Article Methods Ethics approval This study was approved by the Erasmus MC Ethics Committee (MEC- 2017-375, METC 2020-054), allowing for retrospectively collected data and residual material from tissue collected for routine diagnostic pur- poses to be used, waiving the need for explicit written informed con- sent. Human tissues and patient data were used according to the Code for Proper Secondary Use of Human Tissue and the Code of Conduct for the Use of Data in Health Research as stated by the Federation of Dutch Medical Scientific Societies. All animal studies were approved by the Institutional Animal Care and Use Committee at Mispro (South San Francisco) or the animal welfare committee (IvD) of the Erasmus MC (Rotterdam), with approval by the CCD (Centrale Commissie Dier- proeven) under the permit number AVD101002017867. Patient inclusion From patients with melanoma who underwent a sentinel LN procedure at the Erasmus MC Cancer Institute between 2005 and 2017, individuals with or without nodal metastasis (that is, positive TDLNs) were identi- fied. For comparative purposes, we selected patients with melanoma who either presented with early (stage II, RFS ≤ 48 months; stage III, RFS ≤ 24 months) distant disease recurrence or did not develop disease recurrence (stage II, RFS > 96 months; stage III, RFS > 60 months) after the sentinel LN procedure. To avoid including false-negative TDLNs, we only included stage II patients with a negative TDLN who developed metastatic disease within the regional LNs (similar to the TDLN basin) after 9 months or longer. Patients with melanoma did not receive neo- adjuvant or adjuvant treatment (which was not routinely administered during the inclusion period), excluding effects of immunotherapy on TDLN contexture or disease outcome. We also selected patients with resectable stage IB–III NSCLC who underwent surgical resection at the Erasmus MC Cancer Institute between 2018 and 2021, with either distant disease recurrence (>6 months and ≤60 months after surgery) or who did not develop disease recurrence (>60 months after surgery). Adjuvant chemotherapy for NSCLC was allowed but is known to improve overall survival only minimally after surgery 48 . Patients with melanoma or with NSCLC were excluded if they had synchronous malignancies of other origin, chronic infections, active autoimmune disease or other disease aetiologies requiring immunosuppressive treatment (includ- ing prednisone or equivalent >5 mg), or if they received neoadjuvant treatment before resection. Most patients with NSCLC in both prog- nostic groups received adjuvant chemotherapy according to local treatment protocols. Tissue sample processing and ROI selection Formalin-fixed paraffin-embedded (FFPE) LN samples were retrieved from the Pathology department at the Erasmus MC (standard protocols for diagnostic work-up) and tissue sections were prepared at a thickness of 4 μ m and 5 μ m for IMC and IHC, respectively. ROIs (500 × 500 μ m) were selected at four distinct LN anatomical locations: cortex, paracor- tex, peritumoural and intratumoural. ROI selection was guided by (i) IHC staining for SOX10 (tumour), CD3 (T cells) and CD20 (B cells), to prevent an exclusive focus on the highly prevalent B cell follicles; and (ii) haematoxylin and eosin (H&E), CD3, CD20 and SOX10-stained 5- μ m sections from each sample. ROI selection was performed under the supervision of an experienced certified pathologist. Tissue microar- rays (TMAs) were created using the fully automated TMA Grand Master (3DHISTECH) system. From the paracortex of TDLNs from patients with stage III melanoma, 2-mm-diameter cores were selected from the above-mentioned ROIs and embedded in paraffin. Design of the IMC antibody panel IMC antibodies were either acquired pre-labelled with metal isotopes (Standard BioTools) or labelled in-house using MaxPar conjugation kits (Standard BioTools) according to the manufacturer’s protocol. Antibodies were titrated on TDLN material containing metastases and were evaluated by an experienced pathologist to determine optimal antibody concentrations. Details of antibodies and dilutions are pro- vided in Supplementary Table 5. IMC tissue staining For IMC, the antibody staining protocol was adapted from Standard BioTools. FFPE LN material was sliced at a thickness of 4 μ m. Slides were baked for 2 h at 60 °C before being dewaxed in fresh xylene (Sigma-Aldrich, 534056) for 20 min. Slides were then dehydrated in descending grades of ethanol (ethanol 100%, Boom, 84901622.5000) (100%, 95%, 80%, 70%) for 5 min each and washed in sterile water for 5 min by gentle agitation. Slides were inserted in preheated antigen retrieval solution at 96 °C (Dako, S2367) and incubated for 30 min with loose lids. After incubation, slides were cooled down to 70 °C before being washed in sterile water for 10 min in 50-ml Falcon tubes by gentle agitation. Slides were then inserted in Epredia Shandon plastic cover plates (Epredia) and moved to the Sequenza Immunostaining Center System (Epredia). Slides were washed twice with phosphate-buffered saline (PBS) by filling the Sequenza cover plates and allowing the fluid to flow through the system. Next, the tissue was blocked using 3% bovine serum albumin (BSA) in PBS for 45 min at room temperature. Mean- while, the antibody cocktail was prepared in 0.5% BSA in PBS. To each slide, 150 μ l antibody cocktail was added, after which the Sequenza Immunostaining Center System was covered with a lid and incubated at 4 °C overnight. The next day, slides were washed with 0.2% Triton X-100 (Thermo Fisher Scientific, WD319895) in PBS until the fluid ran through the system. The slides were washed twice with PBS in the same manner before being stained with 150 μ l Intercalator-Ir in PBS for 30 min at room temperature. Finally, the slides were washed with sterile water before being dried at room temperature. Slides were stored in a dry place until acquisition. Details of antibodies and dilutions are provide in Supplementary Table 5. Image acquisition For IMC, images were acquired on the Hyperion Imaging System using CyTOF software v.7.1 (Standard BioTools). Laser strength was deter- mined using representative tissue samples. The machine was tuned before every acquisition and reference energy was calibrated once a month. All intra- and peritumoural ROIs from patient P002 were excluded from further analysis owing to abnormal staining patterns. Cell segmentation To generate single-cell data from IMC images, segmentation was per- formed in accordance with the Bodenmiller IMC segmentation pipe- line 49 , including the use of CellProfiler v.4.2.5 50 and Ilastik v.1.4.0 51 . In brief, MCD and .txt files were converted into individual tiff files using the IMC preprocessing script. Hot pixels were filtered by comparing the intensity of each pixel against the maximum intensity of the 3 × 3 neigh - bouring pixels; if differences were larger than a threshold of 50 (default settings), the pixel intensity was clipped to the maximum intensity in the 3 × 3 neighbourhood. Next, images were cropped in CellProfiler for use in supervised pixel classification using Ilastik, which was trained to recognize the nucleus, cytoplasm and background using selected markers (DNA, CD45, CD20, CD3, SOX10, and ICSK1–3 from the Cell segmentation kit III). We trained Ilastik on 50 × 50- μ m cropped images of multiple ROIs from each patient. In brief, the average intensity across all channels is calculated and multiplied by 100 to help identify back- ground regions. Next, the average expression was clipped to a range between 0 and 1. Ilastik stacks were upscaled by factor 2 and cropped to 50 × 50 pixels. After feature reduction, cell masks were generated, and images were segmented using CellProfiler to extract single-cell staining intensity data. This segmentation strategy was validated using Steinbock (v.0.16.3)—a toolkit for the processing of multiplex tissue