Non essential amino acids (neaa)

Lung adenocarcinoma cells A549 (CCL-185, ATCC, Manassas, VA, USA) and H460 (HTB-177, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified eagle medium (DMEM; CAT#01–055, Biological industries, Beit HaEmek, Israel). Meanwhile, human embryonic lung fibroblasts HFL-1 (CC-Y1584, EK-Bioscience, Shanghai, China) were cultured in Ham’s F-12 (CAT#01–095, Biological industries). All media were then supplemented with 10% fetal bovine serum (FBS; GIBCO™, CAT# 10270106, Life Technologies, San Jose, CA, USA), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, which were all purchased from Biological industries (Kibbutz Beit Haemek, Israel). Non-essential amino acids (NEAA; CAT# X0557, 1: 100, Biowest, Logan, Utah, USA) were employed for HFL-1 culture. Afterwards, the cells were cultured in a humidified incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37 °C with 5% CO₂.
A549 cells were transfected with the HA-BIRC5 expression vector (pcDNA3-HA-BIRC5) and Myc-DR5 expression vector (DR5 Myc-tag) or corresponding empty vector (pcDNA) following the instructions of the Lipofectamine 2000 transfection reagent (CAT# 11668019, Invitrogen, Carlsbad, CA, USA). Cells were transfected with 1 μg of pcDNA3-HA-BIRC5 and DR5 Myc-tag or the corresponding empty vector pcDNA. After a 24-h period of transfection, the cells were treated with NPs for subsequent analyses.

Another part of material, obtained from 5/10 tumor specimens following mechanical disintegration (as described above), was further established according to the protocol developed by Xie et al. [16 (link)]. Briefly, minced tumor tissue was incubated in 1:1 mixture of StemPro Accutase and TrypLE (10 min, 37 °C; Invitrogen). Following filtration through 70 μm cell strainer cells were centrifuged (80 xg, 90 s) and subsequently suspended in 1:1 mixture of Neurobasal medium (Life Technologies) and DMEM/F12 (Biowest), supplemented with N2, B27, Antimitotic-antimycotic, Glutamax (1x; all Life Technologies), NEAA (1x; Biowest), bFGF (40 ng/mL, Peprotech) and EGF (5 ng/mL, Peprotech). Approximately 5–7 days later, formed spheres were treated with StemPro Accutase, washed with culture medium and seeded onto 6-well plates previously coated with poly-l-ornithine (10 μg/mL; 2 h, RT; Sigma Aldrich) and further with mouse laminin (10 μg/mL; 30 min, 37 °C; Sigma Aldrich). Depending on proliferation rate, such adherent cultures were passaged with StemPro Accutase to a new culture dish every 7–14 days. For further immunofluorescence analyses, spheres were passaged onto 4 well plates with coverslips and left to adhere and let cells migrate out of sphere.

Human STK31 full length cDNA was cloned into vector pEGFP-N1. AZ521, a human colorectal cancer cell line, was cultured in DMEM medium (Sigma) plus 10% MEM (GIBCO) and 1% NEAA (Bio-West). HCT116, a human colorectal cancer cell line, was maintained in RPMI-1640 medium (GIBCO). All media were supplemented with 10% FBS (Bioscience), 100 units/ml penicillin, and 100 μg/ml streptomycin (GIBCO). For transient transfection, cells were grown up to 80% confluence and transiently transfected with STK31-GFP using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Pulmonary microvascular endothelial cells (HPMVECs, passages 4 to 6) were seeded 1:1 in 0.1% gelatin-coated 96-well ibidi culture slides (96W10idf PET; Applied BioPhysics) for electrical cell-substrate impedance sensing, as previously described (109 (link)). In short, HPMVECs were maintained in culture in Endothelial Cell Medium (ScienCell) supplemented with 1% penicillin–streptomycin, 1% ECGS, 5% FCS, and 1% NEAA (Biowest). From seeding onward, electrical impedance was measured at 4,000 Hz every 5 min. HPMVECs were grown to confluence. After 72 h, ECM was removed and replaced by either complete ECM with DMSO or 1 μM entospletinib. After 2.5 h of pretreatment, the medium was removed and replaced by the macrophage-conditioned media stimulated for 6 h as described above with poly(I:C) or in combination with patient serum. Three technical replicate measurements were performed for each condition. For every experiment, HPMVECs and macrophages obtained from different donors were used.

The human non-small cell lung cancer cell line H358 was purchased from ATCC (ATCC-CRL-5807; ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; c.c.pro, Oberdorla, Germany), 2 mM L-glutamine (Biowest, Nuaillé, France), 1% non-essential amino acids (NEAA, Biowest), and 1% penicillin-streptomycin (Biowest) and maintained at 37 °C in a humidified atmosphere with 5% CO₂. When confluent, cells were washed with phosphate-buffered saline (PBS, Biowest) and then harvested using trypsin-EDTA (Biowest).

Human embryonic kidney 293 cells (HEK293, CRL-1573) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and the neuroblastoma cell line IMR-32 from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). HEK293-GFP cells were obtained from GenTarget (SC001, San Diego, CA, USA). Human kidney fibroblasts were purchased from Innoprot (P10666, Derio, Bizkaia, Spain). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biowest, Nuaillé, France) containing 10% fetal bovine serum (FBS; c.c.pro, Oberdorla, Germany), 2.5 mg/mL of glucose (Biowest, Nuaillé, France), 2 mM of L-glutamine (Biowest, Nuaillé, France), 1% non-essential amino acids (NEAA, Biowest, Nuaillé, France), and 1% penicillin-streptomycin (Biowest, Nuaillé, France) and maintained at 37 °C in humidified atmosphere with 5% CO₂. When confluent, the cells were washed with phosphate buffered saline (PBS, Biowest, Nuaillé, France) and then harvested using trypsin-EDTA (Biowest, Nuaillé, France).

PAEC passage 4-6 cells were seeded 1:1 in 0.1% gelatin coated 8-well (8W10E) or 96-well (96W10idf PET) IBIDI culture slides for electrical cell-substrate impedance sensing (ECIS), as previously described (34 (link)). Cells were maintained in culture in endothelial cell medium (ECM, ScienCell, #1001) supplemented with 1% Penicillin/Streptomycin, 1% endothelial cell growth supplement (ECGS), 5% FBS and 1% non-essential amino acids (NEAA, Biowest, #X055-100), with medium change every other day. From seeding onwards, electrical impedance was measured at 4000Hz every 5 min. Cells were grown to confluence and after 72 hours, ECM medium was removed and replaced by supernatant of alveolar macrophage-like monocyte-derived macrophages stimulated for 6 hours as described above with poly(I:C), or in combination with patient serum. Within every experiment triplicate measurements were performed for each condition. For every experiment PAECs and macrophages obtained from different donors were used.