Matrigel

For xenograft experiments, mouse prostate stem cells (5 × 10⁶) were mixed with an equal volume of Matrigel and injected subcutaneously into the flank region of Nu/J (nude) male mice (Jackson Laboratories). Tumor volume was measured twice weekly. All animal studies were performed at NYU School of Medicine. The animal research was approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC), protocol number IA16-01775.

For orthotopic xenografts, cells were resuspended in 25 µl of media. The resuspended cells were then mixed with 25 µl of Matrigel (Corning, NY) for the engraftment. After mice randomization, ~ 50 µl of Matrigel-media mix was then orthotopically injected into the bladder wall of 6–8-week-old NOD/SCID/IL2rγnull (NSG) (Jackson labs, USA) female mice. To measure the tumor growth and metastasis, a total of 200,000 control or SMARCB1 KO (C16 or C45) or rescue cells (Fig. 2B–D and Supplementary Fig. 4A and 4C) or SMARCB1 KD cells (Left panel of Supplementary Fig. 4G) were injected into the bladder wall. 100,000 cells of  SMARCB1 KO with shCtrl or STAT3 KD (Fig. 4) were injected into the bladder wall. T24 SMARCB1 KO  therapeutic experiments 50,000 cells  (Fig. 5) were injected into the bladder wall. T24 control cells for therapeutic experiments, a total of  500,000 cells (Supplementary Fig. 12), were injected into the bladder wall. After seven days of the implantation of cells for orthotopic in vivo experiments, wound clips were removed, and the bioluminescence imaging (BLI) signal was measured. The measurement of BLI signal was performed by injecting 100 µl of luciferin (15 mg/ml in PBS; GoldBio, USA) substrate via the retro-orbital route in anesthetized mice. The weight of the mice was measured and recorded weekly. BLI signal measurements were plotted using GraphPad Prism v10.

A498 cells (3×10⁶) overexpressing an empty vector (EV), RKIP (RKIP), short hairpin control (Ctrl ShRNA) or RKIP ShRNA were resuspended into a MEM/Matrigel mixture (1:1 volume) and subcutaneously implanted into the flank of NOD/SCID mice (The Jackson Laboratory) with each group containing 5 mice. Tumor volume was calculated according to the formula L × W² × 0.52, where L and W are the longest and shortest diameters respectively [58 (link)]. All animal work was performed according to protocols approved by the McMaster University Animal Research Ethics.

SKmel147 cells were infected with doxycycline (DOX)-inducible shNTC or shZNF180 lentivirus and selected with 2 μg/ml puromycin for 48 h. Cells were induced with 2 μg/ml DOX for 3 days, trypsinized, washed with PBS, then suspended in sterile PBS at a concentration of 2 × 10⁶ cells per 150 µl, and maintained on ice until injection. Immediately before injection, cell aliquots were mixed with Matrigel (Becton Dickinson). Totally, 150 µl of cell/Matrigel (1:1) suspensions were injected subcutaneously in the right flank of NOD/Shi-scid/IL-2Rgamma null (NSG, Jackson labs #005557) 20-weeks-old male mice (n = 12 per group). Mice were fed DOX-containing food (200 mg/kg weight). When primary tumors were palpable (6 days post injection), length (l) and width (w) were measured with calipers, 3 times weekly over a period of 13 days. Tumor volume was calculated using the formula (l × w²)/2. Tumor weight was measured at endpoints. Animal experiments were conducted in accordance with guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of NYU (protocol # S16-00051).

MDA-MB 231 (1 × 10⁶ cells) with 50% Matrigel™ were injected into Number 4 mammary gland of 4–5 weeks old NSG (Jackson Laboratory). Tumors were measured by a caliper. When tumors reached a size of ∼100 mm³ the mice were randomly distributed into two groups (ten mice in each group): untreated control and NSC260594 (30 mg/kg bodyweight). NSC260594 was suspended in sesame oil and administrated by oral gavage once daily for two weeks. Tumor volume measured twice a week after the initial injection, and the volumes were calculated using the formula (π x length x width1 x width2 /6). Mice were euthanized when they became moribund, or when they lost 20% weight. All organs examined for the presence of tumors and metastases at autopsy. Data grouped and plotted using GraphPad Prism 8.

SM3 PanIN cells were infected using control lenti-eGFP (LPP-EGFP-Lv105-025-C) or lenti-cxcl10 (LPP-Mm03214-Lv105-100) lentiviral particles (Genecopoeia, Rockville, MD) with 5 μg/ml of polybrene (Santa Cruz Biotechnology, Dallas, TX) at 70–80% confluency. Infected cells were incubated at 4°C for 2 hr and then at 37°C overnight. On the following day, media containing lentiviral particles and polybrene was removed and replaced with fresh media to allow the cells to recover. Next day, selection media containing 2.5 µg/ml puromycin was added to the cells and was replaced every 2–3 days. After 11 days of selection, cells were harvested and embedded in Matrigel (Corning, Corning, NY) with regular media without puromycin. After 2 days, cells formed duct-like PanIN organoid structures. Using a non-enzymatic cell dissociation reagent (Corning), PanIN organoids were harvested and mixed with activated primary pancreatic stellate cells at a ratio of 1:4. Cell mixture was resuspended in phenol-red free Matrigel, and 50 µl (25,000 organoid cells + 100,000 activated primary stellate cells) was injected directly into the pancreas of athymic nude (Foxn1^nu) mice (Jackson laboratory, Bar Harbor, ME). Wound was closed using 4–0 Vicryl sutures and 7 mm wound clips. After 1 week of recovery, wound clips were removed, and ultrasound imaging was performed to ensure implantation of cells.