Human tumour dissociation kit

Subcutaneous tumours from mice were dissociated using a human tumour dissociation kit (Miltenyi Biotec, 130-095-929) with an enzyme cocktail and a gentleMACS dissociator with heaters (Miltenyi Biotec). The cell suspension was then strained through a 40 µm cell strainer (Corning, 352340). Samples were treated with 1× Red Blood Cell Lysis solution (Miltenyi Biotec, 130-094-183) for 10 min at 4 °C, and then spun down at 300g for 5 min and resuspended in 0.5% BSA in PBS. Last, mouse cells were removed from the sample using a Mouse Cell Depletion kit (Miltenyi Biotec, 130-104-694) following the manufacturer’s protocol.

Reference chemicals benzo[a]pyrene (BaP, B1760), ethyl methanesulfonate (EMS, M0880), 1,2-dimethylhydrazine hydrochloride (DMH, D161802), etoposide (Etop, E1383), phenformin hydrochloride (Phen HCl, PHR1573), potassium bromate (KBr, 309087), glycidamide (GA, 04704), and ethyl nitrosourea (ENU, N3385) were purchased from Sigma Aldrich (St. Louis, MO). Test chemical stock solutions were prepared in anhydrous DMSO (Sigma Aldrich, St. Louis, MO) or in water.
Cell recovery solution (Corning, Tewksbury, MA) and human tumour dissociation kit (130-095-929, Miltenyi Biotec, Bergisch Gladbach, Germany) were used for the purpose of 3D tissue dissociation as previously described [31 (link)].
Comet assay kit consisting of low-melt agarose (4250-050-02, 1% low melting agarose in PBS), cell lysis solution (4250-050-01), pre-coated 2-gel agarose comet slides (CometSlide™ 4250-200-03), and electrophoresis apparatus (4250-050-ES) were sourced from RnD systems (Minneapolis, MN). Low-melt agarose was reused up to 10 times after melting. TE buffer was obtained from Promega (Madison, WI). Alkali DNA unwinding (300 mM NaOH, 1 mM EDTA) and electrophoresis solutions (200 mM NaOH, 1 mM EDTA) were prepared fresh for use on the day of experiment. SYBR Green DNA stain was procured from Thermo Fisher Scientific (Waltham, MA).

PDX samples have been chopped and digested with a Human Tumour Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer protocol. Human cells were isolated using a Mouse Cell Depletion Kit (Miltenyi Biotec). Cells were plated, and after 24–48 h treated with a drug in 96-multiwell plates.

Ovarian tumours were dissociated into single-cell suspensions using the MACS Human Tumour Dissociation Kit (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. Briefly, tumour biopsies were cut into 2–4 mm³ fragments using a sterile scalpel and transferred into a C-tube (Miltenyi Biotec, Germany) with RPMI-1640 (Lonza, Slough, UK) and the appropriate volumes of enzymes H, R and A (Human Tumour Dissociation Kit, Miltenyi Biotec, Germany). The sample was placed onto the GentleMACS dissociator and subjected to three pre-set dissociation programs according to the manufacturer’s instructions. This was interjected by two 30-min incubations at 37 °C under continuous rotation using the MACSmix Tube Rotator (Miltenyi Biotec, Germany). Following disaggregation, the cell suspension was passed through a 100-µm strainer, and centrifuged at 400g for 5 min. The supernatant was discarded and the remaining cell pellet was thoroughly resuspended and cells were counted.

The ex vivo drug treatment protocol was performed as previously described (Bruna et al, 2016 (link)). Briefly, frozen patient‐derived tumour xenografts (PDTXs) were thawed and dissociated into single cell suspensions by combining mechanical and enzymatic dissociation using the soft tumour dissociation protocol on a GentleMACS Dissociator and the human tumour dissociation kit (Miltenyi Biotec, Cat ID 130‐093‐235) according to the manufacturer instructions. Single cells were plated at ~40,000 cells/ml in 50 µl per well in 384 well plates and dosed 72 h after plating. The selected drugs were added to the wells after 24 h of seeding using Echo Liquid Handler 550 (Labcyte). Cell viability was assessed using CellTiterGlo 3D Cell Viability (Promega) 6–10 days after dosing following manufacturer specifications and normalised against blank wells and control wells treated by Dimethyl Sulfoxide (DMSO, Sigma). Plates were read on the Pherastar plate reader using the Luminescence module.

Tumour samples were harvested from recipient mice, minced with scissors, and dissociated using the human tumour dissociation kit (Miltenyi Biotech) and gentle MACS dissociator (Miltenyi Biotech). Single cells were incubated with ACK lysis buffer (Invitrogen, Carlsbad, CA, USA) for 5 min at 4 °C, followed by incubation with cell surface protein-specific antibodies for 30 min at 4 °C. Then, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Kit (BD Biosciences) and incubated with intracellular protein-specific antibodies for 30 min at 4 °C (Supplementary Table 1). The samples were resuspended in autoMACS Rinsing Solution with MACS buffer, followed by analysis with Fortessa-X20 (BD Biosciences) and data analysis using FlowJo v10.2 (Tree Star Inc., Ashland, OR, USA). Figures were drawn using GraphPad Prism 8.4.3 (GraphPad Software, San Diego, CA, USA).