Cultured cell lines.
The cell lines OVCAR8, OVCAR8-ULK1KO, HeyA8, and HeyA8-ULK1KO were grown in RPMI-1640 medium (Wisent), while FT190 and FT190-ULK1KO were grown in DMEM/F12 medium (Life Technologies). All growth media were enhanced with 10% fetal bovine serum. The OVCAR8 and HeyA8 cells were procured from the American Type Culture Collection. Adherent cells were sustained on tissue culture-treated polystyrene (Sarstedt, Newton, NC, USA), and spheroids were kept in Ultra-Low Attachment (ULA) cluster plates (Corning, NY, USA). The immortalized human fallopian tube secretory epithelial cell line FT190 (cite) was generously provided by R. Drapkin from the University of Pennsylvania, Philadelphia, PA. The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada) authenticated all cell lines through short tandem repeat analysis and routinely examined them for mycoplasma using the Universal Mycoplasma Detection Kit (30-1012K; ATCC).
Generation of ULK1 KO cell lines:
CRISPR/Cas9 (Santa Cruz Technology, sc-400516-KO-2 Lot# C3016) was used to ablate ULK1 gene expression in OVCAR8, HEYA8, and FT190 cells. Briefly, cells were seeded at 100-150k per well of a 6-well plate and transfected the following day. Cells were lifted four days post-transfection and sorted using fluorescence activate cell sorting (FACS) into 96-well plates. Clones were left to grow for a minimum of two weeks, where colony formation was subsequently observed. Colonies were harvested and plated into 6-well plates, and then 10cm plates upon reaching confluency. Cells were harvested for protein lysates and screening for ULK1 loss via western blot and passaged for continued culturing and subsequent pooling of clones.
Generation of Nuclight GFP cell lines:
Cells were transduced with Incucyte® Nuclight Green Lentivirus (EF1a, Puro) (Sartorius, #4624) following the manufacturer's instructions. After transduction, cells were cultured in complete media supplemented with puromycin to select for successfully transduced cells. The transduced and puromycin-selected cell population was subjected to fluorescence-activated cell sorting (FACS) to isolate GFP (green fluorescent protein) -positive cells. The isolated GFP + cells were then expanded and used for further analysis or experimentation.
Generation of mCherry-eGFP-LC3 cell lines:
Parental and ULK1KO cells were transfected using an mCherry-eGFP-LC3B autophagy reporter plasmid (Addgene, #22418). After transfection, cells were grown in complete media containing G-418 for two weeks to select those with successful reporter plasmid integration. Following the selection phase, the cells were cultured in complete media without G-418 for another four weeks, allowing for growth and recovery. Cells were then sorted using FACS to identify double positive cells (GFP+, mCherry+).
Generation of luc2tdTomato cell lines:
Cells were transduced with pCDH-EF1-Luc2-P2A-tdTomato (plasmid #72486, Addgene) following the manufacturers instructions. After transduction, cells were subjected to FACS to isolate tdTomato + cells. Cells were cultured in complete media for growth and recovery. Cells were then subjected to another round of FACS to select cells varying intensities of tdTomato expression.
Antibodies and reagents:
Antibodies against ULK1 (#8054S, 1:1000), p62 (#5114S, 1:1000), LC3B (#2775S, 1:1000), p-Beclin1 (#5410S), and Beclin1 (#3738S) were purchased from Cell Signaling Technology. Anti-ULK2 antibody (AB97695, 1:1000), ATG16L1 (AB187671), p-ATG16L1 S30 (AB19016, 1:100), and mCherry (AB167453, 1:500) was purchased from Abcam. Anti-actin antibody (A2066, 1:25000) was purchased from Millipore. Antibody against tubulin (T5168; 1:40000) was purchased from Sigma. HRP-conjugated antibodies against mouse IgG (NA931; 1:10000) and rabbit IgG (NA934; 1:10000) were purchased from GE Healthcare. All antibodies were diluted in tris-buffered saline-Tween 20 containing 5% bovine serum albumin.
Immunoblot analysis:
The Bio-Rad Mini-PROTEAN II Electrophoresis System was employed for immunoblotting following the manufacturer's guidelines, utilizing in-house prepared gels (30% acrylamide/bis solution 37.5:1, catalog number 1610158; Bio-Rad). Densitometric analysis was conducted using the Image Lab 6.05 software suite (Bio-Rad).
Preparation of wholecell lysates:
For assessment of all proteins, 4, 8, 24, 48, 72-hour whole-cell lysates were generated from adherent cells cultured at a density of 0.75-1× 106 cells in 10 mL medium (10 cm dish), or spheroid cells cultured at a density of 1–3× 106 cells in 15 mL medium (35 mm ULA well). Seeding numbers were chosen to obtain acceptable protein yields for each cell line.
Wholecell lysates. Adherent cells cultivated on tissue culture-treated plates or dishes were gathered by removing the medium, washing twice with cold PBS, and then scraping into a modified RIPA buffer. Spheroids (a minimum of 1.5 × 10^6 cells per sample) were collected by transferring the cell suspension into an ice-cold conical tube, followed by centrifugation using a swinging bucket rotor (800 × g at 4°C for 4 minutes) to form a pellet. The medium was then aspirated, and the pellet was resuspended in at least 10 mL of cold PBS. This process was repeated by resuspending the pellet in cold PBS once more, followed by centrifugation and aspiration of the PBS. The resulting cell pellets were lysed using modified RIPA buffer, vortexed, exposed to one freeze-thaw cycle, and finally clarified through centrifugation (maximum × g at 4°C for 20 minutes).
