Fetal bovine serum (FBS) and culture medium were from Invitrogen Life Technologies (Carlsbad, CA). Plasticware for cell cultures was from Falcon (Becton Dickinson, Franklin Lakes, NJ). The protein content was assessed with the BCA kit from Sigma Aldrich. (St. Louis, MO). Cis-diammineplatinum (II) dichloride, docetaxel, elesclomol were purchased by Sigma Aldrich. [10-(2,5-dihydroxy-3,4-dimethoxy-6,ethylphenyl)decyl]triphenyl-phosphonium, monomethanesulfonate (mitoquinol or mitoQ) was from Cayman Chemical (Ann Arbor, MI).
Human NSCLC cells (NCI-H1650, NCI-H1385, NCI-H460, NCI-H522, NCI-H661, NCI-H2126, NCI-H23, NCI-H1703, NCI-H1435, NCI-H596, NCI-H2286, NCI-H1437, NCI-H1651, NCI-H2085, NCI-H2342, NCI-H2073, NCI-H1793, NCI-H2170, NCI-H1299, NCI-H2066, NCI-H2347, NCI-H1734, NCI-H1563, NCI-H441, NCI-H1975, A549, Calu-3, NCI-H2228) were purchased from ATCC (Manassas, VA). Cells were maintained in the respective medium supplemented with 10% v/v FBS, 1% v/v penicillin-streptomycin, 1% v/v L-glutamine, in a Heracell incubator (ThermoFisher, Waltham, MA) with different pO2. The experimental conditions were set as it followed: 24 h at 20% O2 (normoxia), 24 h at 1% O2 (hypoxia), 12 h at 1% O2 followed by 12 h at 20% O2 (hypoxia/normoxia), 12 h at 1% O2 followed by 12 h at 20% O2 and 12 h at 1% O2 (hypoxia/normoxia/hypoxia or intermittent hypoxia).
After the growth in normoxic or hypoxic conditions indicated, cells were incubated 48 h in normoxia (20% O2), with increasing concentrations (from 1 × 10− 9 to 1 × 10− 5 M) of cisplatin or docetaxel. Cell viability was assessed by the ATPlite Luminescence Assay System (PerkinElmer, Waltham, MA), as per manufacturer’s instructions, using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments, Winooski, VT) to measure the relative luminescence units (RLUs). The RLUs of untreated cells were considered as 100% viability; the results were expressed as a percentage of viable cells versus untreated cells. The IC50 was calculated applying the [inhibitor] vs. normalized dose-response equation (GraphPad Prism 9 software, https://www.graphpad.com/).
1 × 104 cells were washed in phosphate-saline buffer (PBS), pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM EDTA, centrifuged at 300×g for 10 min, incubated 20 min at room temperature in the dark with 250 μl of Inside Fix reagent (Inside Stain Kit, Miltenyi Biotec., Bergisch Gladbach, Germany), centrifuged at 300×g for 5 minutes, washed with 1 ml of Inside Perm (Inside Stain Kit), centrifuged at 300×g for 5 minutes, and incubated 30 minutes at room temperature with the following antibodies (all from Miltenyi): anti-CD243/ABCB1 antibody (PE-Vio® 770-conjugated); anti-MRP1/ABCC1 antibody (PE-conjugated); anti-ABCA1(DyLight 488-conjugated). Cells were washed with 1 ml of Inside Perm reagent, centrifuged at 300×g for 5 minutes and read using a Guava® easyCyte flow cytometer (Millipore, Billerica, MA), equipped with the InCyte software (Millipore).
PCR arrays and qRT-PCR
Total RNA was extracted and reverse-transcribed using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). The PCR arrays were performed on 1 μg cDNA, using customized Plus PCR Arrays (Bio-Rad Laboratories) pre-coated with primers for the ABC transporters or the transcription factors indicated in the Results section, as per manufacturer’s instructions. Data analysis was performed with the PrimePCR™ Analysis Software (Bio-Rad Laboratories). The qRT-PCR was performed with the IQ SYBR Green Supermix (Bio-Rad Laboratories). The primer sequences, designed using the qPrimerDepot database (http://primerdepot.nci.nih.gov/), were reported in the Additional File 1 (Supplemental Table S1). The relative quantification was performed by comparing each PCR product with the housekeeping PCR product S14, using the Bio-Rad Software Gene Expression Quantitation (Bio-Rad Laboratories).
