124 - Multiplex Immunofluorescence, Immunohistochemical, and Mass Spectrometry Evidence for Localized Chemical Modifications in Tissue Using a Swine Model

Purpose: To investigate the effects in tissue of an exploratory new method called thermoembolization using a multimodality, multiparametric approach.

Materials and Methods: Thermoembolization was performed in 3 outbred swine (2M/1F) under an approved protocol. Via common femoral artery access, the liver was catheterized using a 5F reverse curve catheter and a 2.8F microcatheter, and a single lobar branch of the hepatic artery was treated using 500-600 µL of a 1 mol/L solution of 2-propylpentanoyl chloride in ethiodized oil. Animals were imaged and necropsied after 7 days and specimens were excised for analysis. Samples of treated tissue were formalin fixed and paraffin embedded (FFPE) for analysis. Sections were processed for H&E staining as well as chromogenic immunohistochemistry (IHC) for cleaved caspase 3 (CC3) as a marker for apoptosis. Samples were further analyzed using multiplex immunofluorescence (MIF) and for mass spectrometry imaging (MSI). MIF staining was performed using a Leica Bond RX (Leica Biosystems, Deer Park, IL, USA) with an Opal 7-color kit (Akoya Biosciences, Marlborough, MA, USA) for FOXP3, CD8, CD4, CD45, Granzyme B, and CD3 with DAPI as a nuclear stain. For MSI, FFPE sections were dewaxed and treated sequentially with matrix (9-aminoacridine) followed by trypsin and peptidyl-N-glycase using an HTX M5 robotic sprayer (HTX TECHNOLOGIES, CHAPEL HILL, NC). Imaging was performed after each enzymatic treatment using a timsTOF Pro2 mass spectrometer (Bruker Daltonics, Billerica MA) in negative ion mode.

Results: H&E sections revealed large, confluent areas of coagulative necrosis with complete loss of cytoskeletal detail. With IHC, increased staining for CC3 was observed at the margins. With all markers for IHC and MIF poor staining was noted in the regions corresponding to the coagulative necrosis on H&E. MSI molecular ion images for tryptic peptides and glycans likewise showed multiple mass markers that were present in normal tissue yet absent in the areas of coagulative necrosis. In a complementary fashion, new mass markers were identified in the treated areas but not present in unaffected tissues.

Conclusion: The combination of necrosis, apoptosis, focal absence of affinity for multiple antibodies in IHC and MIF, and the altered profiles for molecular ion images in MSI compared to normal tissue suggest that fundamental chemical modifications occurred in the affected areas. These results support further study of in vivo chemistry for clinical application.