Abstract 2037: Spatial omic changes of malignant pleural mesothelioma following treatment using tumor-treating fields
Authors: Lou Emil , Ladner Katherine , Wainer-Katsir Kerem , Deniz Karina , Porat Yaara , Brant Boris , Davidi Shiri , Padmanabhan Yuki , Wong Phillip , Nain Amrinder , Steer Clifford J. , Giladi Moshe 
Published: June 2022

Abstract
Background: Application of alternating-electric fields as cancer-directed therapy known as Tumor-Treating Fields (TTFields) has been shown to be effective by exerting dipole alignment forces on polar microtubule subunits and dielectrophoretic forces in the cytokinetic furrow. This effect results in disruption of mitosis at the cellular level, and at the clinical level with significantly prolonged overall survival of patients with glioblastoma and malignant pleural mesothelioma (MPM). The molecular alterations that occur at the genomic and transcriptomic levels following TTFields treatment are unknown. We applied a spatial omics approach to elucidate spatial intratumoral effects of TTFields at the molecular level in regions of interest (ROI) in a mouse model of MPM.
Methods: Eight Balb/C mice were injected with AB1 MPM cells until sizable tumors were observed (2-3 weeks). TTFields were applied, with heat sham used as a negative control. After 14 days (6 days of treatment, 2 days of rest, then 6 more days of treatment) tumors were resected, fixed and paraffin embedded. Following the Nanostring GeoMx protocol, thin sections of all eight tumors were placed on a slide, and incubated with both Ki-67 antibodies, and a GeoMx Mouse Cancer Transcriptome Atlas RNA probe set. Using Ki-67 staining as a guide, 12 ROIs were selected across each tumor to capture intratumoral heterogeneity, including the core and periphery of each tumor The DSP barcoded RNA probes were cleaved, sequenced and analyzed. We compared differential expression of subset gene classes of TTFields vs heat sham treated tumors; results were further stratified into Ki-67 high and low subsets for each sample. Results were assessed by Gene Set Enrichment.
Results: Sham-treated tumors grew to over 300 mm3; TTFields-treated tumors averaged 100 mm3, confirming anti-tumor effect. Gene Set Enrichment Analysis uncovered upregulation of genes associated with interferon-alpha and -gamma responses in TTFields-treated tumors, which also displayed downregulation of pathway components associated with glycolysis, mTOR signaling, oxidative phosphorylation, cell invasion, hypoxia, and TNF-alpha signaling. Spatial analysis detected a heterogeneous response and differential expression between different portions of tumors.
Conclusions: TTFields application induces clear patterns of differential expression in the transcriptome of treated tumors, including an increasingly immune-stimulated tumor microenvironment that also affects molecular pathways critical to cellular proliferation and invasion. These findings point the way toward improved understanding of timing and sequences of TTFields in relation to systemic cytotoxic, targeted, and immune modulation forms of therapeutic strategies. Our finding of upregulated immune response implicates TTFields as a potential synergistic tactic when coupled with immunotherapeutic approaches.
Citation Format: Emil Lou, Katherine Ladner, Kerem Wainer-Katsir, Karina Deniz, Yaara Porat, Boris Brant, Shiri Davidi, Yuki Padmanabhan, Phillip Wong, Amrinder Nain, Clifford J. Steer, Moshe Giladi. Spatial omic changes of malignant pleural mesothelioma following treatment using tumor-treating fields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2037.