Promoter and transcription factors analysis
The promoter sequences were identified from the Eukariotic Promoter Database (EPD; https://epd.epfl.ch//index.php), using as inputs: https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=ABCB1_1 (RefSeq NM_001348945) for ABCB1, https://epd.epfl.ch/search_EPDnew.php?query=ABCC1&db=human (RefSeq NM_004996) for ABCC1, https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=ABCA1_1 (RefSeq NM_080282) for ABCA1. The transcription factors predicted to bind ABCB1, ABCC1 and ABCA1 promoters (Additional File 1; Supplemental Table S2) were identified using the TRANSFAC software v 8.3 (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) .
Cells were lysed in the MLB buffer (125 mM Tris-HCl, 750 mM NaCl, 1%v/v NP40, 10%v/v glycerol, 50 mM MgCl2, 5 mM EDTA, pH 7.5, supplemented with the protease inhibitor cocktail set III (Sigma Aldrich), 25 mM NaF, 1 mM NaVO4, 10 mg/ml aprotinin), sonicated and centrifuged at 13000×g for 10 minutes at 4 °C. 50 μg of proteins were subjected to immunoblotting and probed with the following antibodies: anti-HIF-1α (clone 54, BD Transduction Laboratories), C/EBP-β (clone C-19, directed against the common C-terminus of LIP and LAP, Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by a peroxidase-conjugated secondary antibody. Anti-actin (clone C-4, Sigma Aldrich) was used as a control of equal protein loading. The proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). In co-immunoprecipitation assays, 100 μg of whole cell lysates were immuno-precipitated at 4 °C overnight with the PureProteome Protein A/G Mix Magnetic Beads (Millipore) as per manufacturer’s instruction, in the presence of the anti-C/EBP-β antibody (clone C-19, diluted 1/50), then blotted with the anti-HIF-1α antibody as reported above.
RNA immunoprecipitation (RNA-IP)
Total RNA was isolated using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Sigma Aldrich), as per manufacturer’s instructions. 2 μg RNA were immuno-precipitated for 3 h at 4 °C using an anti-HIF-1α antibody (PA1–16601, Invitrogen Life Technologies, Milan, Italy). A blank was prepared by incubating the samples without the antibody. The immunoprecipitated RNA was retro-transcribed using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories). RT-PCR was performed with the IQ SYBR Green Supermix (Bio-Rad Laboratories), using primers upstream and downstream the hypoxia-response elements (HREs) contained in C/EBP-β LAP and LIP, respectively (Additional File 1; Supplemental Table S1). The expression in normoxic cells was considered as 1. The levels of LAP and LIP mRNA in the other experimental condition were expressed as fold-enrichment versus normoxic cells, using the Bio-Rad Software Gene Expression Quantitation (Bio-Rad Laboratories).
Chromatin immunoprecipitation (ChIP)
Cells were lysed and sonicated as reported previously . 200 μl of samples were used as inputs. The remaining lysates were pretreated for 2 h at 4 °C with the Magna ChIP™ Protein A + G Magnetic Beads (Sigma Aldrich), as per manufacturer’s instructions. Samples were incubated overnight with the anti-C/EBP-β antibody (clone C-19, Santa Cruz Biotechnology Inc.) or without antibody, as a blank. The recovered DNA was washed, eluted with the elution buffer (0.1 M NaHCO3, 1% w/v SDS), heated at 65 °C for 6 h and incubated with proteinase K for 1 h at 55 °C. Samples were cleaned by Qiaquick spin columns (Qiagen, Venlo, The Netherlands). The CAAT sites on ABCB1, ABCC1 and ABCA1 promoters were identified using the Transfac® Database (http://genexplain.com/transfac/). Inputs and immunoprecipitated samples were analysed by RT-PCR, using primers designed to amplify the sequence around the CAAT sites in ABCB1, ABCC1 and ABCA1 promoters, as well as nonspecific primers, used as negative internal controls (Additional File 1; Supplemental Table S1).
