Quantification of longitudinal tissue pO(2) gradients in window chamber tumours: impact on tumour hypoxia

We previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surfac e, and that the pO(2) of tumour arterioles is lower than in comparable arte rioles of normal tissues. On average, the vascular pO(2) in vessels of the upper surface of these tumours is lower than the pO(2) of vessels on the fa scial side, suggesting that there may be steep vascular longitudinal gradie nts (defined as the decline in Vascular pO(2) along the afferent path of bl ood flow) that contribute to vascular hypoxia on the upper surface of the t umours, However. we have not previously measured tissue pO(2) on both surfa ces of these chambers in the same tumour. In this report, we investigated t he hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO(2) sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap windo w chambers. Fischer-344 rats had dorsal flap window chambers implanted in t he skin fold with simultaneous transplantation of the R3230AC tumour. Tumou rs were studied at 9-11 days after transplantation, at a diameter of 3-4 mm ; the tissue thickness was 200 mu m. For magnetic resonance microscopic ima ging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was inject ed i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 mu m. Intravital microscopy was used to visualize the position and number of arterioles ent ering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO,, Blue and green light excitations o f the upper and lower surfaces of window chambers were made (penetration de pth of light similar to 50 vs >200 mu m respectively). Arteriolar input int o window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green ligh t excitation (P < 0.03 for 10th and 25th percentiles and for per cent pixel s < 10 mmHg), In contrast, illumination of the fascial surface with blue li ght indicated less hypoxia compared with illumination of the tumour surface (P < 0.05 for 10th and 25th percentiles and for per cent pixels < 10 mmHg) . There was no significant difference in pO(2) distributions for blue and g reen light excitation from the fascial surface nor for green light excitati on when viewed from either surface, The PLI data demonstrates that the uppe r surface of the tumour is more hypoxic because blue light excitation yield s lower pO(2) Values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial sur face showed higher pO(2) distributions compared with the tumour surface. Th ese results suggest that there are steep longitudinal gradients in Vascular pO(2) in this tumour model that are created by the limited number and orie ntation of the arterioles. This contributes to tumour hypoxia. Arteriolar s upply is often limited in other tumours as well, suggesting that this may r epresent another cause for tumour hypoxia, This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia i n tumours.