PBMC were separated using Ficoll-Hypaque (Sigma-Aldrich) and stained with anti -CD19 PE and -

Transcription

1 Supplemental Methods CLL cell preparation and culture PBMC were separated using Ficoll-Hypaque (Sigma-Aldrich) and stained with anti -CD19 PE and - CD5 FITC antibodies (Miltenyi Biotec). When the CD19 + /CD5 + cells were < 95%, CLL cells were purified by negative selection 1 and cultured in RPMI-1640 medium with 10% of fetal calf serum (FCS) and penicillin/streptomycin (all from Sigma-Aldrich). A humidified hypoxia incubator chamber (StemCell Technologies, Vancouver, Canada) was used for hypoxic cultures. Nurse-like cell (NLC) generation NLC were generated by culturing PBMC from CLL patients under normoxic or hypoxic conditions. 2 Cell morphology and numbers were determined by Giemsa staining. Phenotype was determined by immunocytochemistry. For HPLC experiments, AIM V serum-free medium (ThermoFisher Scientific) was used. Reagents AMP, adenosine, inosine, HPLC-grade reagents (acetonitrile, ammonium acetate ph 3.0 and methanol), CGS21680 (the A2A receptor agonist), SCH58261 (the A2A receptor antagonist) and EHNA hydrochloride (erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride) were purchased from Sigma-Aldrich and fludarabine and ibrutinib from Selleckchem. Detection of hypoxia using pimonidazole hydrochloride (PIM) Cells were incubated with PIM (400 µm, 2 hours, 37 C; Hypoxyprobe, Inc), fixed with 4% paraformaldehyde (PFA, 10 min, room temperature), permeabilized with 0.1% saponin (20 min, room temperature), saturated with goat serum (10 min, 4 C), and stained with a FITC-conjugated mouse anti-pim antibody (1:200, 1 hour, 4 C, Hypoxyprobe, Inc). Fluorescence was analyzed by

2 FACS or confocal microscopy. For confocal microscopy studies, cells were spun onto glass slides and counter-stained with DAPI (4,6 diamidino-2-phenylindole; ThermoFisher Scientific). Antibodies used for immunohistochemistry Primary antibodies used for immunohistochemistry and immunofluorescence studies were: rabbit polyclonal anti-caix (#NB ), anti-a2a (#NB ; both 1:500), mouse monoclonal anti- HIF-1α (clone H1alpha67, #NB , 1:50, all from Novus Biologicals), anti-ki-67 (#ab66155, 1:100, Abcam), anti-cd2 (#HPA003883, 1:750, from Sigma-Aldrich), rabbit monoclonal anti-foxp3 (clone SP97, #NBP , 1:100, Novus Biologicals), goat polyclonal anti-cd23/fc (#AF-123, 10 µg/ml), anti-il-10 (#AF-217, 1 µg/ml) and anti-pd-1 (#AF-1086, 2.5 µg/ml, all R&D Systems), goat monoclonal anti-cd3ε (clone M-20, #sc-1127, 1:500, Santa Cruz), mouse monoclonal anti-cd3 (clone PS1, #NCL-L-CD3-PS1, 1:200), anti-cd20 (clone L26, #NCL-L-CD20-L26, 1:50, both from Novocastra), anti-ki-67 (clone MIB-1, #M7240, 1:100, Dako), anti-cd68 (clone KP1, #ab955, 1:200, Abcam), and anti-cd4 (clone UMAB64, #UM800010, 1:100, OriGene). The choice between anti- CD23 or anti-cd20 to stain neoplastic B cells was dictated by the need to obtain the best antibody combination for double or triple stainings. Secondary antibodies used for tissue immunofluorescence were: AlexaFluor 488-conjugated goat anti-mouse and anti-rabbit IgG (1:50 and 1:100, respectively), AlexaFluor 633-conjugated goat anti-mouse IgG (1:100, all from ThermoFisher Scientific), DyLight 488-conjugated bovine anti-goat IgG and DyLight 594-conjugated donkey anti-rabbit IgG (1:100, both from Jackson Immunoresearch). Antibodies used for immunofluorescence Antibodies used for immunofluorescence were: anti-cd68 AlexaFluor488 (clone Y1/82A, #333812), -CD163 AlexaFluor647 (clone RM3/1, #326508, both from Biolegend), -CD206 AlexaFluor488 (clone 19.2, # , ebioscience), -CD73 (#HPA017357, Sigma-Aldrich), -perk1/2 (clone 20A,

