Multiplex Immunofluorescence for Immune Cell Profiling

Multiplex immunofluorescence (mIF) was performed using an Opal Multiplex fIHC kit (PerkinElmer, Inc., Waltham, MA, USA) as previously described by our group and other studies (20 (link), 20 (link), 27 (link)–36 (link)), on FFPE tissue sections. TMA sections of 4 μm thickness were incubated with primary antibodies against CD38 and CD68, followed by appropriate secondary antibodies (Supplementary Table 2). Following this, a fluorophore-conjugated tyramide signal amplification buffer (PerkinElmer, Inc.) was applied, and DAPI was used as a nuclear counterstain. Images were acquired using a Vectra 3 pathology imaging system microscope (PerkinElmer, Inc.) and analyzed using inForm software (version 2.4.1; PerkinElmer, Inc.) (28 (link), 37 (link), 38 (link)). Maximally selected rank statistics (39 (link)) were applied using the maxstat R package to find optimal cut-off points for variables with good survival outcome prediction (Cutoff for CD38⁺ macrophages = 56.9%, whereas cutoff for CD68 immune infiltrates = 19.2%).