Abstract 2444: Anticancer effects of antimaia in a highly metastatic, orthotopic, immunologically competent model of breast cancer.
Authors: Yonezawa Tomohiro , Chen KaunHui E. , Duong David , Ma Lisa , Ghosh Mrinal , Kawaminami Mitsumori , Walker Ameae M. 
Published: August 2014

Abstract
Traditional testing of candidate therapeutics has examined the ability of drugs to shrink pre-formed, primary xenografts in immunocompromised animals. While shrinkage of primary tumors can be crucial for inoperable tumors, the vast majority of primary tumors are surgically excised. Moreover, patient death results from cells that escaped the primary tumor. In new environments, these metastatic escapees may respond entirely differently to the drug, especially in the presence of an intact immune system. To test a novel anti-breast cancer drug, we have therefore used a highly metastatic, syngeneic model (4T1) that develops in an immunologically and endocrinologically normal animal. The drug, for brevity named Antimaia, is ∼10kDa, readily synthesized, and designed to inhibit expression of the long form (LF) of the prolactin receptor (PRLR). Studies in vivo demonstrated inhibited expression of LF PRLR without any effect on expression of the short forms of the PRLR or the growth hormone receptor in normal liver and ovary and the primary tumor. Treatment of 4T1 cells in vitro inhibited the ability of cells within the population to form mammospheres (100/well in controls and 0/well in Antimaia-treated, when defining a mammosphere as a colony &gt;50μm) indicative of an effect on stem-like cells within the population. This anti-stem cell effect was duplicated when stem cells were identified on the basis of cell markers (Lin-CD29hiCD24+ AldH+), but stem cell elimination was still ongoing at 15 days. With the 4T1 model, orthotopic injection of 1x106 cells produces multiple, grossly-visible metastases in the lung and liver by 14 days. Antimaia treatment (100 pmoles/h/mouse via Alzet minipump) at the time of orthotopic injection reduced both gross and microscopic metastases without effect on primary tumor weight. However, histological examination of the primary tumors nevertheless showed only a rim of viable cells in the Antimaia-treated group. Morphometric analysis quantified the area of central tumor death as 3 fold that seen in the two control groups. Since in vitro stem cell death was still ongoing at 15 days, the next goal was to examine the effect of longer term treatment in vivo. To do this, it was necessary to initiate primary tumors with fewer cells to be compliant with standards of animal care. When 1x 103 cells were orthotopically injected and treatment continued for 29 days, 4 of 6 primary tumors in the Antimaia group (and 0 of 6 in the control group) showed a flattened growth curve after initial tumor formation. Micrometastasis histological analyses are ongoing, but ex vivo analysis of immune cells from the livers has shown an increase in tumor-specific IFNγ+ CD4+ cells that correlates with effects of treatment on final tumor weight. Analysis of liver enzymes at 14 and 29 days showed no evidence of toxicity. We conclude that Antimaia holds great promise as a breast cancer therapeutic. CBCRP 171B-0053.
Citation Format: Tomohiro Yonezawa, KaunHui E. Chen, David Duong, Lisa Ma, Mrinal Ghosh, Mitsumori Kawaminami, Ameae M. Walker. Anticancer effects of antimaia in a highly metastatic, orthotopic, immunologically competent model of breast cancer. [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 2444. doi:10.1158/1538-7445.AM2013-2444