Abstract B54: Location of tumor burden influences tumor and vascular architecture, necrosis, and nanoparticle delivery
Authors: Kalra Jessica , Baker Jennifer , Kyle Alastair , Minchinton Andrew , Bally Marcel 
Published: May 2016

The gene and protein expression profile of a metastatic lesion can vary from the primary tumor. As a result, behavior of a secondary lesion can differ in the context of drug uptake and sensitivity as compared to the parent tumor. A better understanding of phenotypic variations that arise as a result of location of disease burden may be able to shed light on why widely used subcutaneous and orthotopic preclinical models fail to predict clinical success of drug candidates evaluated in the metastatic setting. These data can be used as a guide for preclinical study design in drug discovery. Our objective was to examine metastatic lesions from a wide range of organs in order to establish patterns of microarchitecture that may influence drug biodistribution and therefore drug efficacy in vivo. We hypothesize that metastatic lesions are phenotypically unique tumors with heterogeneous microenvironments which results in variable drug delivery and therefore drug efficacy. The investigations described compare tumor and vascular architecture of an orthotopic tumor with metastatic lesions. To study this, female NCr nude mice were inoculated with Her2/neu positive human breast cancer cells (JIMT-1) transfected with a fluorescence protein (mkate). Animals were inoculated either in the mammary fat pad (o.t.) to replicate a primary tumor, or directly into the left ventricle (i.c.) to establish systemic disease. Tumor development was monitored using in vivo fluorescence imaging (IVFI). Once metastases were established, animals were dosed with a custom formulated fluorescent labelled liposome (LipoDiR). The distribution of LipoDiR was imaged using IVFI. Subsequently, animals were sacrificed, mammary fat pad tumors and organs with metastatic lesions were excised and imaged ex vivo for mkate and LipoDiR. Tumors within organ tissues were further analyzed via multiplex immunohistochemical staining. Tumor sections were stained with antibodies against Her2/neu, smooth muscle actin (SMA), Collagen IV (CIV), a blood vessel marker (CD31), and a marker of hypoxia (CAIX) in order to examine the variability of tumor and vascular architecture. JIMT-1mKate cells proved to be a valuable Her2/neu positive cell line with the ability to form consistent systemic disease in the lung, liver, brain, kidney, ovaries, and adrenal glands after i.c. inoculation. LipoDiR, was successfully used to image distribution of nanoparticles in vivo, ex vivo and in tissue sections at the microscopic level. Although LipoDiR pharmacokinetics were comparable between the two models (o.t. and i.c.), orthotopic tumors showed a significant enhanced permeability and retention (EPR) effect while most organ tumors were unable to accumulate drug over a 24 hours. When comparing the tumors collected from the mammary fat pad to disseminated tumors, vessel density (CD31), vessel maturity (CIV), vessel perfusion (DiR), hypoxia (CAIX) and tumor marker expression (Her2/neu) were highly variable. Interestingly, tumors seeded within an organ did not take on the organ architecture, nor did they appear similar to the primary tumor. Rather, metastatic lesions showed considerable variability suggesting that each secondary tumor is a distinct disease entity. Our data suggests that more heterogeneity in tumor architecture and protein expression exists in metastatic lesions than has been previously appreciated. This variability is fairly profound even between tumors found in the same organ and leads to a significant impact on nanoparticle distribution. These studies highlight a need to investigate candidate drugs in multiple disease models that recapitulate the most aggressive disease. Not only will this improve the ability of preclinical studies to predict clinical success of experimental drugs, it will also help to concentrate research efforts on those drugs with the ability to engender better outcomes for patients with the most refractory disease.

Citation Format: Jessica Kalra, Jennifer Baker, Alastair Kyle, Andrew Minchinton, Marcel Bally. Location of tumor burden influences tumor and vascular architecture, necrosis, and nanoparticle delivery. [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 B54.