Experimental metastasis assay

The number of tumor cells injected affects lung colony formation, as does cellular viability, the tumor embolus size and composition. The number of experimental metastases is proportional to the number ofviable emboli injected into the circulation (11, 12). However, the number of experimental metastases is also dependant on the size of tumor emboli (number of tumor cells per clump). Therefore, viability and clump (embolus) size influence the outcome of experimental metastasis assays (13). 

The mechanics for experimental metastasis assays are similar to those for the spontaneous metastasis assay. As above, volume and viscosity of the inoculum are important parameters. Volumes should never exceed 0.2 ml because plasma blood volume exceeds the normal range ( 10%) such that the distribution pattern of cells is altered. Tumor density is even more critical for intravascular injections since introduction of emboli can cause death because of vascular obstruction. Therefore, the same concentration maximums reported above are also applicable for this type of assay. 

Box GM, Eccles SA (2011) Simple experimental and spontaneous metastasis assays in mice. Methods Mol Biol 769 311-329... 

The very nature of the metastatic process dictates that the organization of this chapter differ from other chapters dealing with in vitro studies. At the outset, it is important to acknowledge that there are as many variations for how to study metastasis in vivo as there are people doing them. While the chapter will conclude with specific technical recommendations, most of the chapter will outline the theoretical considerations and rationale for experimental design and interpretation of metastasis assays. 

Experimental pulmonary metastasis was assessed by tumor cell injection into the lateral tail vein of mice. C57BL/6 mice were injected intravenously (IV) with B16-BL6 melanoma (5 X 10 ) admixed with or without chitin derivatives. The mice were killed 14 days after tumor inoculation, and the number of lung tumor colonies was counted. In a spontaneous pulmonary metastasis assay, mice were inoculated subcutaneously with B16-BL6 cells (5 X 10 ) in the right hind footpad. The primary tumors were surgically removed on day 21 after tumor inoculation. The injection of chitin derivatives was performed on various days before or after the amputation. Mice were killed 14 days after the amputation, and the number of lung tumor colonies was counted. An outline of the experiment is shown in Scheme 3. 

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