Quantitative angiogenesis in a syngeneic tumor spheroid model

The aim of this work was to develop a system for noninvasive, in vivo, and in situ study of tumor angiogenesis in awake mice. Tumor spheroids were prepared from Lewis lung carcinoma cells prelabeled with methylrhodamine. A transparent chamber consisting of two titanium frames was implanted into the dorsal skin of CB6 mice. One layer of the skin was removed in a 15-mm area and covered with a coverslip. A few days later, the coverslip was removed and one to three tumor spheroids (diameters, 500-900 microns) were placed over the upper tissue layer. The selected fields were recorded under trans- and epi-illumination using video microscopy. Separate fluorescence filter sets were used to visualize the FITC-labeled plasma and the rhodamine-labeled tumor spheroids. The dual labeling technique allowed precise identification of the tumors and the study of tumor and microvessel growth for up to 14 days. The tumor area and morphometric parameters of tumor vessels were measured from recorded images. After implantation, tumor cells formed well-defined tumor foci. Venular and capillary dilation and tortuosity were observed in the surrounding tissue 1-2 days after implantation, followed by the appearance of buds and sprouts. After that, vascular networks developed around and within the spheroid. During the first week, angiogenesis was very intense: at Day 6, vascular density and tumor area reached 81 and 19% of their respective maximum values. Vascular densities at Days 3, 6, 10, and 14 were 106 +/- 59, 147 +/- 62, 183 +/- 108, and 173 +/- 38 cm-1, respectively. Tumor volume increased exponentially, with a doubling time of 2 days. Similar results were obtained in nude mice. The model allows detailed repeated observations of angiogenesis and permits quantitative evaluation of tumor growth and angiogenesis in vivo. It is applicable for mechanistic studies as well as therapeutic and pharmacokinetic studies of angiostatic and cytotoxic anti-tumor agents.