Abstract 3145: TRIPODD: a novel fluorescence imaging platform for in situ quantification of drug distribution and therapeutic response

Abstract

Abstract Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells as well as signaling pathway cross talk. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this interaction network and identify therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of molecularly targeted therapeutics seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is inevitable due to acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved molecularly targeted therapeutic efficacy, we have developed a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell drug target availability and protein expression with preserved tumor spatial context. TRIPODD combines intracellular paired agent imaging (iPAI) to quantify drug target interactions and oligonucleotide conjugated antibody cyclic immunofluorescence (cyCIF) to characterize proteomic response to therapy. Importantly, iPAI and cyCIF data is collected on the same tissue sections facilitating spatial registration of the two datasets. iPAI employs spectrally distinct, fluorescently labeled targeted and untargeted drug derivatives, which correct for untargeted uptake and facilitate quantitative in situ assessment of drug target engagement. cyCIF exploits in situ hybridization of complementary oligos for biomarker labeling, while oligo modifications facilitate signal removal for sequential rounds of fluorescent tagging. cyCIF is capable of generating multi-parametric images to quantify protein expression, distribution and phosphorylation. To date, we have quantified and evaluated our iPAI toolbox using a suite of functional assays, where in vitro screening confirmed biological functionality. Subsequent in vivo validation studies successfully performed ratiometric iPAI quantification after systemic iPAI probe administration, followed by sequential cyCIF imaging on the same xenograft tissue sections for single-cell quantification of drug target availability, EGFR pathway signaling and cell viability. TRIPODD will enable an improved mechanistic understanding of clinically-relevant treatment regimens through spatially resolved single-cell quantification of drug concentration and proteomic response to identify mechanisms of resistant subclonal population outgrowths driving resistance. Citation Format: Nathan P. McMahon, Allison Solanki, Jocelyn A. Jones, Lei G. Wang, Kenneth M. Tichauer, Kimberley S. Samkoe, Summer L. Gibbs. TRIPODD: a novel fluorescence imaging platform for in situ quantification of drug distribution and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3145.