Background The advent of the spatial omics era has enabled us to acquire vast amounts of transcriptomics and proteomics data. The abundance of a transcript, however, does not necessarily display strong correlation with protein expression levels, so a holistic approach to sample analysis is pivotal. For the first time, we hereby combined two state-of-the art technologies for detecting RNA expression and protein interactions to better understand tissue phenotypes from a functional perspective. HCR™Pro RNA-FISH (Molecular Instruments) enables high-resolution, multiplex detection of transcripts without requiring harsh protease-digestion steps. In situ proximity ligation assay (isPLA, Navinci) informs about protein functional states by sensitively detecting protein-protein interactions. Both technologies can be fully and robustly automated on the Leica BOND RX stainer. As a proof of principle, we focused on the PD1/PD-L1 inhibitory pathway, detecting the PD1 and PD-L1 transcripts, as well as the interaction between them on a protein level.

Methods Human tonsil FFPE tissue sections, which are well known to be positive for PD1 and PD-L1 expression, were used for the combined HCR™Pro RNA-FISH/isPLA staining on the Leica BOND RX stainer. In brief, after standard antigen retrieval, the HCR™Pro RNA-FISH steps were performed to detect PD1 (AlexaFluor 488) and PD-L1 (AlexaFluor 555) transcripts via tyramide signal amplification (TSA). Next, to remove HRP, another round of antigen retrieval was performed, followed by isPLA with the Naveni® PD1/PD-L1 BOND RX HRP kit to detect the PD1/PD-L1 interaction. The isPLA signal was additionally amplified by TSA (Atto647N). Slides were imaged on the Olympus V200 automated slide scanner.

Results Due to the conserved spatial aspect, it was possible to visualize the levels of RNA expression and localization, as well as the location and presence of protein interactions. Correlations or discrepancies between transcripts and functionally active proteins can be observed in situ, providing a unique insight on immune and tumor biology.

Conclusions Combining the HCR™Pro and isPLA methods on a single slide is a novel development that provides possibilities for deeper and better understanding of the tissue milieu. Additionally, recent data (Strell C. et al, unpublished) demonstrated that even when PD1 and PD-L1 are expressed in non-small cell lung cancer patient samples, the PD1/PD-L1 pathway is active in only about half of the patients, as determined by isPLA, and highly correlates with positive outcomes from immune checkpoint therapy. This underscores the importance of detecting PD1 and PD-L1 on several levels and understanding their dynamics to better stratify patients.