Inform advanced image analysis software inform 2.4.1

Opal™ 7-Color Multiplex IHC Kit (Akoya Biosciences, NEL861001KT) was employed to perform multiplex staining. The protocol was referred to the manufacturer’s construction. FFPE sections were incubated at 65°C for at least 18 h as preprocessing. The slide underwent a serial deparaffinization step and then immersed to quench peroxidase followed by washing. The following steps were repeated for multiple-marker staining. The slides were successively treated for primary antigen retrieval, blocking of unspecific binding, secondary antibody conjugating, and stripping. After completing multiple stainings, the slides were scanned on the PerkinElmer Vectra 3^® Polaris™ platform and imaged on the inForm Advanced Image Analysis software (inForm 2.4.1; Akoya Biosciences, USA) with the DAPI (Akoya Biosciences) filter set. The antibodies and reagents are listed in Table S1.

Multiplex staining of primary tissues was performed by using Opal^TM 7-color multiplex IHC kit (Akoya Biosciences, NEL861001KT) according to the manufacturer’s instruction^{65 (link)}. Briefly, the slides were baked for approximately 1 h at 68 °C followed by de-paraffinization and rehydration. For each staining cycle, the slides were treated with the retrieval of antigen, blocking, and primary antibodies incubation, followed by HRP-conjugated secondary antibody incubation and Opal tyramide signal generation (Detailed information of antibodies and corresponding Opal fluorophores were provided in Supplementary Table S1). Then the slides were stripped with a retrieval solution as required before the next round of staining. The above process was repeated until all markers were completed. Once all markers were labeled, slides were counterstained with DAPI (Akoya Biosciences) and scanned using the PerkinElmer Vectra3® Polaris^TM platform. The multispectral images obtained were unmixed using the inForm Advanced Image Analysis software (inForm 2.4.1; Akoya Biosciences, USA).