Dog osteogenic sarcoma microvasculature observed with a Spälteholz technique

A well-distributed, patent microvascular network is essential for adequate, uniform delivery of chemotherapy into solid tumors. This network has not been evaluated in osteogenic sarcoma. Spälteholz tissue clarification was used to observe the microvasculature of canine humeri bearing osteogenic sarcoma. Freshly amputated limbs, obtained from therapeutic amputation, were infused with a micron-sized carbon particle solution, frozen, and then cut into sagittal and axial 0.5-mm thick sections. They were photographed, then radiographed using high resolution Faxitron xray, chemically treated to clarify the tissue, and then rephotographed. Microvasculature was identified by the localization of carbon particles, which were unaffected by the clarification process, within the clarified sections. Clarified section photographs were digitized to gray scale levels and analyzed using IMAGE software; levels are directly related to capillary density. Faxitron and original images were registered to the clarified images to identify tissue regions. Multiple regions of interest from normal muscle, fat, bone, and tumor regions were selected and averaged. The microvasculature of the tumor was inhomogeneous, whereas its density was considerably lower than normal adjacent muscle and bone (range, 56-72% lower). These findings suggest that insufficient microvascular density and distribution may provide additional explanation for the poor response of solid tumors to chemotherapy and radiation therapy.