Background The tumor microenvironment hosts a myriad of cellular interactions that influence tumor biology and patient outcomes. Multiplex immunofluorescence (mIF) provides the ability to investigate a large number of these interactions in a single tissue section, and has been shown to outperform other testing modalities for predicting response to immunotherapies.¹ Multispectral imaging (MSI) improves the capabilities of mIF by providing the ability to spectrally unmix fluorescence signals. This increases the number of markers that can be probed in the same scan and allows for separation of true immunofluorescence signals from tissue autofluorescence background. Here, we apply MSI to explore spatial interactions observed in lung cancer samples using an end-to-end translational workflow based on the Phenoptics^TM platform. The workflow includes a pre-optimized 7-color staining panel kit along with a pre-configured analysis algorithm for cell phenotyping. Using tissue microarrays (TMA), we demonstrate the heterogeneity of spatial interactions observed among different lung cancer samples and the improved sensitivity of detection afforded by unmixing multispectral scans.

Materials and Methods Formalin-fixed paraffin-embedded (FFPE) lung cancer TMA contained 120 cores (1.5 mm diameter, US Biomax, Inc., Derwood, MD). The TMA was stained using the MOTiF^TM PD-1/PD-L1 Panel: Auto Lung Cancer Kit and pre-optimized protocol for the Leica BOND RX^TM. Whole slide 7-color MOTiF multispectral scan was acquired on Vectra Polaris® using pre-defined parameters. Phenochart^TM software was used to identify cores for analysis. Scans were unmixed and analyzed with inForm® software using a pre-configured algorithm tailored to the MOTiF^TM PD-1/PD-L1 Panel kit. With this algorithm, cells are assigned phenotypes using intensity thresholds for CD8, PD-1, FoxP3, CD68, and PanCK signal levels, subject to pre-defined marker priority rules. The rules limit co-positivity to any combination of CD8, FoxP3, and PD-1, but no combinations of those markers with CD68 or PanCK, and no combination of CD68 with PanCK. When threshold levels generate excluded combinations, priority is given to calls for CD8/FoxP3/PD-1 over CD68, which in turn has priority over PanCK. To explore the dynamic range of PD-L1, it was assessed via expression level (signal intensity), not phenotyping. Spatial analyses and visualizations were performed in R ² using the phenoptr and phenoptrReports packages³, and custom scripts.

Results The pre-optimized Opal Polaris 7-Color PD-1/PD-L1 Lung Cancer Panel Kit was able to visualize the panel targets (PD-L1, PD-1, CD8, CD68, FoxP3, and Cytokeratin) across the variety of lung cancer samples in the TMA. Cell phenotyping and spatial analyses revealed core-to-core variations in cell densities and proximities among different markers. Measurement of the dynamic range of PD-L1 expression across different cores also revealed the improved sensitivity in PD-L1 detection provided by unmixing.

Conclusions The end-to-end Phenoptics staining, imaging, unmixing, and spatial analysis workflow described here provides a robust and sensitive platform for exploring the immune landscape within the tumor microenvironment.

Disclosure Information V. Goubert: None.