1 μg pcDNA4/TO expression vectors (Invitrogen Life Technologies) for C/EBP-β LAP and LIP, produced as reported previously , were co-transduced with 1 μg pcDNA6/TR vector (Invitrogen Life Technologies) in 1 × 106 cells. Stable TetON clones were generated by selecting cells with 2 μg/ml blasticidin S (ThermoFisher) and 100 μg/ml zeocin (InvivoGen, San Diego, CA). LAP and LIP induction was activated by adding 1 μg/ml doxycycline (Sigma Aldrich) in the culture medium. LAP and LIP expression was analyzed by immunoblotting 24 h after doxycycline treatment. For silencing, 1 × 106 cells were transduced with 1 μg of a green fluorescence protein (GFP)-lentiviral plasmid containing a non-effective 29-mer scrambled shRNA cassette (Origene, Rockville, MD), two different sequences targeting HIF-1α (TL320380, Origene), two different sequences targeting C/EBP-β (TL320301, Origene). Stably silenced clones were generated by selecting cells with 0.25 μg/ml puromycin (InvivoGen) for 4 weeks. The silencing was verified by RT-PCR and immunoblotting.
Release of IPP
The efflux of IPP was measured by radiolabelling 1 × 106 cells for 1 h with 0.02 mCi of [14C]-IPP (50 mCi/mmol; Amersham International, Piscataway, NJ), extracting lipids, isolating the IPP by thin layer chromatography and counting radiolabelled IPP by liquid scintillation . Results were expressed as nanomoles/ml, according to the relative calibration curve.
Vγ9Vδ2 T-lymphocytes activation and tumor killing
Blood samples were obtained from healthy blood donors (Blood Bank of the AOU Città della Salute e della Scienza, Torino, Italy; DG 767/2015). After isolation on a Ficoll-Hypaque density gradient, peripheral blood mononuclear cells (PBMC) were sorted using the TCRγ/δ+T Cell Isolation Kit (Miltenyi Biotec.). The presence of Vγ9Vδ2 T-lymphocytes was confirmed by staining 5 × 105 isolated cells with anti-TCR Vγ9 (VioBlue conjugated; Miltenyi Biotec.) and anti-CD3 (fluorescein-isothiocyanate - FITC - conjugated, Miltenyi Biotec) antibodies. Samples with > 80% Vγ9+/CD3+ cells were incubated 48 h with 1 μM zoledronic acid (Sigma Aldrich) and 10 IU/ml IL-2 (Sigma Aldrich), to expand Vγ9Vδ2 T-lymphocytes , then 5 × 105 Vγ9Vδ2 T-lymphocytes were cultured overnight with NSCLC cells at 1:1 ratio. After this incubation period, the amount of active and proliferating Vγ9Vδ2 T-lymphocytes was measured by staining cells present in the supernatants with anti-Ki67 (FITC-conjugated) and anti-INF-γ (allophycocyanin - APC - conjugated) antibodies (Miltenyi Biotec.), and quantified with a Guava® easyCyte flow cytometer (InCyte software). Results were expressed as percentage of Vγ9+Ki67+IFNγ+over Vγ9+cells. Vγ9Vδ2 T-lymphocytes killing was measured as reported . After Vγ9Vδ2 T-lymphocytes/NSCLC cells co-incubation, adherent (i.e. tumor cells) were washed twice with PBS, detached by gentle scraping and stained with the Annexin V/Propidium Iodide kit (Sigma Aldrich.), as per manufacturer’s instruction. The fluorescence was acquired using a Guava® easyCyte flow cytometer (InCyte software). The percentage of Annexin V+/Propidium Iodide+ cancer cells was considered as an index of Vγ9Vδ2 T-lymphocytes killing.
