Manager 4

Cryopreserved PDA tissue samples were cut into multiple consecutive sections (20 µm) and then stained with cresyl-violet. Areas of tumor epithelium and stroma were highlighted using brightfield microscopy at 20× magnification. Laser microdissection was performed with the Leica Laser Microdissection V5.0.2.0 software. Sufficient protein concentrations for multiplex analysis could be obtained by the dissection of 30–50 × 106 mm². The tissue was lysed using the BioPlex™ Cell Lysis Kit (Bio-Plex Cell Lysis Kit, BioRad, Hercules, CA, USA; 171304011) according to the manufacturer´s instructions. Serum samples were thawed overnight at 4 °C and then diluted at 1:1 using Sample Diluent (BioRad, Hercules, CA, USA) prior to protein quantification. A two-laser array reader simultaneously quantified all proteins of interest. The concentrations were calculated using Bio-Plex Manager 4.1.1 and a 5-parameter logistic plot regression formula. The Bio-Plex Pro Human Cytokine Screening Panel 48-plex (BioRad, Hercules, CA, USA; 12007283), Bio-Plex Pro Human Cytokine ICAM-1 (BioRad, Hercules, CA, USA; 171B6009M) and Bio-Plex Pro Human Cytokine VCAM-1 (BioRad, Hercules, CA, USA; 171B6022M) were used for the quantification of immunological parameters.

Multiplex cytokine analyses were performed using a two-laser array reader system (Bio-Rad, Munich, Germany) for quantification of multiple cytokines and chemokines. Briefly, serum samples obtained at different time points during disease (Fig. 1A) were processed with respective dilutions, and calibration of investigated analytes was performed according to the manufacturer’s instruction. Bio-Plex Manager 4.1.1 was used for generation of standard curves and concentrations. Further data analyses were performed with R version 4.0.3.Fig. 1

Paraneoplastic leukemoid reaction in melanoma A Progression of absolute leukocyte count, absolute neutrophil count (count/nl), and C-reactive protein (mg/l) during disease course. Time points of blood draw for cytokine analysis (patients 2, 4, and 5) are marked by red arrows. B Correlation of serum G-CSF concentration to absolute neutrophil count (n = 5). A significant correlation was observed (Pearson’s correlation coefficient, R = 0.95, p = 0.014). C Comparison of serum cytokine concentrations between not-leukemoid status and leukemoid reaction (not leukemoid: n = 2, leukemoid: n = 3). D Hierarchical clustering of serum cytokines and blood markers based on euclidean distance calculations. Marker concentrations are normalized as log₁₀ of fold-change to baseline concentrations (n = 2)