M. Papageorge1, T. Liu1, L. Chen1, J. Yoo1 1Tufts Medical Center,Surgery,Boston, MA, USA
Introduction: Communication between colorectal cancer cells and adjacent stromal cells alter the tumor microenvironment to regulate loco-regional disease and cancer progression. However, existing mouse models are limited in their ability to study tumor-stromal cell interactions. Additionally, they often use immunocompromised mice, or lead to tumors that do not grow in the colon. 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.
Methods: Primary mouse myofibroblasts (MFB) were isolated from the colon of C57BL/6 mice and grown in cell culture. Genetically defined (Apc-null; Kras mutant;Trp53-null) primary mouse colon cancer cells (CRC) were suspended in serum-free media (20 μL) at varying concentrations (5×103-2×104 CRC cells) either alone or in combination with syngeneic MFB (2×104 CRC cells + 2×105 MFB). Following isoflurane anesthesia, a colonoscopy was performed on immune-competent 8-10 week-old C57BL/6 mice with endoscopic microinjection of the cell suspension which also contained 20% Spot® to “tattoo” the bowel wall for subsequent evaluations. Microinjections were performed using a Karl Storz Coloview miniendoscopic system, a manufactured 33-gauge needle (Hamilton®) and a 100 μL syringe (Hamilton®). Proper injection into the colon wall was confirmed endoscopically by the development of a submucosal wheal. A repeat colonoscopy was performed weekly for 3 weeks with photo and video documentation of the endoscopic findings. Tumor size is presented as average diameter relative to colon circumference ± SD, n≥3.
Results:A total of 20 mice were injected with a survival rate of 80% (16/20). Endoscopic microinjection of CRC cells led to dose-dependent tumor growth in the distal mouse colon (Table 1) that could be assessed regularly without animal sacrifice. Microinjection of 2×104 CRC cells led to reliable (100%, 5/5 mice) tumor growth that was present at 1 week (0.05±0.06) with persistent growth at week 2 (0.11±0.01) and week 3 (0.11±0.01). Microinjection of 2×104 CRC cells with 2×105 MFB resulted in much larger tumors that persisted over the 3 week time period (0.2±0.06 at week 1, 0.19±0.06 at week 2, 0.35±0.17 at week 3).
Conclusion:Endoscopic microinjection of primary mouse CRC cells is feasible and leads to reliable and reproducible growth of colon tumors beginning 1 week post-injection. Co-injection of primary mouse CRC cells with syngeneic MFB leads to enhanced tumor growth. Co-implantation of CRC cells with syngeneic MFB provides a novel platform to study tumor-stromal interactions in the native colon of immune-competent mice.