3 #612359), -NF-kB p65 (clone 20/NF-kB/p65, #610868, both BD Transduction Laboratories), - pstat3 (clone D3A7, #9145), -pakt (#9271, all Cell Signaling Technology). Reactions were visualized using the following secondary antibodies: AlexaFluor 488-conjugated goat anti-mouse IgG (1:50) and AlexaFluor 488-conjugated goat anti-rabbit IgG (1:100, both from ThermoFisher Scientific). Antibodies used for flow cytometry and Cell Sorting Antibodies used for flow cytometry were: anti-cd19 PE (LT19), -CD19 FITC (LT19), -CD5 FITC (UCHT2), -CD73 APC (AD2), -CD39 PE (ebioa1), -CD73 PE (AD2), -CD3 PE (OKT3), -CD3 APC (OKT3), - CD4 PE (RPA-T4), -CD4 PE-Cy5 (RPA-T4), -CD8 PerCP-Cy5.5 (RPA-T8), -CD14 PE (61D3, all from ebioscience), -CD39 APC (A1), -CD26 PE-Cy5 (BA5b), -CD45 PerCP-Cy5.5 (2D1, all from BioLegend), -CD4 APC-H7 (clone RPA-T4), -CD25 FITC (M-A251), -CD127 PE-Cy7 (HIL-7R-M21), -CD45RA APC-H7 (clone HI100, all BD Pharmingen), -CCR7 FITC (#150503, R&D Systems). Data were acquired using a FACSCanto II cytofluorimeter and processed with FACSDiva v8 (BD Biosciences, Milan, Italy) and FlowJo v10 softwares (TreeStar, Ashland OR). For T lymphocyte isolation, cells were stained using anti-cd19-pe, -CD5-FITC and sorted on a FACSAria III (BD Biosciences), selecting for CD5 + /CD19 - cells. RNA extraction and quantitative real-time PCR (qrt-pcr) RNA was extracted using RNeasy Plus Mini kit (Qiagen, Milan, Italy) and converted to cdna using the High Capacity cdna Reverse Transcription kit (Applied Biosystems). Primers used are listed in Supplemental Table 1. The comparative CT method was used to calculate expression relative to the endogenous control ACTB. 2 Western blot analysis Lysates were resolved by SDS PAGE and transferred to nitrocellulose membranes (Bio-Rad). The following antibodies were used: anti-hif-1α (#NB , Novus Biologicals) and -pan ERK1/2

