R. Plummer1, T. Liu1, N. Ciomek2, J. Yoo1 1Tufts Medical Center,Surgery,Boston, MA, USA 2Tufts Medical Center,Pathology,Boston, MA, USA
Introduction: The myofibroblast (MFB) is an inportant stromal cell of the GI tract that has been implicated in the pathophysiology of colorectal cancer (CRC). Angiogenin (ANG), a secreted 14-kDa member of the ribonuclease superfamily, enhances cell growth and survival. We recently reported that ANG regulates cell signaling events within the MFB, but the impact of MFB-derived ANG on CRC has not been previously studied. Our goal was to evaluate the role of MFB-derived ANG on CRC growth in a novel mouse model involving orthotopic implantation of primary CRC and MFB cells via murine colonoscopy.
Methods: After isoflurane anesthesia, a colonoscopy was performed with endoscopic microinjection of a cell suspension that contained genetically defined (ApcΔ /Δ ;Kras G12D/+;Trp53Δ /Δ ) primary mouse CRC cells (1×104-4×104) into 8-10 week-old wild-type (WT) and ANG-knockout (ANG-KO) C57BL/6 mice. In a second set of experiments, CRC cells (4×104) were injected with primary syngeneic WT or ANG-KO MFB (2×105) into WT and ANG-KO C57BL/6 mice. A follow-up colonoscopy was performed weekly for 4 weeks with photo and video documentation. Tumor size was graded based on tumor diameter relative to colon circumference (grade 1=just detectable; grade2=1/8; grade3=1/4; grade 4=1/2). Endoscopic data was correlated with anatomic/histologic findings.
Results: A total of 37 mice were injected with a survival rate of 94%. Injected CRC cells successfully implanted in colon submucosa of WT immune-competent mice and grew in a dose-dependent fashion (Week 1: 5×103 – grade 0, 1×104 – grade 1, 4×104 – grade 1.4, n=17 total) that persisted over 4 weeks. However, injected CRC cells at all concentrations failed to grow in ANG-KO mice over 4 weeks (grade 0, n=8). Compared to injection of CRC cells alone, co-injection of CRC with WT MFB in WT mice led to enhanced tumor growth (Week 1: grade 3.3, n=4). Tumor growth was unchanged following co-injection of CRC with WT MFB in ANG-KO mice (Week 1: grade 3.1, n=4). However, there was significantly diminished tumor growth following co-injection of CRC with ANG-KO MFB in ANG-KO mice (Week 1: grade 1.5, n=4).
Conclusion: Orthotopic implantation of CRC leads to dose-dependent tumor growth in WT mice but no growth in ANG-KO mice, suggesting that ANG is required for tumor growth. WT MFB enhances tumor growth when orthotopically co-injected with primary CRC in WT mice. This effect can be sustained when host ANG is absent (ANG-KO mice) but MFB-derived ANG (WT MFB) is still present. However, the loss of both host ANG (ANG-KO mice) and MFB-derived ANG (ANG-KO MFB) resulted in markedly reduced tumor formation and growth. These results suggest that MFB enhances CRC growth through a mechanism that involves MFB-derived ANG.