Microscopy:
Brightfield images of spheroids seeded in 24-well ULA plates were captured using a Leica DMI4000B inverted microscope. Immunofluorescent images of mCherry-eGFP-LC3 and Nuclight GFP spheroids were captured using an IncuCyte S3 Live-Cell Analysis System (Sartorius).
Spheroid viability assays.
For bulk spheroids. Cells were placed in 24-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 × 10^5 cells per well in 1 mL of medium. Spheroids were then gathered into chilled microcentrifuge tubes and centrifuged at 500×g for 3 minutes to form pellets. After aspirating the medium, the pellets were washed with 500 µL of PBS (avoiding cell uptake into the pipette tip), centrifuged once more as previously described, and resuspended in 50–250 µL of trypsin/EDTA. The suspension was incubated at 37°C with periodic gentle agitation every 10 minutes until no aggregates were visible (10–30 minutes). Trypsin was inactivated by adding an equal volume of FBS, followed by the addition of Trypan Blue dye (in a volume equal to the combined trypsin/EDTA and FBS), and gentle mixing via pipetting. Cell counting was carried out using a TC20 Automated Cell Counter (Bio-Rad).
For single spheroids. Cells were seeded in a 96-well round bottom ULA plate at a density of 2000 cells per well in 100 µL of medium. For alamarBlue assays, cells were incubated with a final dilution of 1:10 for 4, 24, or 48-hours and fluorescence was measured using a Biotek plate reader. For CellTiter-Glo (Promega, G7572) and Caspase-Glo 3/7 (Promega, G8092) Assay’s, 100 µL of reagent was added and the plate was frozen at -80°C. After 24-hours, plates were incubated in the dark for 60 min on a plate rocker. Wells were transferred to a 96-well opaque white plate and luminescence was read on a Biotek plate reader.
Doubling time:
Cells expressing Nuclight GFP were placed in 96-well standard-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 × 10^5 cells per well in 200 µL of medium. Fluorescent images were captured at 4-hour intervals in the IncuCyte S3 Live-Cell Analysis System (Sartorius). Growth curves and doubling time calculations were generated in GraphPad Prism 9. Adherent and spheroid doubling time calculations were completed using the Green Object Count and Green Mean Intensity feature, respectively.
Carboplatin and paclitaxel treatments:
Paclitaxel (5mM in DMSO) was purchased from Cayman Chemical Company (10461) and stored at -20°C. Carboplatin (27mM in saline) was received from the London Regional Cancer Program and stored at 4°C.
To determine adherent carboplatin and paclitaxel IC50 values, 2000 cells in 100 µL media were seeded in a 96-well plate. The next day, cells were treated individually over a 12-point concentration gradient. After 72 hours of treatment, cell viability was determined using the alamarBlue Cell Viability Reagent (Invitrogen CAT# DAL 1025) according to the manufacturer’s instructions. To determine spheroid carboplatin and paclitaxel C50values, 2000 cells in 100 µL media were seeded in a 96-well ULA plate to induce spheroid formation. After 72-hours, cells were treated individually over a 12-point concentration gradient for an additional 72-hours. Following treatment, viability was determined by alamarBlue viability assay. Viability was assessed 4- and 48-hours post alamarBlue incubation for carboplatin and paclitaxel, respectively. IC50 values were calculated using GraphPad Prism 9.
Xenotransplantation assays:
NOD/SCID female mice (8–10 weeks old; Charles River Laboratories) were inoculated by intraperitoneal injection with 150 µL of PBS containing the following numbers of cells: OVCAR8/OVCAR8-ULK1KO, 2 × 106; HeyA8/HeyA8-ULK1KO, 1 × 106). For survival analyses (OVCAR8, OVCAR8-ULK1KO, HeyA8, HeyA8-ULK1KO cells), mice were monitored daily after intraperitoneal injection and euthanized using standard criteria for humane endpoints (i.e., lethargy, hunched posture, impaired breathing, extreme weight loss, excessive ascites). Mice received weekly injections of D-luciferin (Perkin Elmer, #122799) at 75mg/kg in 100µL of PBS to monitor tumor progression via bioluminescent imaging using our IVIS Lumina S5 system. Mice were provided chow (Envigo, #2919) and water ad libitum throughout the study. All animal experiments were approved by Institutional Animal Care and Use Committee of the University of Western Ontario (London, Ontario, Canada) and carried out in accordance with the approved guidelines.
IHC quantification and scoring
IHC analysis was performed using the Fiji distribution of ImageJ30. Ki67-positive nuclei were masked using the Trainable Weka Segmentation plugin31, and masked regions were counted using a minimum particle area of 120 pixels. Cleaved caspase-3 staining in xenograft tumor sections was evaluated using the “IHC Profiler” plugin for ImageJ as described previously32. Positive caspase-3 staining reflects “high-positive” and “positive” as defined by IHC profiler.
Statistical analysis:
Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). Specific analysis details are described in figure legends.