4 (#610124, BD Transduction Laboratories). Bands were detected with an HRP-conjugated goat antimouse IgG antibody (PerkinElmer). An anti-actin HRP-conjugated antibody was used as a loading control (sc-1616, Santa Cruz). Blots were developed using enhanced chemiluminescence (GE Healthcare). Images were acquired with the ImageQuant LAS 4000 ChemiDoc (GE Healthcare). Detection of hypoxia in a human CLL xenograft model In brief, CD3 + T cells from total 2 CLL patient PBMC were activated with CD3/CD28 Dynabeads (ThermoFisher Scientific) and IL-2 (30 U/ml, PeproTech) in vitro for 10 days. Autologous activated T cells and CLL PBMC were then injected through retro-orbital veins into NOD/SCID/gamma chain -/- (NSG) mice. Spleens were collected 5 weeks after tumor cell injection. Two hours before sacrificing, animals were intraperitoneally injected with PIM (100 mg/kg), and PIM incorporation was evaluated by flow cytometric analysis and immunohistochemistry using a FITC-conjugated anti-pim antibody and HRP-conjugated anti-fitc secondary antibody (Hypoxyprobe, Inc). Immunohistochemistry and immunofluorescence microscopy Sections were deparaffinized and endogenous peroxidase activity blocked with a 6% of hydrogen peroxide solution. Epitope retrieval was performed in 10 mm citrate buffer ph 6 (40 min, 98 C). Primary antibodies used for immunohistochemical and immunofluorescence studies are listed above. Anti-goat HRP-conjugated kit (Biocare Medical), anti-mouse and anti-rabbit HRPconjugated antibodies and 3,3 -diaminobenzidine (EnVision System, Dako) were used to visualize the reaction. For immunofluorescence, tissue sections were incubated with the secondary antibodies indicated in SM. Where indicated samples were counter-stained with AlexaFluor 568- conjugated phalloidin (1:100), DAPI (1:30,000) and mounted in SlowFade Gold reagent (all from ThermoFisher Scientific).

5 Slides were analyzed using an AXIO Lab.A1 (Zeiss), equipped with a Canon EOS600D reflex camera and the images were acquired using the ZoomBrowserEX software (Canon). Quantification of brown signal was measured with the LAS Application Suite software (Leica Microsystems). Immunofluorescence studies were performed using a TCS SP5 laser scanning confocal microscope equipped with 4 lasers, a 20x/0.5, oil immersion 40x/1.25 and 63x/1.4 objective lenses (Leica Microsystems). Images were acquired using the LAS AF version Lite 2.4 software (Leica Microsystems) and processed with Adobe Photoshop (Adobe Systems). Pixel intensities were analyzed using the ImageJ software (downloadable at Immunocytochemistry of NLC cultures For Giemsa staining, cells were first cultured on glass coverslips, fixed with ice-cold methanol (5 min, room temperature; Sigma-Aldrich) and stained with Giemsa (30 min, room temperature; Merck). For the immunocytochemistry analysis, adherent cells were stained with the primary and secondary antibodies indicated in SM. Cells were then fixed with 4% PFA, permeabilized with 0.1% saponin and counter-stained with AlexaFluor 568-conjugated phalloidin (1:100) and DAPI (1:30000, both from ThermoFisher Scientific). Fluorescence was measured by confocal microscopy and pixel intensity was calculated using the ImageJ software. HPLC measurement of AMP, adenosine and inosine Purified CLL cells (2 x 10 6 /0.5 ml) and NLC from normoxic and hypoxic cultures were pre-treated with the adenosine deaminase inhibitor EHNA hydrochloride (10 µm, 30 min, 37 C), and incubated (30 min, 37 C) in AIM V medium with 200 µm AMP. At the end of the incubation time, supernatants were ultra-filtered and 50 µl of each sample were injected into the HPLC system. Chromatographic analysis was performed using a Waters Alliance 2965 Separation Module equipped with silica-based, reversed-phase Atlantis dc18 column (5 µm, 4.6 x 150 mm) and Atlantis dc18 Guard column (5 µm, 4.6 x 20 mm; all from Waters). Separation of AMP, adenosine