Electron transport chain (ETC) activity
Mitochondria were isolated from 10 × 106 cells, lysed in 0.5 ml lysis buffer (5 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, 1.8 mM ATP, 1 mM EDTA, pH 7.2), supplemented with Protease Inhibitor Cocktail III, 1 mM phenylmethylsulfonyl fluoride and 250 mM NaF. Samples were centrifuged at 650×g for 3 minutes at 4 °C, the supernatants were re-centrifuged at 13000×g for 5 minutes at 4 °C. The pellets, containing mitochondria, washed with lysis buffer, were resuspended in 0.25 ml resuspension buffer (250 mM sucrose, 15 mM K2HPO4, 2 mM MgCl2, 0.5 mM EDTA). 50 μl aliquots were sonicated and used for the measurement of protein content. 10 μg of each sonicated sample were analyzed by SDS-PAGE and immunoblotting with an anti-porin antibody (clone 20B12AF2, Abcam, Cambridge, UK) to confirm the presence of mitochondrial proteins in the extracts. The electron efflux from complex I to complex III, taken as an index of the mitochondrial respiratory activity , was measured on 50 μg of non-sonicated mitochondrial samples, re-suspended in 0.2 ml of buffer A (5 mM KH2PO4, 5 mM MgCl2, 5%w/v BSA; pH 7.2) and 0.1 ml of buffer B (25%w/v saponin, 50 mM KH2PO4, 5 mM MgCl2, 5%w/v BSA, 0.12 mM oxidized cytochrome c, 0.2 mM NaN3, which blocks complex IV allowing the accumulation of reduced cytochrome c; pH 7.5). The reaction mix was allowed to equilibrate for 5 minutes at room temperature. The cytochrome c reduction reaction was monitored for 5 minutes after adding 0.15 mM NADH, reading the absorbance changes at 550 nm by a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments). Results were expressed as nanomoles of reduced cytochrome c /min/mg mitochondrial proteins.
O2 consumption rate (OCR)
25 × 104 cells were seeded in 96-well microplates (Nunc, Rochester, NY). After 24 h, the Resipher oxygen sensing lid (Lucid Scientific, Atlanta, MA) was positioned upon the plate . Cells were subjected to these culture conditions: 36 h in normoxia, 36 h in hypoxia, 18 h in hypoxia followed by 18 h normoxia, 12 h hypoxia followed by 12 h normoxia and 12 h hypoxia, to monitor the O2 consumption over the same period. Live OCR was monitored continuously for 36 h by measuring the flux of O2 diffusing into the cells from the air above the well. The measurement was performed by sensing the O2 concentration gradient across a range of heights throughout the media and then calculating the flux of O2, according to Fick’s first and second laws . Data were analyzed using the Resipher web application (Lucid Scientific).
ATP levels in mitochondrial extracts were measured with the ATP Bioluminescent Assay Kit (FLAA; Sigma Aldrich), as per manufacturer’s instructions. Results were expressed as nanomoles/mg mitochondrial proteins.
1 × 106 cells were washed with PBS, detached by gentle scraping and incubated for 30 minutes at 37 °C with 2 μM of the fluorescent probe JC-1 (Biotium Inc., Hayward, CA), centrifuged at 13000×g for 5 minutes and re-suspended in 0.5 ml PBS. The red fluorescence (λ excitation: 550 nm, λ emission: 600 nm), index of polarized mitochondria, and the green fluorescence (λ excitation: 485 nm; λ emission: 535 nm), index of depolarized mitochondria fluorescence, were read using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments). The relative fluorescence units (RFUs) were used to calculate the percentage of green (depolarized)/red (polarized) mitochondria, considered an index of damaged mitochondria .
Mitochondrial permeability transition pore (mPTP) activity
The opening of the mPTP, a second index of mitochondria depolarization and damage, was measured with the Mitochondrial Permeability Transition Pore Assay Kit (BioVision, Milpitas, CA), as per manufacturer’s instructions, using a Guava EasyCyte flowcytometer (Millipore), equipped with the InCyte software (Millipore). 1 × 105 unstained cells were used to set the threshold of autofluorescence and subtracted from the stained cells. Results were expressed as percentage of fluorescent cells over total cells.
Total and mitochondrial ROS
10 × 106 cells were washed with PBS and detached by gentle scraping. One 50 μl aliquot was sonicated and used to measure cellular proteins. The remaining cells were treated for 30 min at 37 °C with 5 μM of the ROS-sensitive fluorescent probes 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA) (ThermoFisher) or with 5 μM MitoSOX (ThermoFisher), to measure total and mtROS, respectively. The RFUs were converted into nanomoles ROS/mg proteins, according to a titration curve performed with serial dilutions of H2O2.
