Nanoscale spatial profiling of DLL3, SEZ6, and B7-H3 in small cell lung cancer using a rapid expansion microscopy assay.

Abstract

e15194 Background: Small cell lung cancer (SCLC) remains an aggressive malignancy with limited therapeutic options. Recent advances have expanded the treatment landscape beyond chemotherapy and immune checkpoint inhibitors, most notably with the DLL3-directed bispecific T-cell engager tarlatamab, which has demonstrated clinically meaningful activity in relapsed disease and is now moving into earlier-line trials. In parallel, antibody–drug conjugates (ADCs) targeting SEZ6 and B7-H3 (CD276) have shown promising early clinical signals. For these emerging targets, both expression level and subcellular localization may influence therapeutic efficacy, particularly for ADC payload delivery and T-cell engagement. However, SCLC diagnostic specimens are frequently limited, and conventional immunohistochemistry lacks the multiplexing capacity and spatial resolution needed to extract this information. We evaluated a rapid, clinically optimized expansion microscopy (ExM) approach to enable multiplexed nanoscale profiling from scarce SCLC tissue. Methods: We applied a streamlined Magnify ExM protocol with a total turnaround time of approximately 6 hours, designed for compatibility with routine pathology workflows. Key steps were partially automated using an Opentrons Flex platform to improve reproducibility. Formalin-fixed paraffin-embedded (FFPE) SCLC tissues were physically expanded to enable super-resolution imaging on standard diffraction-limited microscopes. To allow simultaneous tri-marker analysis on limited tissue, we used Proteintech FLEXABLE 2.0 antibody labeling kits, enabling concurrent staining with three rabbit primary antibodies (CD276, SEZ6, DLL3) on the same tissue section without species cross-reactivity. Tissue microarrays from 24 SCLC patients (ages 28–75; stages I–IIIA) were analyzed. Results: Magnify ExM preserved tissue architecture and protein integrity while achieving effective spatial resolution of ~60–70 nm. Multiplexed imaging revealed marked inter- and intra-patient heterogeneity that was not detectable by conventional methods. Distinct nanoscale clustering patterns of B7-H3 and SEZ6 were observed, with potential implications for ADC internalization, while DLL3 demonstrated variable surface-to-Golgi localization ratios that may differentiate suitability and response for ADCs versus T-cell engagers. FLEXABLE 2.0 enabled dense multiplexing on rare biopsies, substantially increasing data yield per specimen. Conclusions: This rapid Magnify-based assay enables clinically feasible, nanoscale spatial profiling of emerging SCLC targets from limited tissue. By resolving subcellular organization of CD276, SEZ6, and DLL3, this approach provides a path toward potentially more informed patient stratification for ADC- and bispecific-based therapies while maximizing diagnostic value from scarce clinical specimens.