Study on the Mechanism of ADC Drug Evaluation Based on Immune Co-culture of Lung Cancer Organoids

A case-control study was conducted to systematically evaluate the therapeutic efficacy and underlying mechanisms of antibody-drug conjugates (ADCs) in non-small cell lung cancer (NSCLC), utilizing an integrated patient-derived organoid (PDO)-immune cell co-culture platform. Focusing on HER2-positive and TROP2-positive NSCLC cases, a comprehensive research pipeline was established, comprising three core components: the construction of a PDO-immune co-culture model, multidimensional tumor killing assessment, and mechanistic dissection of cellular internalization.

Clinically resected tumor tissues and malignant pleural effusion specimens were harvested to generate PDOs, which were rigorously validated for histological fidelity and phenotypic stability via H&E staining and TTF-1 immunohistochemistry; cases were subsequently stratified based on HER2/TROP2 expression intensity. The functional integrity of the co-culture system was confirmed through flow cytometric analysis of immune cell purity and activation status, coupled with ELISA quantification of cytokines to verify effective immune-tumor crosstalk.

Pharmacodynamic evaluations were performed using ATP-based viability assays, PDO viability imaging, and Caspase-3/7 apoptosis detection. These assays simulated clinically relevant peak plasma concentrations (C max) to directly reflect in vivo drug exposure, while also assessing the synergistic potential of "ADC + Immuno-oncology" combination strategies to optimize clinical dosing regimens. Mechanistically, pHrodo dye tracking was employed to visualize and quantify cellular internalization and phagocytosis, complemented by single-cell RNA sequencing to delineate transcriptional profiles and identify specific subpopulations sensitive to ADC therapy. Furthermore, high-sensitivity Olink proteomics and multiplex fluorescence immunohistochemistry provided "cellular-molecular-spatial" evidence of immune activation and intracellular trafficking dynamics.

Collectively, the data revealed that distinct ADC responses correlated with target expression and the immune microenvironment, precisely characterizing the molecular signatures of sensitive cell subpopulations and their enhanced endocytic activity. These findings provide critical molecular targets and a theoretical basis for patient selection and the rational design of next-generation ADC therapies in HER2/TROP2-positive lung cancer.

Inclusion criteria

• Age ≥ 18 years.
• Availability of patient-derived organoids (PDOs) with matched autologous tumor-infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from non-small cell lung cancer (NSCLC) cases.
• Patients currently undergoing or scheduled to receive Trastuzumab deruxtecan (T-DXd) therapy who meet clinical eligibility criteria.
• Provision of written informed consent.
• PDOs exhibiting strong positive HER2 and TROP2 expression by immunohistochemistry (IHC) assigned to the experimental group.
• PDOs exhibiting weak positive HER2 and TROP2 expression by IHC assigned to the negative control group.

Exclusion criteria

• PDOs derived from patients with pathologically confirmed small cell lung cancer (SCLC).
• Unavailability of matched autologous PDOs, TILs, or PBMCs.
• Presence of any contraindications to T-DXd treatment.
• Presence of other serious comorbidities resulting in an estimated survival of <3 months.
• Pregnant or breastfeeding women.