A Quantitative in vivo Method of Analyzing Human Tumor‐induced Angiogenesis in Mice Using Agarose Microencapsulation and Hemoglobin Enzyme‐linked Immunosorbent Assay

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Abstract
This study was conducted to develop a quantitative assay system for use in the in vivo evaluation in mice of angiogenesis induced by human tumor cells. The human epidermoid carcinoma cells, A431 cells, were cultured on microcarriers. Microcarrier‐attached A431 cells (A431‐MC) were micro‐encapsulated with agarose hydrogel to isolate them from the immune system of the C57BL/6 mice after subcutaneous dorsal midline implantation. The agarose hydrogel‐mjcroencapsulated A431 cells (Aga‐A431 cells; diameter=300 (μm) survived for at least 10 days in vitro, and the proliferation profile of the Aga‐A431 cells was indistinguishable from that of non‐microencapsulated A431 cells. The Aga‐A431 cells were subcutaneously injected into mice with an 18‐gauge needle. Ten days later, few vessels had formed at the site implanted with cell‐free agarose beads, whereas notable angiogenesis was observed at the site implanted with Aga‐A431 cells. The degree of angiogenesis was evaluated by measurement of the hemoglobin content in the implanted site using a mouse hemoglobin (mHb) enzyme‐linked immunosorbent assay (ELISA) system. This mHb‐ELISA system has the advantages of great simplicity and reproducibility. The measured mHb content of new blood vessels at the site implanted with agarose beads was in good agreement with the amount of angiogenesis observed under a stereoscopic microscope. This assay system enabled us to evaluate the angiogenesis induced by xenogeneic cells, such as human tumor cells. Thus, our novel method may be useful for the study of the angiogenic potential of various human tumor cells and in research on the anti‐angiogenic properties of various agents.