Abstract 2128: Simple Western Size (SWS) in combination with precision cut cancer tissue slices: An excellent patient-derived platform to support drug development
Authors: Grabinski Nicole , Bernoth Kristina , Piller Mirja , Petersen Moiken , Krüger Jana , Timm Olivia , Juhl Hartmut , David Kerstin A. 
Published: August 2019

The goal of personalized medicine is to provide individual patients with the most appropriate treatment. This approach strongly depends on extensive characterization of individual tumors and their sensitivity to therapeutics. We previously have shown that our drug testing platform based on Precision Cut Cancer Tissue Slices (PCCTS) is applicable to analyze individual responses of patient's tumor tissue to defined compounds such as checkpoint modulators or bispecific antibodies. PCCTS from tumors of CRC and NSCLC cases were generated using a Krumdieck Slicer and incubated in triplicates with immunomodulatory compounds such as OKT3® (Muromonab) or Nivolumab with and without autologous PBMCs. After incubation in 24-well plates, tissue slices were snap frozen and subsequently lysed for analysis of protein expression by Simple Western™ Size (SWS) analysis. In addition to the tissue slices, immune cells were collected from each well and characterized by flow cytometry. Cytokines as an important read-out for immune cell stimulation were analyzed using the Meso Scale Discovery® technology. Combining the different technologies enables a comprehensive characterization of drug effects in individual patient samples. The SWS technology by ProteinSimple™, a highly standardized capillary western blot, using minute amounts of protein (ng), has become extremely useful for the low amount of protein generated from small, 500 µm tissue slices. We developed protocols for several signaling molecules such as PD-1, PD-L1, pAkt, Akt, pErk, Erk, and pS6RP running on a Peggy Sue™ instrument. In addition, a SWS assay for the detection of cleaved caspase-3 has been applied for the detection of apoptosis. All targets have been analyzed in duplicates within one run, a very efficient procedure for the limited amount of material available. PCCTS treated with OKT3® or Nivolumab showed significant changes in the expression level of pERK, pAKT, and pS6RP in the cases analyzed. An increase of cleaved caspase-3 was detectable after treatment compared to untreated PCCTS. These data are in accordance to the down-regulation of signaling molecules. The analysis of cytokines in the supernatants using a ten-plex panel from Meso Scale Discovery® confirmed the activation of T-cells after OKT3® treatment. In all patients a significant increase of IFN gamma, IL-2, and TNF alpha was measured. Treatment with Nivolumab resulted in an increase of IFN gamma.

This model of PCCTS represents a unique opportunity to test immunemodulatory compounds in a fully human, patient-derived model that is close to the in vivo situation. In combination with sensitive methods such as SWS a detailed characterization of compound induced molecular intracellular signaling pathways is possible and enables a better understanding of the mechanism of action in the process of compound development.

Citation Format: Nicole Grabinski, Kristina Bernoth, Mirja Piller, Moiken Petersen, Jana Krüger, Olivia Timm, Hartmut Juhl, Kerstin A. David. Simple Western Size (SWS) in combination with precision cut cancer tissue slices: An excellent patient-derived platform to support drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2128.