6 and inosine was carried out using a method based on a binary mobile phase which consists of 7 mm ammonium acetate (Buffer A), ph 3, and acetonitrile (Buffer B) with a flow rate of 1 ml/min and UV detection set at 260 nm. Peak identities were confirmed by using standard compounds and concentrations were calculated by comparing the peak area of the samples with calibration curves for the peak areas of each standard compound. All concentrations measured on CLLderived supernatants are expressed as nmoles/10 6 cells. For the NLC experiments, adenosine concentrations are expressed as nmoles/mg of protein, because of the variability of cell number in different wells. Cyclic AMP (camp) measurement camp intracellular levels before and after A2A cross-linking in the different culture conditions listed were determined using a commercially available kit (GE Healthcare). Apoptosis assays CLL cells (3 x 10 5 /0.2 ml) were plated in 96 well plates and preincubated for 24 hours in normoxic or hypoxic conditions. After pretreatment with SCH58261 (10 µm, 30 min, 37 C), cells were incubated 48 hours in the presence of 5 µm fludarabine or 10 µm ibrutinib in normoxic and hypoxic conditions. Proliferation assays T-cell proliferation assays were performed after CFSE labeling of purified PBMC. Cells (3 x 10 5 /0.2 ml) were then plated in 96 well plates, pretreated with CGS21680 and SCH58261 (both 1 µm, 30 min, 37 C) and activated with an anti-cd3 monoclonal antibody (0.5 µg/ml) and anti-cd28 (1 µg/ml, clone CD28.2, ebioscience). After 72 hours of incubation under normoxic and hypoxic conditions, PBMC were stained with anti-cd4 and -CD8 mabs (as indicated in the flow cytometry section) and the percentage of CFSE + T cells was evaluated. Chemotaxis assay

7 Freshly isolated PBMC from normal donors (1 x 10 6 cells/well) were resuspended in 100 µl RPMI medium plus 0.5% bovine serum albumin (BSA, Sigma-Aldrich) and plated in the upper chamber of 6.6 mm diameter transwell culture insert in bare polycarbonate with a 5 μm pore size (Corning Incorporated). The lower chamber of each well contained 600 µl of NLC spent media derived from normoxic and hypoxic cultures with or without A2A agonist and antagonist. RPMI medium plus 0.5% BSA and 10% FCS were used as negative and positive internal control, respectively. After 4 hours of incubation (37 C, 5% CO 2 ), 3 aliquots of 100 µl were recovered from each lower chamber. Samples were analyzed by flow cytometry counting for 30 seconds under a defined flow rate in order to obtain the relative number of total transmigrated cells. The remaining 300 µl of the lower chamber were labeled with anti-cd19 FITC, -CD3 APC and -CD14 PE and used to determine the percentage of transmigrated monocytes, B and T cells. Migration index (MI) was calculated as number of CD14 + cells transmigrating in presence of NLC spent medium per number of CD14 + cells transmigrated in presence of RPMI-1640 medium plus 0.5% BSA. Measurement of cell metabolism Freshly purified CLL cells (5 x 10 6 cells/well) were plated 48 hours under normoxia and hypoxia in the presence or absence of the A2A antagonist SCH58261 (10 µm). At the end of the culture, CLL lymphocytes were counted and plated (5 x 10 5 cells/well) in XFp plates (Seahorse Bioscience) for ECAR measurement. T cells (1.5 x 10 5 cells/well) were plated in XFp plates (Seahorse Bioscience) and ECAR was measured using Glycolysis Stress Test kit (Seahorse Bioscience) in basal condition and in response to 10 mm glucose, 1 μm oligomycin and 50 mm of 2-Deoxy-D-glucose (2-DG). Data were analyzed using Wave software (Seahorse Bioscience) and expressed as mean ± SEM from 4 separate experiments. n = 3 replicated per sample.

8 Statistical analyses Data are reported as box plots, where the top and bottom of the rectangle define the third and first quartile, respectively, and the whiskers define the maximum and minimum values. The line inside the rectangle represents the median value. Data were analyzed with GraphPad Prism v6 (GraphPad Software) software. Differences were considered significant when P-values were Results are shown as means ± SD.

