Single Cell-Resolution Imaging of Drug Resistance via Toxic Metal Pre-exposure in Lung Patient Tumor Microenvironment

SUMMARY Kentucky’s Appalachian population has by far the highest lung cancer incidence and mortality rates in the US, where toxic metal exposure can be an important co-morbidity. Despite improved lung cancer patient survival via targeted therapy, major challenges remain such as variable efficacy, resistance development, and severe toxicity. Metabolic heterogeneities in the Tumor MicroEnvironment (TME) are crucial contributors to these challenges. To greatly improve patient care and outcomes, there is an urgent, but unmet, need to understand how patients’ TME metabolism interacts with other unexplored factors such as toxic metal buildup to promote cancer progression and therapeutic resistance. Lack of proper cancer models to recapitulate individual patient’s TME hinders this effort. Our non-small cell lung cancer (NSCLC) patient-derived organotypic tissue cultures (PD- OTC) are unique models that retain the native 3D architecture, microenvironment, and metabolic functions of original tissues. We have mapped extensive metabolic networks and key oncometabolic proteins in 105 matched pairs of cancerous (CA)/non-cancerous (NC) lung PD-OTC using Stable Isotope-Resolved Metabolomics (SIRM) coupled with Reverse Phase Protein Array (RPPA) analyses. Using Digital Spatial profiling of transcripts (DSP- T) and proteins, we saw diverse pro- and anti-tumor TME responses of lung PD-OTC to immune activators. Harnessing Laser Ablation coupled with Inductively Coupled Plasma Mass Spectrometry revealed for the first time co-localization of chromium (Cr) and oncometabolic proteins in Appalachian NSCLC patient tumor tissues at single-cell (10 µm) resolution. Metal carcinogen Cr and co-morbidity metal lead (Pb) are known exposure agents in Appalachian Kentucky, and were shown to dysregulate cell metabolism and drug resistance to promote cancer development. We thus hypothesize that Cr/Pb pre-exposure promotes NSCLC resistance to therapy by eliciting pro-tumor/resistance events within the TME such as the IL6/STAT3 signaling pathway. We will test this hypothesis by exploring the linkage of Pb/Cr pre-exposure to oncometabolic/pro-tumor signaling/drug resistance markers in NSCLC patients’ TME. This is best achieved by developing a novel platform – ACCLAIMS (ACcumulated elements and Cyto-proteomics by Laser Ablation Imaging Mass Spectrometry) – for high-plex in situ imaging at single-cell resolution (10 µm) of various toxic, drug, and TME reporter metals in patients’ OTC or reposited tissues. We will fulfill our goals with two specific aims (SAs). SA1: To establish ACCLAIMS platform for high-plex in situ single cell-resolution imaging of Pb/Cr/Pt and pro-tumor/drug resistance protein markers in NSCLC patients’ TME. Antibodies against these markers will be tagged with lanthanides for optimal detection of cell type specific pro-tumor/Pt resistance markers along with Pb/Cr/Pt by ACCLAIMS in Pt-resistant OTC or reposited tissues. SA2: To validate the induction of pro-tumor/Pt resistance response in SA1 by Pb/Cr in NSCLC patient-derived organoids (PDO). A successful outcome will reveal a new drug resistance mechanism for cancer therapy that contributes to Appalachian health disparity. Project Number: 1R21CA307890-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Teresa Fan | Institution: UNIVERSITY OF KENTUCKY, LEXINGTON, KY | Award Amount: $395,973 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 CTH-N (56)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11282294