A microphysiological system model of therapy for liver micrometastases

Abstract

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability.

Keywords: Micrometastasis; chemotherapeutics; liver; mammary carcinoma.

Figure 1. Cellular composition of the liver

Highly specialized parenchymal and non-parenchymal cell types populate the liver. The hepatocytes form the parenchymal portion, while the non-parenchymal portion comprises multiple cellular types, predominantly liver sinusoidal endothelial, Kupffer and stellate cells.

Figure 2. LiverChip experimental schematic to probe for function and tumor outgrowth

Day 0, the LiverChip system (Zyoxel, Ltd.) is seeded with hepatocytes and non-parenchymal cells. Day 1, the medium is changed to serum free maintenance medium. Day 3, the hepatic tissue has formed and is seeded with RFP⁺ breast cancer cells (MCF-7 or MDA-MB-231). Cytochrome P450 activity is assessed on day 3 and 13. Medium was changed every 2 days and effluent samples taken routinely. To monitor the influence of circadian rhythms, sampling frequency is increased to at least 7 times per day. The hepatic niche is maintained for a minimum of 15 days. Assays to assess hepatocyte health and function, cytokine profiles as well as tissue morphology and cellular phenotype are performed on the tissue and effluent samples.

Figure 3. The LiverChip microphysiological system

A) Aerial view of the LiverChip plate. Cells are seeded into the scaffold area, fluid flows up through this region across the oxygenation channel and is then recirculated via the reservoir. B) Functional and healthy hepatic tissue is formed and maintained for 15 days in the LiverChip. i – Hepatocyte injury markers, AST and ALT, continue to decrease as tissue formation matured in the LiverChip system (n = 2 donors). Following isolation and seeding into the system, the levels are high but as the cells establish tissue they drop to near undetectable levels. ii, iii – Hepatocytes still produce albumin (day 15) and maintain cytochrome P450 activity (day 3 and 13) over the culture period (n= 2 donors). C) An array of microdispensers can be mounted over the LiverChip system providing for precise delivery of drug, hormone and nutrient boluses as small as 100 nL. D) When used in conjunction with custom-made software and electronic controllers, programmable diurnal control of drug concentrations within the bioreactor can be achieved.

Figure 4. General overview of the systematic approach for analyzing communication networks in the early micrometastatic microenvironment

The cues, signals and responses are color coded. Adapted from (74).