Inter-Metastatic Heterogeneity of Tumor Marker Expression and Microenvironment Architecture in a Preclinical Cancer Model

Abstract

Background: Preclinical drug development studies rarely consider the impact of a candidate drug on established metastatic disease. This may explain why agents that are successful in subcutaneous and even orthotopic preclinical models often fail to demonstrate efficacy in clinical trials. It is reasonable to anticipate that sites of metastasis will be phenotypically unique, as each tumor will have evolved heterogeneously with respect to gene expression as well as the associated phenotypic outcome of that expression. The objective for the studies described here was to gain an understanding of the tumor heterogeneity that exists in established metastatic disease and use this information to define a preclinical model that is more predictive of treatment outcome when testing novel drug candidates clinically. Methods: Female NCr nude mice were inoculated with fluorescent (mKate), Her2/neu-positive human breast cancer cells (JIMT-mKate), either in the mammary fat pad (orthotopic; OT) to replicate a primary tumor, or directly into the left ventricle (intracardiac; IC), where cells eventually localize in multiple sites to create a model of established metastasis. Tumor development was monitored by in vivo fluorescence imaging (IVFI). Subsequently, animals were sacrificed, and tumor tissues were isolated and imaged ex vivo. Tumors within organ tissues were further analyzed via multiplex immunohistochemistry (mIHC) for Her2/neu expression, blood vessels (CD31), as well as a nuclear marker (Hoechst) and fluorescence (mKate) expressed by the tumor cells. Results: Following IC injection, JIMT-1mKate cells consistently formed tumors in the lung, liver, brain, kidney, ovaries, and adrenal glands. Disseminated tumors were highly variable when assessing vessel density (CD31) and tumor marker expression (mkate, Her2/neu). Interestingly, tumors which developed within an organ did not adopt a vessel microarchitecture that mimicked the organ where growth occurred, nor did the vessel microarchitecture appear comparable to the primary tumor. Rather, metastatic lesions showed considerable variability, suggesting that each secondary tumor is a distinct disease entity from a microenvironmental perspective. Conclusions: The data indicate that more phenotypic heterogeneity in the tumor microenvironment exists in models of metastatic disease than has been previously appreciated, and this heterogeneity may better reflect the metastatic cancer in patients typically enrolled in early-stage Phase I/II clinical trials. Similar to the suggestion of others in the past, the use of models of established metastasis preclinically should be required as part of the anticancer drug candidate development process, and this may be particularly important for targeted therapeutics and/or nanotherapeutics.

Accumulation of liposomes in metastatic tumor sites is not necessary for anti-cancer drug efficacy

“…To measure the distribution of DiR labelled liposomes in organs, the lung, liver, kidney, adrenal glands, ovaries and brain were harvested from each animal (n = 6) and imaged ex vivo or prepared for LSC of [3H]–CHE (see Methods). The selection of organs examined was based on previous studies which showed that tumors consistently developed in these sites following intracardiac injection of JIMT-1 cells [ 1 ]. The AUC 0–48 h was calculated for each organ collected from animals injected with the [3H]–CHE/DiR labelled liposomes (Fig.…”

“…JIMT-1 mkate cells were injected into the left ventricle of animals to seed tumors systemically. Animals were subsequently imaged twice weekly using IVFI to assess tumor development throughout the body (see Methods and reference 1; [ 1 ]). Similar to previous studies, mice consistently developed JIMT-1 mkate tumors in the lung (6 animals), liver (6), kidney (3), ovary (6), adrenal gland (6) and brain (4).…”

“…NCr nude mice were selected because the tumor models derived following injection of JIMT-1 human breast cancer cells require the use of an immune compromised mouse. The JIMT-1 cells consistently develop into tumors following IC and OT inoculation in this strain of mice [ 1 ]. For comparison purposes studies completed in tumor free mice were also done using the NCr mice.…”

“…Previously, our group assessed the heterogeneity of Her2/neu expression and the vasculature within the tumor microenvironment (TME) of tumors that developed at multiple sites following intracardiac injection of Her2/neu positive, trastuzumab-insensitive JIMT-1 cells [ 1 ]. When these cells are implanted, they establish tumors that recapitulate some of the intra- and inter-tumor heterogeneity that is observed clinically.…”

A Two-Photon Microimaging-Microdevice System for Four-Dimensional Imaging of Local Drug Delivery in Tissues

“…While the microimaging system can visualize the tissue response in a 2D manner, it integrates the fluorescence signals accumulated over the penetration depth of around 300 µm, and thus lacks three-dimensional (3D) imaging capabilities that are critical to the understanding of the drug-tissue interactions. Studies have shown that uneven drug exposure may significantly contribute to the resistance of cancers to chemotherapy [ 6 , 7 , 8 , 9 ]. Measurement of the drug penetration, distribution, and efficacy relies on advanced 3D microscopy techniques.…”