Abstract 403: Acquired resistance to pegylated liposomal doxorubicin
Authors: Kai Megumi , Tanei Tomonori , Liu Yan Ting , Saito Yuki , Ferrari Mauro , Yokoi Kenji 
Published: August 2015

Abstract
Background
Pegylated liposomal doxorubicin (PLD) is advantageous over doxorubicin because of its preferential accumulation to tumor through their leaky vessel walls. However, the development of acquired resistant to PLD often results in insufficient clinical outcome. To date, the mechanisms of acquired resistance to PLD have not yet been explored. While free chemotherapeutics can penetrate and diffuse across biological barriers, such as vessel walls including endothelial cells and basement membrane (composed mainly of type IV collagen) which provide a formidable barrier to PLD. We have previously shown that Matrix metallopeptidase 9 (MMP-9), the enzyme to degrade type IV collagen, and its inhibitor, metallopeptidase inhibitor 1 (TIMP-1) play a crucial role to control vessel leakiness. Our objective is to seek the mechanisms of acquired resistant to PLD by comparing the contexts of biological barriers in tumor microenvironment.
Materials/Methods
4T1 murine breast cancer cells were inoculated into mfp of BALB/c mice. Mice were treated with PLD (6.5 mg/kg) through intravenous injection when tumor volumes had reached a size of approximately 100-200 mm3. The tumors after the initial treatment and tumors which progressed after second and third treatments were subjected as naïve (control) and resistant tumors, respectively. Mice were sacrificed after 24 hours of PLD injection. Serum levels of MMP-9 and TIMP-1 were measured by ELISA. Immunofluorescence (IF) staining was performed to evaluate the expression of efflux pump associated p-glycoprotein (P-gp), MMP-9, endothelial cells and type IV collagen. Positive area was quantified by ImageJ. Statistical differences were tested by t-test. P-value &lt; 0.05 was considered to be statistically significant.
Results
The tumor volume in all the mice was reduced after initial PLD injection. Then, tumor started to grow again after 30 days and became resistant to the second/third injection of PLD. Although the expression of P-gp was not increased in the resistant tumors, PLD accumulation was significantly less compared to control tumor (p = 0.016). Endothelial cells in the resistant tumors were covered more tightly by type IV collagen as to those in control tumors (p&lt; 0.01), which indicates a reduction in vasculature permeability. MMP-9 expression was reduced in the resistant tumors, suggesting less degradation of basement membranes. The ratio of MMP-9/TIMP-1 in the serum of resistant tumor bearing mice decreased significantly as compared to that in the control tumor mice (p&lt;0.01).
Conclusion
The acquired resistance to PLD can be developed by reducing drug accumulation to tumor as a result of changing in microenvironment such as vasculatures and basement membranes, which is controlled by MMP-9/TIMP-1. Furthermore, serum levels of MMP-9/TIMP-1 can be used to monitor the development of resistance.
Citation Format: Megumi Kai, Tomonori Tanei, Yan Ting Liu, Yuki Saito, Mauro Ferrari, Kenji Yokoi. Acquired resistance to pegylated liposomal doxorubicin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 403. doi:10.1158/1538-7445.AM2015-403