Background The emergence of an increasing number of immunotherapy biomarkers and the importance of spatial context within the tumor microenvironment (TME) has resulted in a need for multiplex IHC assays. The newly developed SignalStar™ Multiplex IHC technology by Cell Signaling Technology® was utilized to simultaneously label and visualize 8 targets aimed at characterizing T cell exhaustion in formalin-fixed, paraffin-embedded cancer tissues of various types.

Methods The first 8-plex panel consisted of antibodies targeting exhausted and effector T cell markers: CD8?, PD-1, LAG3, TIM-3, Tox/Tox2, TCF1/TCF7 and Granzyme B in the context of the tumor, as marked by Pan-Keratin. Using the SignalStar™ assay in both tumor microarrays and whole tissues, all 8 site-specific, oligo-conjugated antibodies were applied simultaneously in one incubation step. A network of complementary oligonucleotides with fluorescent dyes (channels: 488, 594, 647, 750 nm) amplify the signal of up to 4 oligo-conjugated antibodies in the first round of imaging, followed by removal and amplification of 4 additional antibodies in the second round of imaging. The images were aligned computationally with open-source software to generate the complete 8-plex image. The signal of each target within the panel was compared to chromogenic staining as well as quantified with respect to signal intensity and frequency. PD-L1 was subsequently validated in tissues with high, medium, and low expression levels, and were then substituted into the 8-plex panel without additional optimization. The SignalStar™ antibody staining was compared to the chromogenic gold standard, as well as to other multiplexing methodologies such as tyramide signaling amplification (TSA), the usage of primary antibodies with fluorescent secondary antibodies and direct conjugates.

Results Our data demonstrate sufficient amplification of the first four targets, followed by complete removal of fluorescent signal and subsequent staining and amplification of the remaining 4 targets. Each target within the panels showed staining comparable to that of the unconjugated antibody when detected with DAB. We also demonstrate that this assay affords flexibility in that the panel can be redesigned without optimization. Finally, this assay affords a level of amplification such that signal intensity is comparable to that produced by TSA and substantially greater than that produced by fluorescently conjugated secondary antibodies and direct conjugates.

Conclusions Ultimately, this technique proves to be a highly sensitive and specific tool to enable the multiplexing of antibodies and could be beneficial for researchers studying T cell exhaustion within the immunosuppressive landscape of the TME.