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Although strong evidence is available suggesting that microenvironmental parameters play a role in lymphogenic or hematogenic metastasis, the underlying mechanisms are still unclear and further investigations of this topic are needed. For such a study however, an appropriate model of metastasis for in vivo analysis of this process would be required. An in vivo model of a solid tumor (rat DS sarcoma) has therefore been established to enable monitoring of the steps involved in tumor metastasis. Rat DS sarcoma cells were transfected with the pTracer-SV40 plasmid, containing the super-GFP and zeocin resistance genes. DS sarcoma cells showing high and stable expression of GFP (DSGFP cells) were selected by cell sorting and in vitro culturing with zeocin. To establish in vivo growth, DSGFP cells were subsequently injected intraperitoneally (i.p.) without additional selection by zeocin and GFP expression was monitored by flow cytometry. Using DSGFP ascites cells, solid tumors were implanted subcutaneously into the hind foot dorsum of rats. The expression of GFP was assayed by fluorescence microscopy. The detection of circulating DSGFP sarcoma cells in the blood was performed using the PCR technique. GFP expression in vitro was stable for more than 40 passages. Cell sorting, however, did not enable selection of a DSGFP cell population with a higher long-term stable GFP expression. After i.p. cell implantation, GFP expression in DSGFP ascites cells was maintained over at least 19 passages. Solid tumors implanted by injection of DSGFP ascites cells showed stable GFP expression. The growth rate of solid DSGFP sarcomas was slightly slower compared to that of non-transfected cell lines. The detection limit for circulating DS sarcoma cells in blood was 100 DSGFP cells/ml whole rat blood. Micrometastases in loco-regional lymph nodes, lung and liver were detectable by immunohistology and real-time PCR. This in vivo model showing stable expression of GFP could be useful for analyzing the mechanisms of metastasis, particularly where micrometastases or circulating tumor cells are to be identified.

The aim of this study was to assess the anti-tumor effect of photodynamic therapy (PDT) using a novel bacteriochlorophyll derivative, palladium-bacteriopheophorbide (TOOKAD) on tumor growth, perfusion and oxygenation. Rat DS-sarcomas were treated with either TOOKAD-PDT (2 mg/kg, i.v., immediate illumination) or one of the control treatments (sham-treatment, illumination without photosensitizer, or photosensitizer without illumination). The light source was an infrared-A irradiator fitted with appropriate filters, so that the wavelengths applied (665-800 nm) included the absorption maximum of TOOKAD at 763 nm. Tumor volume was monitored for 90 days after treatment or until a target volume (3.5 ml) was reached. TOOKAD-PDT dramatically inhibited tumor growth with 92% of tumors not reaching the target volume within the observation period. In further experiments, tumor perfusion was assessed using laser Doppler flowmetry. Upon TOOKAD-PDT treatment, a rapid, pronounced decrease in perfusion was seen, down to levels corresponding to only 3% of initial values. Tumor oxygenation monitoring revealed parallel decreases, with levels corresponding to anoxia being reached. The significant anti-tumor effects presented in this report, taken together with the chemical and pharmacokinetic properties of the novel photosensitizer TOOKAD, underline the therapeutic potential of this approach in which flow stasis and induction of anoxia are key elements.

The aim of this study was to assess pivotal factors determining the oxygenation status of malignant tumors, and, for comparison, of normal tissues. For this purpose, microvascular patterns, blood flow data, O2 consumption rates and O2 extractions were correlated to the median pO2 values of isografted rodent and xenografted human tumors, and of various normal tissues. No correlations were found between median pO2 values and vascular patterns, O2 consumption rate or blood flow rate. When O2 extraction, however, was considered (a comprehensive parameter which encompasses all of the above factors), a strong inverse correlation was found between this parameter and the median pO2 values, both for malignant tumors (r2=0.917) and normal tissues (r2=0.959). At similar O2 extraction values, the median pO2 values in the solid tumors were substantially lower than those in normal tissues, a finding which most probably reflects the chaotic microcirculation found in tumors, the loss of flow regulation which in normal tissues meets the metabolic demands of the cells, and in some cases, tumor-associated anemia.

The oxygen deficiency seen in solid tumors is predominantly caused by an insufficient O2 supply as a result of inadequate tumor perfusion. The aim of this study was to analyze whether a number of vasodilator drugs might be suitable to increase tumor perfusion and consequently improve the oxygenation status of experimental tumors. Rats with s.c. DS-sarcomas were treated with either Na+-nitroprusside (7-25 microg x min(-1) x kg(-1) BW) or nifedipine (10 microg x min(-1) x kg(-1) BW). Red blood cell (RBC) flux was assessed continuously using laser-Doppler flowmetry and mean tumor pO2 was measured polarographically using O2-sensitive catheter electrodes. Systemic application of the vasodilator drugs resulted in a dose-dependent decrease in MABP. In parallel, tumor perfusion was reduced linearly with falling MABP resulting in a decrease in RBC flux by approximately 40%. Resistance to flow did not change during the infusion indicating that these drugs have no impact on tumor vessel diameter. With decreasing tumor perfusion, tumor pO2 was reduced parallel to the MABP drop. This effect was more distinct with Na+-nitroprusside than with nifedipine due to the more pronounced fall in MABP. The vasodilator drugs studied are not suitable for dilation of tumor vessels either because the tumor vasculature lacks contractile wall elements or because the vessels are already maximally dilated.

Differentiation of a tumor plays an important role in terms of biological aggressiveness. The question arises as to whether this is reflected in differences in the metabolic and energetic status of solid tumors. The aim of this study was to analyze the influence of clonal tumor cell differentiation on the microenvironment of rat rhabdomyosarcomas. Two distinct lines of a rhabdomyosarcoma (BA-HAN-1) with different histomorphological properties were used (line F1, co-existence of mononuclear stellate cells and multinuclear myotube-like giant tumor cells; G8, polygonal, mononuclear tumor cells). Solid tumors were grown s.c. on the hind food dorsum of Lewis rats. Tumor oxygenation was measured using O2-sensitive needle electrodes. For determining tumor blood flow, the 133Xe clearance method was used. Global glucose and lactate concentrations were measured enzymatically, global ATP, ADP, and AMP were analyzed by HPLC. The regional distribution of metabolic and energetic parameters within the tumors was analyzed using quantitative bioluminescence and image analysis. Tumor growth rate was significantly different between the two lines. The volume doubling time was 2.5 days for the F1 and 3.0 days for G8 tumors. No differences in blood flow were seen between the two lines investigated, oxygenation was slightly poorer, glucose and ATP levels slightly higher, and lactate concentration somewhat lower in the F1 line as compared to the G8 line. From these differences - although marginal - it is concluded that the G8 line presumably relies on glycolysis whereas the F1 line seems to prefer oxidative glucose turnover. Despite these different metabolic profiles between the two tumor lines, the histopathology of the rhabdomyosarcomas seems to be only of limited significance for the tissue oxygenation status as already postulated for various tumors in the clinical setting.