Background Inhibitory pathways such as PD1/PD-L1 can be hijacked as immune escape routes by cancer cells in the tumor microenvironment (TME). Their blockade is thus an attractive, widely applied immunotherapeutic approach, but its efficacy has been limited, even for patients positive for diagnostic biomarkers. This underscores the importance of a holistic view of the TME and a functional assessment of key interactions rather than expression profiling alone. Here, we introduce a new method based on the in situ proximity ligation assay (isPLA) – the Immuno-oncology triplex panel (IOplex) – which enables the parallel detection of the PD1/PD-L1 and CD8/MHC-I interactions together with the ultraprecise capture of CD3 expression in human cancer tissues.

Methods Formalin-fixed, paraffin-embedded (FFPE) tissue sections were baked, deparaffinized and epitopes were unmasked. Thereafter, staining was performed with IOplex (Navinci). To detect the interactions between PD1/PD-L1, CD8/MHCI, and the CD3+ T cells, three pairs of Navenibodies were simultaneously used. In short, IOplex comprises tissue blocking, followed by primary Navenibody incubation, ligation and rolling circle amplification (RCA), in which each Navenibody pair results in a distinct RCA product. The RCA products for PD1/PD-L1, CD8/MHCI, and CD3+ cells were detected with oligonucleotides labeled with Atto647N, Atto550 and AlexaFluor488, respectively. Imaging was carried out on the Olympus VS200 scanner.

Results PD1/PD-L1, CD8/MHC-I interactions and CD3 expression as assessed by multiplex isPLA were observed in healthy tonsil tissue, which is positive for all markers, and absent in the negative control (muscle). Other examined tissues included samples from head-and-neck cancer, lung cancer, Hodgkin lymphoma and melanoma. We demonstrated that the lung cancer tissues were immune-infiltrated, with varying degrees of PD1/PD-L1 activation involving CD3+ cells. In Hodgkin lymphoma, we observed high numbers of activated T cells (CD8/MHC-I+), as well as abundant PD1/PD-L1 interactions. One melanoma sample was immune-cold and negative for signal, whereas the other had limited activation of immune checkpoint (IC) inhibitory pathways and presence of active immune cells.

Conclusions Understanding the dynamics of IC within the TME can enhance the development of effective strategies to overcome immune escape mechanisms employed by cancer cells. Functional studies that show protein involvement in key pathways such as PD1/PD-L1 and CD8/MHC-I offer a more biologically relevant view of the TME than traditional IHC. The simultaneous visualization of these three functional markers enhances the possibilities of meaningful spatial profiling. Further research is needed to explore the therapeutic potential of protein interactions as superior biomarkers, advancing cancer immunotherapy, and improving patient outcomes.