Colorectal cancer is a common malignant tumor that occurs in the colon, predominantly in the junction between the rectum and sigmoid colon. It  ranks as the third most common gastrointestinal tumor with the highest incidence in the age group of 40 to 50 years, with a male-to-female ratio of 2-3:1. Colorectal cancer mainly comprises adenocarcinoma, mucinous adenocarcinoma, and undifferentiated carcinoma, with various morphologies including polypoid or ulcerative forms. Susceptible populations include individuals with chronic colitis, colon polyps, and obese males.

Orthotopic animal models of colorectal cancer, involving In-situ transplantation of colon cancer cells, can simulate the occurrence and development of human colon cancer to some extent, providing a valuable platform for researching the occurrence, development, biological characteristics, and treatment of human colon cancers.

Introduction to Colorectal Cancer Orthotopic Transplantation Model

Compared to heterotopic transplantation, orthotopic transplantation colorectal cancer models can better reflect the real conditions of the tumor and enhance the reliability of the tumor model. In the orthotopic or in-situ transplantation model, metastasis can be induced, indicating the interaction between tumor cells and organ-specific factors, which is associated with the development of colorectal cancer. In-situ transplantation can also simulate the tumor immune microenvironment. Therefore, most results based on heterotopic models need further validation through orthotopic transplantation models to assess results in a more clinically-relevant tumor microenvironment. Colorectal cancer's in-situ growth better replicates the tumor microenvironment for tumor cell growth in vivo and provides a more accurate prediction of drug efficacy for preclinical evaluations.

Exploration of Colonic In-Situ Surgical Modeling Sites

The thin and delicate colonic wall in mice makes in-situ injection surgery in the colon wall challenging. In human anatomy, the area where blood vessels, lymphatics, and nerves enter and exit the intestinal wall is referred to as the mesenteric border. Here, the intestinal wall is enclosed by two layers of peritoneum, forming the mesenteric triangle. In mouse anatomy, the equivalent of the "mesenteric border" in human anatomy is called the "intestinal mesenteric attachment site." However, in mouse anatomy, there is no concept of the "mesenteric triangle." The triangular area formed by the colonic wall and the two layers of peritoneum at the cecal mesenteric attachment site in mice is defined as the "cecal mesenteric triangle." In this region, the colonic wall lacks serosal covering, and blood vessels, lymphatics, and nerves enter and exit. An orthotopic implantation mouse model of colorectal cancer is established by surgically exposing the cecum of mice and in-situ implantation of fluorescently labeled colon cancer cells into the cecal mesenteric triangle.

The tissue within the cecal mesenteric triangle is loose, and the resulting blister formation following injection is distributed in a striated pattern along the cecal mesenteric attachment site. This formation has low tension, making it less prone to leakage. The tumor is located on one side of the cecal mesentery and is distributed along the axis of the cecum. Additionally, the cecal lumen is spacious, reducing the likelihood of intestinal obstruction in the short term.

Notably, the colonic wall at the cecal mesenteric triangle lacks serosal covering, which facilitates the growth of implanted colon cancer cells. Mesenteric arteries, veins, and lymphatic vessels in the cecal mesentery triangle enter and exit the intestinal wall, providing a rich blood supply that outflows into the portal venous system. Tumors in this area are more likely to invade mesenteric blood vessels and lymphatic vessels, leading to distant metastasis of colon cancer, with a particularly higher incidence of liver metastasis.

Tumor tissue in the colorectal cancer model can metastasize to multiple locations within the abdominal cavity, including the liver, spleen, diaphragm, and mesenteric lymph nodes. The metastatic pathways align with the characteristics of human colorectal cancer metastasis, enabling a complete simulation of the natural metastatic process of colorectal cancer.

Case Study of Colorectal Cancer Orthotopic Transplantation NKG Model

⮚ HCT116-luc cell line

Figure 1. In vivo Fluorescence Imaging, Fluorescence Intensity Changes, and Survival Curve of HCT116-luc Colorectal Cancer Cells Colon Orthotopic Transplantation Tumor NKG mouse model.

The results indicate that HCT116-luc readily forms tumors on Cyagen’s NKG mice, with the highest fluorescence signal reaching up to 1010. The maximum survival period for the mice is 27 days, with a median survival of 26 days, indicating a short survival duration. The tumor tissue metastasizes within the abdominal cavity, and after a certain period of proliferation (D25), internal calcifications occur in the tumor tissue, corresponding to a decrease in the fluorescence signal in the mice.

⮚ HCT15-luc cell line

Figure 2. In vivo fluorescent imaging of HCT15-luc colorectal cancer cells transplanted in the colon of NKG mice, fluorescence intensity change chart, and survival curve chart in mice.

The results indicate that HCT15-luc easily forms tumors on NKG, with a maximum fluorescence signal intensity reaching 108. The highest survival period for mice was 39 days, with a median survival of 35 days, indicating a relatively short survival duration, and tumor tissue metastasis occurred within the abdominal cavity.

⮚ DLD-1-luc cell line

Figure 3. In vivo fluorescent imaging of DLD-1-luc colorectal cancer cells transplanted in the colon of NKG mice, fluorescence intensity change chart, and survival curve chart.

The results indicate that DLD-1-luc easily forms tumors on NKG, with a maximum fluorescence signal intensity reaching 109. The highest survival period for mice was 63 days, with a median survival of 59 days, indicating a relatively longer survival duration, and tumor tissue metastasis occurred within the abdominal cavity.

Transplantation Model Services: Orthotopic, NKG Immunodeficient Mice, and More

Cyagen’s NKG mice are a type of severe immunodeficient mouse developed by Cyagen by deleting the Il2rg gene from the NOD-Scid strain. This strain lacks mature T, B, and NK cells, has reduced complement activity, and weak macrophage phagocytosis of human cells. As a result, NKG mice can efficiently engraft human hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), patient-derived xenografts (PDX), or adult stem cells and tissues.

Cyagen has successfully transplanted over 200 human cell lines into immunodeficient mice, including subcutaneous, intravenous, heterotopic, and orthotopic/in-situ implantation sites.  This has resulted in the development of numerous orthotopic transplantation models involving various cancers such as colorectal cancer, lung cancer, and liver cancer. If you have any orthotopic or other transplant modeling needs, please feel free to contact us for a free consultation!