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

Changes in ATP assay of tumor patient-derived organoids (PDOs) under treatment with ADCs and various drug combinations

The single high-concentration ADC design was formulated to mimic the peak plasma concentration achieved after clinical administration, thereby directly reflecting the cytotoxic potency of the drug at effective concentrations. The combination therapy assessment focuses on the current trend of "ADC + immuno" strategies in oncology, providing ex vivo data to support optimization of clinical dosing regimens.

Real-time high-content imaging reveals efficient internalization of pHrodo-conjugated ADCs

Kinetic analysis of ADC internalization was performed using a high-content imaging system. Briefly, cells were incubated with pHrodo-conjugated ADCs at 37°C. Real-time fluorescence imaging was conducted at 15-minute intervals for 24 hours to track the internalization efficiency. Quantitative data regarding cellular uptake were analyzed using the integrated high-content analysis software."

Integrated analysis of ADC IC50 determination and scRNA-seq data post co-culture

We observed a wide range of sensitivities to ADC treatment across different PDO models, as reflected by varying IC50 values (ranging from X nM to Y nM). To uncover the mechanistic basis for this heterogeneity, we performed scRNA-seq on tumor cells following the co-culture and ADC treatment. Unsupervised clustering revealed distinct tumor cell subpopulations, including a cluster characterized by high proliferative signature and another marked by stress-response pathways.

Multiplex immunofluorescence (mIF) and high-sensitivity Olink proteomics for inflammatory cytokines.

To characterize the immune landscape, we performed multiplex immunofluorescence (mIF) staining on tissue sections to analyze the spatial distribution of immune cells. Additionally, high-sensitivity Olink proteomics was utilized to quantify a panel of inflammatory cytokines in the culture supernatant.

Fluorescence imaging of PDO viability and apoptosis assessment via caspase-3/7 activity in pharmaco-sensitivity and PDO-immune co-culture assays.

We utilized fluorescent viability dyes to visualize the cytotoxic effects of ADCs in co-culture. Concurrently, caspase-3/7 cleavage was detected as a marker of intrinsic apoptosis induced by the drug payload.

Cytokine secretion profiles (IL-6, TNF-α, IFN-γ) in co-culture supernatants measured by ELISA

The levels of key effector cytokines, including IL-6, TNF-α, and IFN-γ, were quantified in the supernatant of the PDO-immune co-culture system using ELISA kits。

Flow cytometric analysis of T cell activation and exhaustion markers in co-culture assays

Cells harvested from co-cultures were stained with fluorophore-conjugated antibodies against surface markers. T cell activation was defined by the upregulation of CD69 and CD25, while T cell dysfunction was characterized by the expression of inhibitory receptors such as PD-1 and TIM-3.