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

Abstract

Despite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern. Colorectal cancer cases represents 15% of all cancer types. Its poor prognosis and the consequence of its metastatic spread makes colorectal cancer the second most common cause of cancer death in western countries.1 However, genetically modified mouse models of colorectal cancer are scarcely or not metastatic.2,3 Moreover, more metastatic cancer models, such as surgical orthotopic implantation (SOI), experimental or spontaneous metastasis assays, and orthotopic cell injection also show limitations. Thus, although SOI of human colorectal cancer in nude mice yields liver metastasis, 4,5 it does not generate lung metastasis, nor mesenteric or retroperitoneal lymphatic metastasis, 6 and requires the previous expansion of the tumor in subcutaneous xenografts, [7][8][9] which may alter its growth and dissemination capacities. 10 On the other hand, the experimental metastasis assay or spontaneous metastasis assay, consisting of cell injection into the tail vein or footpad, are less physiological and usually generate tumor foci only at one single site. [11][12][13][14][15][16] Moreover, injection of colorectal cancer cells in the ileocolic vein or in the apical lymphoid follicle 12,17,18 limits metastases to liver and lymphatics, varying widely in their rate.We tested whether direct orthotopic cell microinjection (OCMI), between the mucosa and the muscularis externa

Identification of MAGI1 as a tumor-suppressor protein induced by cyclooxygenase-2 inhibitors in colorectal cancer cells

“…As subcutaneous transplantation models are not well suited for studying metastasis, we interrogated the role of MAGI1 on metastasis formation using an orthotopic implantation model (Cespedes et al, 2007). This model produces metastases in almost all clinically relevant sites occurring in human patients: regional and occasionally distal lymph nodes, liver, lungs and peritoneum.…”

A novel model of distal colon cancer in athymic mice

“…Realistic models of colorectal cancer are necessary to study cancer biology and evaluate therapeutic interventions. Currently, it is the most accurate orthotopic representation of human colon cancer is via a cecal injection 6,7,8,9 . However, real-time observation and repeated sampling of implanted tumor is difficult to achieve in those model.…”

“…This use is mainly due to ideal characteristics, which include rapid tumor growth, easy handling, and the ability to accept human cells. Athymic mice have been used as an important biomedical tool 1,2,[3][4][5][6][7][8][9][10] . Athymic mice accept xenogeneic and allogeneic grafts, including those of malignant tumors, because they are immunocompromised.…”

Lymph node-independent liver metastasis in a model of metastatic colorectal cancer

“…Indeed, although these techniques have been utilized to generate mouse models of CRC that reportedly develop metastases in the intestinal lymph nodes, liver and lungs, these techniques also give rise to widespread peritoneal carcinomatosis [14][15][16][17] . It is thus highly plausible that tumour formation in these secondary sites may not be true metastases, but rather a result of peritoneal seeding.…”

“…We therefore sought to transplant a single intact Apc/Kras compound mutant donor tumour within the mucosal layer of a host wildtype C57BL/6J mouse colon, as this would faithfully represent an orthotopic primary colorectal tumour at clinical stage 0 (in situ carcinoma) with the potential to progress to stage IV disease. Several colon orthotopic transplantation techniques have been described to date, including the injection of cancer cell suspensions directly into the rectal mucosa 13 or serosal wall of the caecum 14 , the instillation of tumour cell suspensions into the colonic lumen following electrocoagulation of the mucosa to promote tumour cell uptake 15 , and the surgical implantation of intact tumour fragments onto the serosal side of the caecal wall 16,17 . One drawback of caecum implantation is the fact that tumours are implanted on the serosal side of the caecal wall, thus bypassing the requirement of primary tumour invasion through the mucosa to the serosa for metastasis to occur.…”