Abstract One of the biggest challenges in endoscopic therapeutic approach for early colorectal cancer with submucosal invasion is how to treat regional lymph nodes (LNs) possibly metastasized. The rate of LN metastasis in submucosal invasive colorectal cancer are estimated approximately 10%. Since endoscopic treatment can't clean LNs outside the bowel physically, open or laparoscopic surgical treatment is still gold standard for submucosal invasive colorectal cancer. We previously reported that in an orthotopic rectal tumor mouse model, a telomerase-dependent tumor killing adenoviral agent (OBP-301) spreads to the regional LNs, selectively replicates in cancer cells in metastasized LNs, and inhibits LN metastasis, when administered into the primary advanced rectal tumor. In this study we evaluated the effectiveness of novel less invasive treatment against submucosal invasive colorectal cancer, using local tumor resection such as endoscopic submucosal dissection (ESD) and OBP-301. HCT-116-GFP human colon cancer cells were orthotopically implanted into the submucosal layer of nude mouse rectum with resultant submucosal invasive early rectal tumor. LN metastasis was observed in 78.5% of mice as early as 7 days after cancer cell inoculation, and the rectal tumor of mice with LN metastasis got locally excised with prior peritumoral injection of OBP-301 solution, mimicking ESD technique. Seven days after tumor resection, treatment of OBP-301 showed a significant inhibition in LN metastasis status compared to control group. Moreover, LN metastatic recurrence was not detected in OBP-301 treated group even 4 weeks after tumor resection. The combination of ESD and OBP-301 has promising potential to take place of the conventional surgical approach in treating submucosal invasive colorectal cancer. Citation Format: Satoru Kikuchi, Hiroyuki Kishimoto, Hiroshi Tazawa, Yuuri Hashimoto, Futoshi Uno, Masahiko Nishizaki, Shunsuke Kagawa, Yasuo Urata, Robert M. Hoffman, Toshiyoshi Fujiwara. Novel strategy for eradicating lymph node metastasis of early-stage colorectal cancers using telomerase-dependent replicating adenoviral agent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1570. doi:10.1158/1538-7445.AM2013-1570