and specificity in the validation cohort. Applying SAA >71 ng/ml as an additional risk factor significantly improved the predictive accuracy of the MSKCC model in both independent cohorts. Changes in SAA levels after 6-8 wk of TKI treatment had no value in predicting treatment outcome. CONCLUSIONS SAA but not ApoA2 was shown to be a robust and independent prognosticator for PFS and OS in mRCC patients. When incorporated in the MSKCC model, SAA showed additional prognostic value for patient management.

Long-term thrombin inhibition promotes cancer cell extravasation in a mouse model of experimental metastasis

T . M . H . N IE R S , * L . W. BR ÜG GE MA NN , G. L . VAN SLU IS , * § R . D . L I U , H. H . VERSTEEG ,– H. R . B Ü LLER , C. J . F . VAN N OORDEN,§ P . H . RE ITSMA,– C. A . SPEK and D . J . R IC HEL* *Department of Medical Oncology, Academic Medical Centre, University of Amsterdam, Amsterdam; Centre for Experimental and Molecular Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam; Department of Vascular Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam; §Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam; and –Einthoven Laboratory for Experimental Vascular Research, Leiden University Medical Centre, Leiden, the Netherlands

Prophylactic plasma levels of the low molecular weight heparin nadroparin does not affect colon cancer tumor development in mouse liver

a Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands b Department of Medical Oncology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands d Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands