Rpmi 1640 medium

We conducted 2D under agarose migration assays as described elsewhere [27 (link)]. Briefly, 35 mm glass bottom Petri dishes (ibidi, Martinsried, Germany) were coated with 50 µg/mL of fibronectin (BD) for 1 h at 37 °C, washed twice with sterile water, and dried prior to casting the agarose gel. Next, 2% (w/v) agarose was boiled in plain RPMI1640 medium (Biochrom, Berlin, Germany) without any supplements and mixed with an equal amount of preheated (50 °C) complete RPMI1640 medium to yield a final agarose concentration of 1% (w/v). Finally, 2 mL of the warm and still fluid mixture were quickly added to the culture dish, covered with the dish lid, and allowed to solidify at room temperature.
To assess migration, two wells were punched 1.5 mm apart using a template and a 3 mm sterile skin punch. The prepared culture dishes were then equilibrated for 1 h at 37 °C and 5% CO₂ prior to seeding of the cells. Freshly isolated BMCs (~2000 cells) were seeded into the first well and allowed to settle for 20 min. Subsequently, 10 µg/mL of the chemoattractant LTB₄ (Cayman Chemical, Ann Arbor, MI, USA) was added to the second well, and the dish was carefully placed onto a heated microscope stage (35 °C) with additional CO₂ gassing (5%). Movement of cells was recorded at 1 frame per minute with a Zeiss AxioCAM MRm for a total period of 90 min.

GBM (n = 14) and MM-BM (n = 12) cell lines were established from surgically removed tumor tissues of GBM and MM-BM patients undergoing surgery at the Department of Neurosurgery of University Hospital of Siena, under approval by the Committee on Human Research. Tumor tissues were processed within 60 min following surgical removal and were dissected into fragments by mechanical digestion (1–2 mm³) washed with PBS 1X and cultured, according to the size in T25 cm² or T75 cm² tissue culture flasks. Cells were cultured using RPMI Medium 1640 (Biochrom, Berlin, Germany), supplemented with 20% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany), 2mML-glutamine and 100 µg/µl penicillin/streptomycin (Biochrom, Berlin, Germany) up to the 5th step of culture and with 10% heat-inactivated FBS for subsequent ones. MM cell lines (n = 11), kindly provided by Dr. Roberta Mortarini (Human Tumors Immunobiology Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy) were established as previously described [25 (link)]. Origin of cell lines and their characteristics are described in Additional file 1: Tables S1–S3.

U87-MG, U251-MG, and U373-MG (Uppsala) GBM cell lines were obtained by Sigma-Aldrich (St. Louis, MO, USA) (HPA Culture Collections). The A172 GBM cell line was obtained by American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were not used beyond passage 20. U251 Cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium (Biochrom, Berlin, Germany) containing 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biowest, Nuaillé, France). All other cells and cell lines were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) low glucose (Biowest, Nuaillé, France) containing 10% FCS (Biochrom, Berlin, Germany), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biowest, Nuaillé, France). Primary GBM cells derived from a primary GBM tumor biopsy were obtained from the University Hospital Cologne, genetically characterized and cultured as previously described (Haas et al. 2018 (link)).

In total, 12 mL of whole blood were collected from healthy donors (HD). Blood was drawn directly into S-Monovette 2.7 mL K3E (1.6 mg EDTA/mL; Sarstedt, Nümbrecht, Germany, cat. no. 05.1167.001) gently rocked at room temperature until processing. Peripheral Blood Mononuclear cells (PBMCs) were obtained by Ficoll gradient. As staining control, enzymatic digestion was performed on PBMCs and compared to undigested PBMCs. Cells were washed and resuspended in cold PBS with 0.5% FCS. PBMCs were aliquoted at 1 × 10⁶ cells/tube and labeled as described above in Section 2.3. The gating strategies used in this study are reported in detail in Supplementary Figures from Figures S12–S16.
PBMCs were cultured in RPMI 1640 medium (Biochrom GmbH, Berlin, Germany) with 10% FBS (Biochrom GmbH) and were stimulated with IFN-γ 100 ng/mL (PeproTech, Inc., Rocky Hill, CT, USA) for 48 h.
Pleural fluids were collected and immediately used. Pleural fluid mononuclear cells were obtained by Ficoll gradient and processed, as previously described for PBMCs. Monocytes from peripheral blood and macrophages from pleural fluid were gated as detailed in Supplementary Figure S15.

All cell lines described here were obtained from ATCC (Manassas, VA) and thus no authentication was done. The human glioblastoma/astrocytoma cell line U251 was cultured in RPMI-1640 medium (Biochrom AG, Berlin, Germany). The human high-grade glioma cell line U87, the human embryonic kidney cell line 293T and the human fibrosarcoma cell line HT1080 were cultured in DMEM (Dulbecco's modified Eagle's medium; Biochrom). All media were supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (100 μg/ml). Cells were cultured at 37°C and 5% CO₂.

Human embryonic kidney cells (HEK293) (ATCC CRL-1573) and PBMCs from AML patient were cultured at 37°C and 5% CO2 in DMEM/F-12 and RPMI 1640 medium (Biochrom, Berlin, Germany), respectively, supplemented with 10% fetal bovine serum. 1×10⁶ of HEK293 cells were nucleofected using Amaxa Cell Line Nucleofector® Kit V (Lonza Group, Walkersville, MD) and 4 μg of plasmid DNA, strictly according to the manufacturer's protocol. The stable cell lines expressing NPM1 variants (NPM1wt, NPM1 R2 and NPM1mut, described fully in following chapter) fused with fluorescent GFP tag were established in the presence of G-418 (400 μg/ml) in the media for two weeks. The patient cells were collected by centrifugation and resuspended at 8 × 10⁶ cells/100 μl for primary AML cells in the Human B Nucleofector® Kit solution (Amaxa Biosystems, Cologne, Germany). PBMCs were nucleofected with 4 μg of appropriate plasmid using the U-013 program of the Nucleofection Device II (Amaxa Biosystems). The nucleofected cells were cultured at 37°C for 1 day and used for immunofluorescence staining (described in Confocal imaging section).