Myofibroblasts Enhance Tumor Growth in a Novel Mouse Model of Colorectal Cancer

Abstract

BackgroundCommunication between colorectal cancer and stromal cells alters the tumor microenvironment to regulate locoregional disease and cancer progression. However, colon cancer–stromal cell interactions are difficult to study in vivo. Limitations of existing animal models include the use of immunocompromised mice, the inability to genetically modify a cell population in a single organ system, or a lack of anatomic context. Our goal was to develop a novel mouse model of colorectal cancer that is capable of studying tumor-stromal cell interactions in the native colon of immune-competent mice. MethodsPrimary mouse myofibroblasts were isolated from the colon of C57BL/6 mice and were grown in cell culture. Genetically defined (ApcΔ/Δ; Kras G12D/+; Trp53Δ/Δ) primary mouse colon cancer cells were suspended in serum-free media (20 μL) at varying concentrations (5 × 103 to 4 × 104 cells) either alone or in combination with syngeneic myofibroblasts (2 × 105 cells). After isoflurane anesthesia, a colonoscopy was performed on immune-competent 8- to 10-week-old C57BL/6 mice with endoscopic microinjection of the cell suspension into the submucosal space of the colon wall utilizing a small animal colonoscope. Surveillance endoscopy was used to assess for tumor growth, along with histologic analysis. Tumor size is presented on a grading system based on tumor diameter relative to colon circumference. ResultsA total of 33 mice were injected with a survival rate of 88% (29/33). Endoscopic microinjection of colorectal cancer cells resulted in dose-dependent tumor growth in the distal mouse colon that could be assessed endoscopically without animal sacrifice. Growth curves varied depending on the concentration of injected colorectal cancer cells, with no growth at the lowest concentration of injected cells (5 × 103 cells), progressive growth over 4 wk using 1-2 × 104 cells, while the highest colorectal cancer cell concentration (4 × 104 cells) led to larger tumors at week 1 followed by a steady decline in tumor growth over the 4-wk time period. Combined microinjection of 2 × 104 colorectal cancer cells with 2 × 105 myofibroblasts resulted in much larger tumors that persisted over the 4-wk time period and which were composed primarily of colorectal cancer cells. Immunofluorescence microscopy after coinjection of colorectal cancer cells with green fluorescent protein positive myofibroblasts confirmed that the injected myofibroblasts are present and remain viable over the 4-wk time period. ConclusionsEndoscopic submucosal microinjection of primary mouse colorectal cancer cells is feasible and leads to reliable and reproducible short-term growth of colon tumors in immune-competent mice. Coinjection of primary mouse colorectal cancer cells with syngeneic myofibroblasts leads to enhanced tumor growth. Coimplantation of colorectal cancer cells with syngeneic myofibroblasts provides a novel platform to study tumor-stromal interactions in the native colon of immune-competent mice.