Antioxidant enzymes activity
The activity of cytosolic superoxide dismutase 1 (SOD1) and mitochondrial superoxide dismutase 2 (SOD2) was measured on 10 μg proteins of the respective extracts, obtained as indicated above, in 100 μl PBS containing 50 μM xanthine, 5 U/ml xanthine oxidase, 1 μg/ml oxidized cytochrome c. The rate of cytochrome c reduction, which is inhibited by SOD, was monitored for 5 minutes by reading the absorbance at 550 nm with a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments). Results were expressed as μmoles reduced cytochrome c/min/mg cytosolic or mitochondrial proteins . Catalase and glutathione peroxidase (GPX) were measured on whole cell lysates using the Catalase Activity Assay Kit (Colorimetric/Fluorometric) (Abcam) and the Glutathione Peroxidase Assay Kit (Colorimetric) (Abcam), as per manufacturer’s instructions. The absorbance was converted into nmoles/min/mg proteins, according to the titration curves of the kits.
Reduced glutathione (GSH) and oxidized glutathione (GSSG) measurement
1 × 106 cells were rinsed with 480 μl PBS plus 120 μl of 6.5% w/v 5-sulfosalicylic acid to precipitate proteins, incubated at 4 °C for 1 h and centrifuged for 13,000×g for 5 minutes at 4 °C. Total (GSH + GSSG) glutathione was measured in 20 μl of the lysate by adding 20μlofstock buffer (143 mM NaH2PO4 and 63 mM EDTA, pH 7.4), 200 μl of daily reagent (10 M 5,5’dithiobis-2-nitrobenzoic acid and 2 mM NADPH, diluted in stock buffer), 40 μl glutathione reductase (8.5 U/mL). To measure oxidized glutathione (GSSG), 10 μl of 2-vinylpyridine were added to 200 μl of cell lysate for 1 h, to derivatize GSH. The remaining GSSG was measured on 40 μl of lysates, as described above. The reaction kinetics was followed for 5 minutes, reading the absorbance at 415 nm with a Synergy HT Multi-Detection Microplate Reader. Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were expressed as pmoles glutathione/min/mg cellular proteins. GSH was obtained by subtracting GSSG values from (GSH + GSSG) values . Results were then expressed as GSH/GSSG ratio.
Patient enrolment and immunohistochemical analyses
60 patients with confirmed histological diagnosis of NSCLC, candidate to receive cisplatin/carboplatin as first-line therapy, were prospectively enrolled at San Lugi Gonzaga Hospital, Orbassano, and Città della Salute e della Scienza Hospital, Torino, Department of Oncology, University of Torino, Italy (March 2018–January 2020). Each patient was anonymized and indicated as “unknown patient number” (UPN). The pathological features, the smoking habits, the clinical follow-up (progression free survival, PFS; overall survival, OS) of patients, performed at the Thoracic Oncology Unit, San Luigi Gonzaga Hospital, are reported in the Additional File 1 (Supplemental Table S3). Formalin-fixed paraffin-embedded (FFPE) samples of patients were analyzed for the presence of hypoxic areas using the pimonidazole-based Hypoxyprobe™ Kit (Hypoxyprobe Inc., Burlington, MA), according to the manufacturer’s instructions. The same sections were also stained with an antibody recognizing only the N-terminal portion of C/EBP-β, corresponding to LAP (clone 21A1, ThermoFisher), followed by peroxidase-conjugated horseradish antibody (Dako, Glostrup, Denmark). Nuclei were counterstained with hematoxylin (Sigma Aldrich). C/EBP-β LAP was considered positive when a weak-to strong nuclear or cytosolic positivity was shown. The tumor proportion positivity was recorded. Patients were divided into LAPlow and LAPhigh, if the tumor proportion of LAP staining was respectively below or equal/above the median value. The Ethics Committee of San Luigi Gonzaga Hospital, Orbassano, Italy approved the study (#73/2018).