9 Supplemental References 1. Vaisitti T, Aydin S, Rossi D, et al. CD38 increases CXCL12-mediated signals and homing of chronic lymphocytic leukemia cells. Leukemia. 2010;24: Audrito V, Serra S, Brusa D, et al. Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia. Blood. 2015;125(1):

10 Supplemental Table Table S1. Primers used for RT-PCR analyses. Gene ACTB ADORA1 ADORA2A ADORA2B ADORA3 CCL3 DPP4 ENTPD1 FOXP3 HIF1A IDO1 INFG IL6 IL10 IRF4 LDHA NT5E PDCD1 PKM2 SLC2A1 SLC16A3 SLC29A1 Assay Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _g1 Hs _m1 Hs _m1 Hs _s1 Hs _m1 Hs _m1 Hs _g1

11 SLC29A2 TGFB1 Hs _m1 Hs _m1

12 Supplemental Figures Supplementary Figure S1. The CLL niche is a hypoxic environment where HIF-1α target genes are differentially modulated. (A) Triple staining of a representative CLL LN section with anti-cd23 (green), -Ki-67 (blue) and -CAIX (red). The proliferating (Ki-67 + ) CLL (CD23 + ) cells are intensively CAIX + (Figure S1A). (B) Immunofluorescence staining of a CLL LN section highlighting the blood vessel (CD31 +, white), indicated in the white squares, and the hypoxic cells (CAIX +, red). Box plot represents the quantification of the mean fluorescence intensity of CAIX. An inverse correlation between the intensity of the staining and the distance from the vessels has been found, with cells in perivascular position being significantly less CAIX + than cells in other positions. (C) Immunohistochemical staining with an anti-cd20, HIF-1 and CAIX antibodies of representative spleen sections of NSG engrafted mice with human CLL cells. Original magnification x20. (D) Double and triple staining (from left to right) with anti-human CD20 (green), -CD3 (white) and - CAIX (red) of spleen sections obtained from NSG mice engrafted with human CLL cells. Original magnification x20 (left and middle panels) and x40 (right panel). Scale bars, 75 µm. (E) RT-PCR analysis of GLUT1 (SLC2A1), LDH (LDHA) and PKM2 (PKM2) mrna expression in PB, BM and LN obtained at the same time point from CLL patients (n=12). The Wilcoxon signed rank test was used for statistical analysis. Images were acquired using a TCS SP5 laser scanning confocal microscope with a 20x/0.5 and an oil immersion 63x/1.4 objective lenses (Leica Microsystems) and processed with Adobe Photoshop. Perivascular, PV; PB, peripheral blood; BM, bone marrow; LN, lymph node.

13 Figure S1

14 Figure S2. Setup of hypoxic culture conditions. (A) RT-PCR analysis of HIF-1α expression in CLL cells cultured under normoxic (21% O 2, N) or hypoxic (1% O 2, H) conditions. (B) Western blot analysis of HIF-1α expression on CLL cells after normoxic or hypoxic culture. ERK1/2 levels are shown as protein loading control. (C) Immunofluorescence analysis of CAIX expression on CLL cells under N or H (left panels). Hypoxia was confirmed by PIM treatment of CLL cells (400 µm, 2 hours, 37 C) and staining using a FITC-labeled anti-pim antibody (right panels). Nuclei were counterstained with DAPI and samples analyzed by confocal microscopy. Original magnification x63. Figure S2

15 Figure S3. Correlation between the HIF-1α transcription factor and CD73 expression in the CD73 + CLL subset. (A) RT-PCR analysis of CD73 (NT5E) mrna in CLL patients with CD73 expression <30% after 48 hours of normoxia or hypoxia. (B-C) Pearson correlation between HIF-1α (HIF1A) and CD73 (NT5E) mrna levels in CLL patients over (B) and lower (C) the 30% cutoff value of CD73 expression. Results indicate a positive correlation in the CD73 + CLL subset. N, normoxia (open box); H, hypoxia (gray box). Figure S3

