PC cell line LNCaP (ATCC, CRL-1740) was purchased from American type culture collection (ATCC, Manassas, USA). Cells were cultivated in RPMI (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin and incubated at 37 °C with 5% CO2. Medium was changed twice a week.

All animal experiments were approved by the cantonal veterinary office (Veterinäramt Zürich, license No.244/2016) and performed according to the Swiss animal welfare act. A total of 28 male nude mice (8 weeks old; Charles River Laboratories, Sulzfeld, Germany) were analyzed. Mice underwent castration before tumor formation. After 2 weeks, nude mice were subcutaneously injected with 5.0 × 106 LNCaP cells with a high concentration Matrigel carrier (500 µl, Corning Life Sciences, NY, USA) on both left and right backsides. Drug injection was started once the tumors were formed, 2–3 weeks after tumor cell injections. The mice were divided into four groups (three animals/group/ time point). The treatment groups Vehicle control (For APA; 18% PEG 400, 1% Tween 80, 1% polyvinyl pyrolidone, 65% 20 mM citrate buffer pH:4.0 in 0.5% carboxymethylcellulose sodium salt, all purchased from Sigma Aldrich), APA (10 mg/kg, Janssen Pharmaceutica NV, Belgium), APA (10 mg/kg) + Chl (10 mg/kg, Sigma-Aldrich, Buchs, Switzerland) and Chl (10 mg/kg) were subjected to intraperitoneal (i.p.) injections. Half of the animals of each treatment group (three/treatment) were kept for the duration of 2 and other half for 3 weeks. At the end of the experiments, animals were sacrificed and all samples were assessed for tumor weight and size.

The tumor samples obtained from each mouse were divided into two pieces. One part was snap-frozen for gene and protein analysis. The second part of the tissue was fixed in 10% buffered formalin (Fisher Scientific, Norcross, GA), then processed and finally embedded in soft paraffin (Sargent-Welch Scientific, Skokie, IL). Paraffin sections were prepared (5 μm) and further processed. Haematoxylin and eosin (H&E, Sigma-Aldrich, Buchs, Switzerland) and Masson’s Trichrome (Sigma Aldrich, Buchs, Switzerland) staining were performed according to the manufacturer’s protocol.

Paraffin-embedded tumor samples were first de-paraffinized by treatment with xylene and then rehydrated by passage through a graded series of ethanol. The indirect immunostainings of tissue sections were performed at 4 °C overnight using the following primary antibodies for the autophagy-related proteins: anti-ATG5 (1:100, 0262-100, 7C6, nanoTools, Taningen, Germany), anti-Beclin 1 (1:200, NB110-87318, NanoTools, Taningen, Germany), LC3 (1:100, 0231–100, 5F10, nanoTools, Taningen, Germany), anti-Caspase 3, active (1:100, cleaved, AB3623, Merck, Switzerland), and anti-Ki-67 (1:100, AB9260, Merck, Switzerland). The slides were incubated with the secondary antibodies goat anti-mouse FITC (1:500, BD Biosciences Allschwil, Switzerland), goat anti-rabbit FITC (1:500, Vector Laboratories, Liestal, Switzerland) or Cy3-conjugated goat anti-mouse antibody (1:1000, Sigma Aldrich, Sigma Aldrich, Buchs, Switzerland) at room temperature for 1 h. Subsequently, they were counter-stained with DAPI (4′,6-diamidino-2-phenylindole, 1:200, Sigma Aldrich, Buchs, Switzerland). For negative controls, the primary antibody was omitted. Images were acquired with a Leica fluorescence microscope (CTR 6000).

The harvested tumor samples were pulverized in liquid nitrogen with a mortar/pestle and re-suspended in modified lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, Buchs, Switzerland). Samples were centrifuged for 20 min at 13,000 rpm and the supernatant was collected for protein determination. Total protein was measured using a BCA protein assay kit (Thermo scientific, Lausanne, Switzerland). Protein at 1 mg/mlL concentration was used for the WES using a 12–230 kDa cartridge kit (Protein Simple WES, Germany). Primary antibodies for autophagy-related proteins were mouse anti-ATG5 (1:100, NanoTools, Taningen, Germany), rabbit anti-Beclin1, rabbit anti-P62, and mouse anti-LC3B (all 1:50, Novus Biologicals Europe, Abingdon, United Kingdom). Mouse anti-GAPDH (1:100, Novus Biologicals Europe, Abingdon, United Kingdom) served as internal control. Samples were analyzed using the Compass software (ProteinSimple). Virtual blot and electropherogram of each sample was checked and evaluated. A sharply defined chemiluminescent signal was quantified by the software and the area of each sample was normalized to GAPDH.

Results were analyzed by one-way ANOVA with Bonferroni’s post correction using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, version 7). P values < 0.05 were considered statistically significant. All data presented are expressed as means with corresponding standard error of the mean (± SEM).