Melanoma-specific genetic changes are being identified for targeted therapy. Unfortunately, patients receiving such therapies often develop resistance and experience recurrence. Current data suggest that the tumor-specific microenvironment has a decisive influence on therapy resistance. We therefore established a multiplexed immunofluorescence staining procedure to precisely characterize the tumor microenvironment in melanoma brain metastases (BM).

Co-detection by indexing (CODEX) technology relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 100 biomarkers in situ. It enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in disease and therapeutic design. Hence, CODEX was provided by Akoya Biosciences. To quickly handle a larger scale of samples, a tissue microarray (TMA) was created both of tumor bulk as well as positive and negative surgical margins.

At first, a panel of yet 30 antibodies were conjugated with its definite DNA oligonucleotide sequence (reporter). On our part, these antibodies were previously validated on conventional IHC using normal and gliotic brain along with tonsil tissue, including CD molecules (e.g. CD4, CD8, CD20, CD31, CD68), signal transducing antibodies (e.g. AKT, ERK, EGFR, IGFR and their phosphorylated isoforms) as well as tumor (e.g. HMB-45, Ki67, Melan-A) and neuronal (e.g. GFAP, NeuN, Olig2, PSD-95) markers. Subsequently, TMAs of different BM composition were generated (25 cores (1.5 mm), per slide 6x4 samples + 1 positive control). Within only a single working day TMAs were prepared and CODEX staining protocol fully assembled (single step staining to preserve sample integrity). CODEX run (3 reporter dyes imaged with complimentary barcodes) was performed overnight truly smooth. Lastly, analysis of spatial information has been received via QuPath (open source).

Once individually designed, multiplex fluorescence imaging platform is an easy and especially in case of TMAs fast-performing tool to gain insight into the tumor microenvironment of small sample volumes in particular. For the prediction of possible therapy resistance distinct further studies are warranted to define the potential of high throughput multiplexed imaging in a clinical routine.