1 × 106 NCI-H2228 C/EBP-β LAP-overexpressing cells, mixed with 100 μl Matrigel (Sigma Aldrich), were injected subcutaneously (s.c.) in female NOD SCID-γ (NSG) mice engrafted with human hematopoietic CD34+ cells (Hu-CD34+; The Jackson Laboratories, Bar Harbor, MA). Mice were housed (5 per cage) under 12 h light/dark cycle, with food and drinking provided ad libitum. When indicated, doxycycline (1 mg/ml) was added daily to the drinking water to induce C/EBP-β LAP intratumorally. Tumor growth was measured daily by caliper, according to the equation (LxW2)/2, where L = tumor length and W = tumor width. In a preliminary experimental set, when tumors reached the volume of 50mm3, animals (4/group) were randomized and treated for 6 weeks as it follows: vehicle group, treated with 0.1 ml saline solution intravenously (i.v.), once a week; cisplatin group, treated with 2 mg/kg cisplatin i.v., once a week; mitoquinol (mitoQ) groups, treated with 10, 25, 50, 100, 200 mg/kg daily via oral gavage; cisplatin+mitoQ groups, treated with 2 mg/kg cisplatin i.v., once a week, and 10, 25, 50, 100, 200 mg/kg daily via oral gavage. In a second experimental set, when tumors reached the volume of 50mm3, animals (6/group) were randomized and treated for 6 weeks as it follows: vehicle group, treated with 0.1 ml saline solution i.v., once a week; cisplatin group, treated with 2 mg/kg cisplatin i.v., once a week; mitoQ group, treated with 100 mg/kg daily via oral gavage; cisplatin+mitoQ group, treated with 2 mg/kg cisplatin i.v., once a week, and 100 mg/kg mitoQ daily via oral gavage. In both experimental sets, tumor volumes were monitored by caliper and animals were euthanized at day 49 after randomization with zolazepam (0.2 ml/kg) and xylazine (16 mg/kg). Animal weights were monitored throughout the study. Tumors were excised, weighted, and photographed. Tumor sections, fixed in 4% v/v paraformaldehyde, were stained with hematoxylin/eosin (Sigma Aldrich) or immunostained for: Hypoxyprobe™ Kit, LAP (clone 21A1, ThermoFisher), C/EBP-β LAP (ThermoFisher), ABCB1 (Novus Biologicals, Centennial, CO), ABCC1 (MyBioSource, San Diego, CA), ABCA1 (Abcam), cleaved (Asp175) caspase-3 (Cell Signaling Technology, Danvers, MA), followed by a peroxidase-conjugated secondary antibody (Dako). Nuclei were counterstained with hematoxylin (Sigma Aldrich). Sections were examined with a Leica DC100 microscope. To evaluate the intratumour Vγ9δ2 T-lymphocytes, the tumors were digested with 1 mg/ml collagenase (Sigma Aldrich) and 0.2 mg/ml hyaluronidase (Sigma Aldrich) for 1 h at 37 °C and filtered using a 70 μm cell strainer to obtain a single cell suspension. Infiltrating immune cells were collected by centrifugation on Ficoll-Hypaque density gradient and immunostained with the following antibodies (Miltenyi Biotec): anti-CD3, anti-Vγ9, anti-Ki67, anti-INF-γ, as indicated in the “Vγ9Vδ2 T-lymphocytes activation” section. Cells were quantified with using Guava® easyCyte flow cytometer and InCyte software. Results were expressed as percentage of Vγ9+Ki67+IFNγ+ over CD3+ cells. At 3.5 weeks and immediately after the euthanasia, 200 μl blood were collected to measure the following parameters: red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), platelets (PLT), as indexes of bone marrow function; lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), as indexes of liver function; creatinine, as index of kidney function; creatine phosphokinase (CPK), as index of muscle/heart damage, using commercially available kits from Beckman Coulter Inc. (Miami, FL). Animal care and experimental procedures were approved by the Italian Ministry of Health (#627/2018-PR, 10/08/2018).
All data in the text and figures are provided as means ± SD. The results were analyzed by a one-way analysis of variance (ANOVA), using Statistical Package for Social Science (SPSS) software (IBM SPSS Statistics v.19). p < 0.05 was considered significant. The Kaplan-Meier method was used to calculate the PFS (survival from the beginning of chemotherapy to the first sign of disease’s progression) and OS (survival from the beginning of chemotherapy until patients’ death). Log rank test was used to compare the outcome of LAPlow and LAPhigh groups. The patient and animal sample sizes were calculated with the G*Power software (www.gpower.hhu.de), setting α < 0.05 and 1-β = 0.80. Researchers analyzing the results were unaware of the treatments received.