Mass Spectrometry imaging in combination with Immunohistochemistry allows for identification of signaling and regulatory biomolecules in a Woodchuck HCC tumor model following Transarterial Embolization with Caffeic Acid.

Previous in-vitro and in-vivo studies in the rat and woodchuck HCC models have demonstrated transarterial chemoembolization with caffeic acid (CA-TACE), a monocarboxylate transport inhibitor, to provide improved tumor regression compared to bland embolization alone. However, there are minimal in-vivo mechanistic studies to elucidate the pathways involved that result in this improved outcome. Herein, we describe a novel use of mass spectrometry imaging (MSI) in combination with immunohistochemistry (IHC), to spatially map the pathways regulated by CA-TACE in a woodchuck model of HCC.

Materials and Methods: Woodchucks with HCC resulting from hepatitis infection were embolized to angiographic stasis through a microcatheter using caffeic acid (CA) and lipiodol. A subset of animals was injected with F18-Edaravone, a novel PET radiotracer used to localize reactive oxygen species (ROS) for in-vivo imaging. A subset of animals was euthanized at 0, 1, 4 and 24 hours post-embolization, liver harvested and tumors flash frozen. Liver samples were cryosectioned at 10 uM. MSI data were acquired using a Bruker Solari 2xR FT-ICR mass spectrometer equipped with a dual ESI/MALDI ion source and Smartbeam II Nd:YAG (355 nm) laser. The slides were stained with H&E. Data were processed using FlexImaging and the SCiLs lab software. The ion maps of CA and numerous expected byproducts of ROS were directly overlaid with their corresponding H&E-stained sections for data analysis and co-registration of drug distribution with pathology. IHC was carried out using standard protocols.

Results:

Data presented offers insight into the pathology-specific accumulation of CA in relation to lipids and metabolites involved in cell regulation and signaling. Using multivariate and Receiver Operator Curve-Area Under Curve analysis, we identified marked differences in ion distribution involved in ROS (GSH:GSSH ratios), mitochondrial function (cardiolipins), cell signaling (bile acids), and metabolism (glycolytic metabolites) when comparing non-embolized to embolized liver tissue and accounting for CA distribution. By correlating MSI data with IHC on adjacent sections, we identified CA-TACE driven pathway alterations that corresponded to tumor cell proliferation indexing, ROS and apoptosis.

 

Conclusion:

MSI in combination with IHC offers a unique method to measure the pathology-specific accumulation of regulatory and signaling biomolecules in the tumor microenvironment. Results identified novel mechanistic information of CA-TACE efficacy that will further aid in the development of new treatments for HCC.