Orthotopic Microinjection of Human Colon Cancer Cells in Nude Mice Induces Tumor Foci in All Clinically Relevant Metastatic Sites María Virtudes Céspedes,

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Orthotopic Microinjection of Human Colon Cancer Cells in Nude Mice Induces Tumor Foci in All Clinically Relevant Metastatic Sites María Virtudes Céspedes, Carolina Espina, Miguel Angel García-Cabezas, Manuel Trias, Alicia Boluda, María Teresa Gómez del Pulgar, Francesc Josep Sancho, Manuel Nistal, Juan Carlos Lacal, Ramon Mangues The American Journal of Pathology Volume 170, Issue 3, Pages 1077-1085 (March 2007) DOI: 10.2353/ajpath.2007.060773 Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 1 OCMI into the cecum of immunosuppressed mice. A: View of the micropipette made from Vitrex capillaries with a 250-μm-diameter tip. B: The cecum of anesthetized nude mice is exteriorized through a laparotomy. C: Two million human colorectal cancer cells, per animal, are injected, with a 30° angle under a binocular lens (×3). D: Reddish area depicts the tissue where tumor cell suspension has been injected. The American Journal of Pathology 2007 170, 1077-1085DOI: (10.2353/ajpath.2007.060773) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 2 Site of injection and pattern of tumor growth and invasion. A: The site of injection, 1 week after the OCMI procedure, showed the tumor cells located between the mucosa and the muscularis externa layers of the cecal wall. B: Tumors grow, both tangentially and circumferentially, and protrude into the lumen of the large bowel obstructing it. C–E: Tumors derived from HCT-116 (C), SW-620 (D), or DLD-1 (E) cell lines are poorly differentiated adenocarcinomas with high cellularity, focal areas of necrosis, frequent mitoses, and highly atypical nuclei, showing invasion of all of the cecal layers. F: Tumor cells infiltrate the lymphatics of the cecal wall (white asterisk). A, C–F: H&E stains. The American Journal of Pathology 2007 170, 1077-1085DOI: (10.2353/ajpath.2007.060773) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 3 Lymphatic, hematogenous, and peritoneal tumor spread in nude mice bearing tumors derived from OCMI-implanted cell lines A: Out of the local tumor (black asterisk), tumor cells spread to the mesenteric lymph nodes (white arrow). B: Microscopically, tumor foci in the mesenteric lymph nodes showed a rim of lymphocytes (white asterisk) compressed against the capsule by tumor cells (black asterisk). C: Representative foci of hematogenous dissemination to the liver, observed in the HCT-116 cell line. D: Tumor cells at the lung were found inside peribronchial vessels. E: Tumor cells invading the muscle of the diaphragm from its peritoneal surface. B–E: H&E stains. The American Journal of Pathology 2007 170, 1077-1085DOI: (10.2353/ajpath.2007.060773) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 4 Survival curves of animals from the three studied groups. Survival time was the shortest in HCT-116 tumor-bearing mice, intermediate in SW-620 animals, and the longest in DLD-1 mice. Cumulative survival values were estimated using the Kaplan and Meier method and plotted versus time after injection. The log-rank test showed statistically significant differences in survival among groups (see also Table 2). The American Journal of Pathology 2007 170, 1077-1085DOI: (10.2353/ajpath.2007.060773) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions