Abstract 3702: Unraveling the mechanism of action for research of cancer therapies using AVITI24

Abstract For decades, the mechanism of action towards research of cancer therapies have been deciphered by the isolated analyses of transcriptomics, proteomics, or cell morphology to resolve heterogeneous responses in a series of assays requiring multiple biological samples which limit the ability to capture the full picture of the biological response. Integrating these readouts through multiplexed analysis enables a comprehensive understanding of how gene expression relates to functional outcomes and phenotypic effects, offering a holistic view for research of cellular processes impacted by treatment. Tyrosine kinase inhibitors (TKIs) have significantly advanced the treatment of non-small cell lung cancer (NSCLC) yet achieving consistent and durable patient responses remains a challenge. In this research study, we utilized AVITI24 Teton CytoProfiling, a platform that enables spatial co-detection of RNA, protein, phospho-proteins, and cell morphology at subcellular resolution within a single biological sample. This approach allowed us to resolve complex systemic responses to TKIs in three NSCLC cell lines. Specifically, three NSCLC cell lines were treated with Gefitinib or Osimertinib to investigate therapy-induced molecular and morphological changes in both TKI-sensitive and TKI-resistant cells. Single-cell analysis uncovered significant heterogeneity in RNA and protein expression, with distinct patterns emerging between drug-sensitive and drug-resistant cells. For example, in Gefitinib-treated A549 cells, we observed notable pathway inhibition, including reductions in phosY1068-EGFR (Log2FC -0.108) and phosTY-ERK12 (Log2FC -0.237). Conversely, the resistant cell line NCI-H1975 showed minimal changes alongside other distinct responses within the MAPK pathway. Drug-specific morphological changes further highlighted how these therapies influence cellular architecture in ways that correlate with functional outcomes. And by resolving cellular heterogeneity at the single-cell level, these insights—often obscured by bulk analyses—shed light on the intricate mechanisms of both therapeutic effects and resistance development. Our findings demonstrate the potential of multi-omics platforms like AVITI24 Teton CytoProfiling to address critical challenges in cancer research. By consolidating transcriptomic, proteomic, and morphological data in a single assay with subcellular spatial resolution, this platform facilitates the resolution of multidimensional mechanisms underlying tumor responsiveness and drug resistance. By bridging molecular and morphology insights, our study provides a powerful framework for studying the intricate dynamics of targeted therapies in NSCLC, paving the way for innovative strategies to enhance cancer treatment and personalize patient care. Citation Format: Vivian Dien, Connor Thompson, Daniel Honigfort, Tristin Rammel, Grace Yeo, Marielle Krivit, Ramreddy Tippana, Jane Yao, Sinan Arslan, Matthew Kellinger, Amirali Kia, Michael Previte. Unraveling the mechanism of action for research of cancer therapies using AVITI24 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3702.