M. Oshi¹, M. Okano¹, A. L. Butash¹, K. Takabe^{1  1}Roswell Park Cancer Institute,Department Of Surgical Oncology,Buffalo, NY, USA

Introduction: Although targeted therapies in primary breast cancer have significantly improved the survival rate in the last two decades, the challenge to improve the survival rate in patients with metastatic breast cancer still remains. Pre-clinical models play an important role in developing treatment strategies, but proper breast cancer metastasis models have not been established due to the difficulty and complication of the procedure. We developed the “thoracotomy” method in order to establish a breast cancer lung metastasis model, which is simple and resembles human lung metastasis. 

Methods: All work was performed in female NSG or bulb/c mice of age 8-12 weeks. PDX of lung metastasis model was made from patient-derived breast tumors. PDX breast tumors that had been passaged 3 times in mammary fat pads or lung metastasis tumor generated using 4T1 cell line were used. Tumors were diced to ~1 mm3 pieces using a sharp blade. 

Results: The right middle lobe was selected as an implantation site in order to allow the tumor to invade the lung and not the chest wall. In the “thoracotomy” method, the chest wall incision was made and tumor fragments were implanted using forceps and 8-0 nylon surgical suture. Another approach was to directly inject the minced tumor tissue 1mm below the lateral pleural surface of the middle lobe using a 23G needle. The incision was closed with a 6-0 surgical suture. An intrathoracic puncture was made with a 27G needle to withdraw the remaining air from the chest cavity. After the air had been withdrawn, a completely inflated lung could be seen through the thin chest wall. In the “non-thoracotomy” method, the minced tissue was injected into the mice lung through the chest wall with a 23G needle. One hour post-surgical survival rate was only 30% after “thoracotomy” method (non-fixing suture 9/30, fixing suture 8/30) due to open pneumothorax resulting from excessive wound tension and intercostal muscle cut through. All mice after “non-thoracotomy” method survived, but implantation in the chest wall was observed in 67% (4/6) of cases and the method achieved only 50% (3/6) of the accurate transplantation into the middle lung when performed preliminarily using the cell line.  To increase the survival rate with the “thoracotomy” method, we limited the incision size <10 mm and compared the outcome with the original incision group. Limited incision “thoracotomy” could significantly increase one hour post-surgical survival to 97% (29/30) (<10 mm vs. ≥10 mm: t test P = 0.003). 

Conclusion: By simple modifications of surgical techniques, we are able to establish an orthotopic lung metastasis mice model with almost zero operative mortality. Our orthotopic thoracotomy model has the potential to be a powerful tool for preclinical studies of breast cancer patients with lung metastases.

M. Oshi¹, M. Okano¹, A. L. Butash¹, K. Takabe^{1  1}Roswell Park Cancer institute,Department Of Surgical Oncology,Buffalo, NY, USA

Introduction: Despite the fact that the 5-year survival rate for breast cancer(BC) is outstanding compared to other cancers, there are 40,000 deaths annually due to the disease in the US. The vast majority of the mortality results from distant metastasis and therefore pre-clinical models are essential for development of proper and precise treatment for each patient. Patient-derived xenograft (PDX) maintains the features of the donor tumors such as intra-tumor heterogeneity. However, the establishment of an orthotopic metastatic model is still lacking due to procedural difficulty. We demonstrate our novel methods to develop an orthotopic brain metastasis patient-derived xenograft model (PDMOX) for BC brain metastasis.

Methods: PDMOX were created using metastatic brain tumors from BC patients and implanting them in the brain of NSG female mice aged 8-12m through a frontal bone burr hole into the right caudate putamen. Tumors of ~1mm³ were implanted in 2 different forms: single solid piece or mechanically minced tissue with medium.

Results:In the “manual push” method, a minced tumor mixed with 3µl medium is instilled at a depth of 4 mm by using a 23G needle, and a single solid piece of tumor is implanted by using forceps. In the “pipette tip” method, we utilized either a pipette for minced tissue, or a Hamilton syringe with a tip for solid tissue in order to inoculate tumor at the same depth. One hour post-surgical survival after implantation of minced tumor by “manual push” method was only 37.5% (3/8), whereas 100% (30/30) of the mice inoculated with the “pipette tip” method survived. The advantage of the “pipette tip” method was to minimize mechanical forces during inoculation into the brain by using a pipette or tip as a stopper. All tumors were well engrafted in surviving mice in both methods. With the “manual push” method, more tumors formed on the brain surface rather than within the brain parenchyma when compared to the “pipette tip” method. There was a large variation in tumor growth after “manual push”(Median 20±25.0, range: 12-24d. Tumor volume: median 5.6±21.0, range 2.8-48.7mm³). Although 2 out of 3 mice that underwent the “manual push” method had sudden death, all mice that underwent the “pipette tip” method lived until the tumor grew to 125-200mm³ without neurological symptoms. There was no difference in the time of engraftment and tumor growth rate between solid piece and minced tumor tissue using the “pipette tip” method. The success rate of passage for 2^nd and 3^rd generation was 100% (26/26).

Conclusion:Various surgical techniques used to generate PDMOX BC models showed major differences in the tumors and outcomes. These novel models are expected to become powerful tools for preclinical studies in metastatic BC.