Abstract A04: The role of surgical resection of primary tumor before the appearance of circulating tumor cells based on orthotopic allograft mouse model
Authors: Lee Hak Woo , Han Airi , Yang Ban Seok , Park Jong Tae , Ahn Sung Gwe , Jeong Joon 
Published: May 2016

Breast cancer causes death not because of the primary tumor in the breast but because of metastases in distant sites that gradually cause organ dysfunction. Circulating tumor cells (CTCs) are cells that have detached from the primary tumor or metastatic tumor site and entered the peripheral circulation. Using CTC-mouse model, we tested the hypothesis that curative resection prior to CTC appearance results in repression of tumor metastasis. In method, we implanted 1 x104 GFP expressing 4T1 cells in 4th or 5th mammary fat pad of 8 week-old BALB/cAnNCrl mice (n=69). Enumeration of CTCs was performed using a FACSCalibur flow cytometry system (Becton, Dickinson and Company, Cowley, U.K.). CTCs were considered positive if the number of counted cells is more than 5. First, to test the validity of our CTC count method using FACS, we conducted cell-spiking tests. Using phosphate-buffered saline (PBS) and human blood, 50, 200, 500, 1000 4T1/GFP tumor cells were spiked. The R2 between spiked tumor cells and counted cells were 0.930 for PBS and 0.975 for human blood, respectively. Next, we tested the correlation between CTC counts and tumor volume. In the results, tumor volume and the number of CTCs increased over time and tumor volume showed statistically significant correlation with CTCs (p&lt;0.001). In most cases, CTC-positive mouse died 6 weeks after orthotropic transplantation of 4T1 cell lines. Then, to evaluate the role of surgery in early breast cancer with no CTC, we removed transplanted tumor just before CTCs appeared in circulation (n=35). The smallest size of tumor with observable CTCs was 47.28mm3. After removal of the primary tumor, we evaluated the extent of surgical procedures. If there was any suspected residual tumor tissue in operation bed, the procedure was categorized as R1 resection. If operation bed was not suspected with residual tumor tissue, the resection was categorized as R0 resection. All the experimental animal showed CTCs after primary tumor appeared (n=69). The mice showed no CTCs during follow up period (16 weeks) if R0 resection was successfully conducted (n=25). However, we could detect CTCs during follow up period if incomplete resection (R1 resection) was done (n=10) (p&lt;0.001). Tumor progression was found in the mice with the re-occurrence of CTC, and these animals expired. In summary, we developed orthotopic allograft mouse model with breast cancer and circulating tumor cells, which is most similar with human breast cancer evolution. We provide evidence that R0 resection prior to CTC occurrence can inhibit tumor progression and metastasis. These results showed biologic rationale that the patients with no detectable CTC confirmed by an accurate CTC-count assay may not need any kinds of adjuvant treatment.

Citation Format: Hak Woo Lee, Airi Han, Ban Seok Yang, Jong Tae Park, Sung Gwe Ahn, Joon Jeong. The role of surgical resection of primary tumor before the appearance of circulating tumor cells based on orthotopic allograft mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A04.