16 Figure S4. Hypoxia favors AMP degradation and increases glycolytic capacity of CLL cells. (A) Box plots showing nmol of AMP degraded by 10 6 purified CLL cells after 30 min of normoxic or hypoxic culture in the presence of 200 µm AMP (n=8). (B) Cumulative data of ECAR and maximal glycolysis measured in CLL cells after 48 hours of normoxia or hypoxia. Treatment with the A2A antagonist SCH58261 significantly reverted the effect of hypoxia (n=8). The Wilcoxon signed rank test was used for statistical analysis. N, normoxia (open boxes); H, hypoxia (gray boxes); SCH, SCH Figure S4

17 Figure S5. Hypoxia increases intracellular camp levels following A2A activation. Intracellular camp levels were determined in primary CLL cells from 3 different patients following ligation of the A2A receptor using ADO (100 M) or CGS21680 (10 M) for 60 minutes. Figure S5

18 Figure S6. Hypoxia increases survival of CLL cells in an A2A-dependent way. (A) Contour plots showing the profiles of live and apoptotic cells in a representative CLL patient after 48 hour cultures under normoxic or hypoxic conditions with and without the A2A antagonist SCH58261 (10 µm). Where indicated, ibrutinib (10 µm) or fludarabine (5 µm) were added to CLL cells culture before assessing apoptosis. (B) Western blot analysis of Btk expression in 4 different CLL patients. Cells were cultured in normoxic or hypoxic conditions. Actin was used as loading control. N, normoxia; H, hypoxia; SCH, SCH58261; Ibr, ibrutinib; Fluda, fludarabine. Figure S6

19 Figure S7. The A2A adenosine receptor is expressed by stromal cells within CLL LN. (A) Representative immunohistochemical images of CLL LN sections stained with anti-human A2A. Low magnification shows A2A receptor expression around areas corresponding to the CLL proliferation centers. Original magnification x10 (left panel) and x40 (right panel). Samples were analyzed using an AXIO Lab.A1 microscope (Zeiss, Milan, Italy) equipped with a Canon EOS600D and images acquired with the ZoomBrowserEx software (Canon). (B) Box plots show cumulative data of NLC counts obtained counting 5 randomly chosen fields for each of the 8 different samples analyzed. N, normoxia; H, hypoxia; CGS, CGS21680; SCH, SCH Figure S7

20 Figure S8. Gating strategy used for sorting T lymphocytes from activated CLL PBMC preparations. (A) PBMC were plated (3x10 6 /ml) in the presence of anti-cd3, -CD28 antibodies (2 µg/ml and 1 µg/ml, respectively) and IL-2 (100 U/ml) for 5 days. Cells were then collected from the culture and stained using CD19 and CD5. Sorting was performed using a FACSAriaIII (BD Biosciences) and selecting for CD19 - /CD5 + cells. Purity of sorted cells was confirmed by staining for anti-cd3 (lower dot blot). (B) Representative CFSE profiles of CD4 + cells after 3 days of culture in normoxia or hypoxia in the presence of anti-cd3 (0.5 µg/ml), -CD28 (1 µg/ml) antibodies and the A2A agonist CGS or antagonist SCH58261 (both at 1 µm, added at the beginning of culture). (C) RT-PCR analysis of A2A (ADORA2A) receptor expression on sorted T cells after 5 days of normoxic or hypoxic culture and activated as above described (n=15). N, normoxia; H, hypoxia; CGS, CGS21680; SCH, SCH Figure S8

21 Figure S9. Hypoxia increases VEGF and PD-1 expression in T cells obtained from CLL patients. (A) RT-PCR analysis of VEGF (VEGFA) expression and production in T lymphocytes obtained from CLL patients under normoxic and hypoxic conditions. Where indicated, cells were treated with SCH58261 (10 µm). (B) Double staining of a representative CLL LN section with anti-pd-1 (green) and -CD4 (red). Cells were counterstained with DAPI and section analyzed using a TCS SP5 laser scanning confocal microscope with an oil immersion 63x/1.4 objective lens (Leica Microsystems). Images were processed with Adobe Photoshop. Original magnification x63. Scale bars, 25 µm. N, normoxia; H, hypoxia; SCH, SCH Figure S9