Page 124 - 2021_05-Haematologica-web

F. Arruga et al.
Hypoxia, defined as a condition of low oxygen tension, is a feature of the tumor microenvironment, particularly, but not exclusively, of solid tumors.10,11 Hypoxia activates a transcriptional program ruled by hypoxia-inducible fac- tor (HIF)-1α, which contributes to building local condi- tions that shield the tumor from eventual therapies and that blunt immune responses.12,13 Our previous data indi- cate that the CLL lymph node is a hypoxic environment and that hypoxia acts, at least in part, through the activa- tion of extracellular adenosine production and signaling.14
This work shows the modulation of adenosine signaling and the effects of its blockade in the CLL microenviron- ment using the TCL1 adoptive transfer model, making it a suitable option for combination strategies.
Methods
Adoptive transfer TCL1 mouse model
Ten million cells from different TCL1 leukemic clones were intraperitoneally injected (in 200 mL of phosphate-buffered saline) in 8-week-old C57BL/6 female mice and left to engraft. Where indicated, mice were treated with SCH58261 (Selleckchem) administered intraperitoneally at a dose of 1 mg/kg every other day. Treatment was started blindly 4 days after injection of leukemic cells, following published protocols.15 When showing clear signs of disease or at the end of the treatment schedule, mice were euthanized and organs (peritoneal cavity, spleen, peripheral blood and bone marrow) collected. In experiments aimed at eval- uating progressive hypoxia, mice were intraperitoneally injected with 100 mg/kg pimonidazole hydrochloride (PIM; Hypoxyprobe, Inc.) 2 h before euthanasia. All the in vivo experiments were per- formed following the institutional and national guidelines and approved by the Italian Ministry of Health (authorization n. 118/2018-PR).
Antibodies and reagents
A complete list of antibodies used in flow cytometry is reported in Online Supplementary Table S1.
Immunohistochemistry and confocal microscopy
Formalin-fixed paraffin-embedded spleen sections were stained and analyzed as previously reported.14 Full details are provided in the Online Supplementary Information.
Detection of hypoxia using pimonidazole hydrochloride
Cells purified from mice injected with PIM prior to euthanasia, were fixed, permeabilized and stained with a FITC-conjugated mouse anti-PIM antibody (Hypoxyprobe, Inc). Fluorescence was analyzed by flow cytometry. Confocal analyses of PIM staining were performed as described in the Online Supplementary Information.
Real-time polymerase chain reaction
Quantitative real-time polymerase chain reaciont (qRT-PCR) was performed using the 7900 HT Fast Real Time PCR System (SDS 2.3 software). Primers for Entpd1 (Mm00515447_m1), Nt5e (Mm00501920_m1), Adora2a (Mm00802075_m1), Adora2b (Mm00839292_m1), Adora3 (Mm00802076_m1), Ada (Mm00545720_m1), and Actb (Mm02619580_g1) were from Life Technologies.
High performance liquid chromatography measurement of adenosine production
Nucleotide metabolism was analyzed by high-performance liq-
uid chromatography.16 A full description of the methods can be found in the Online Supplementary Information.
Statistical analyses
Data are reported as box plots, with the top and bottom of the rectangle defining the third and first quartile, respectively, and the whiskers defining the maximum and minimum values. The line inside the rectangle represents the median value. Data were ana- lyzed with GraphPad Prism v6 software (GraphPad Software). Differences were considered statistically significant when P-values were ≤0.05. Results are shown as means ± standard deviation. Statistics were calculated using either the Mann-Whitney test for unpaired data or the Wilcoxon test for paired data, as specified for each graph in the figure legends.
Methods described fully in the Online Supplementary Information include the enzyme-linked immunosorbent assay for protein kinase A (PKA) activity, assays of cell degranulation and cytokines release, as wells as the Phosflow and apoptosis assays.
Results
Leukemia engraftment is accompanied by progressive hypoxia
The TCL1 adoptive transfer model is based on the intraperitoneal injection of leukemic cells derived from TCL1 transgenic mice into normal immunocompetent C57BL/6 recipients. Rapid disease engraftment occurs in 100% of cases: after a period ranging from 2 to 5 weeks, depending on the number of cells transferred and on the leukemic clone, B220+/CD5+ leukemic cells appear in the peripheral blood (Figure 1A). At this moment, the spleen already shows signs of leukemic colonization, with large nodules progressively effacing the normal tissue architec- ture (Figure 1B). Terminally ill animals show signs of widespread disease, with massive colonization of the spleen, bone marrow, peritoneal cavity and peripheral blood. At this stage, no architecture is recognizable in the spleen, which is composed of a sheet of cells, with signif- icant necrosis (Figure 1B). Following the hypothesis that leukemic colonization is accompanied by the progressive onset of hypoxic conditions, we stained for carbonic anhydrase IX (CAIX), an enzyme that is under the direct transcriptional control of HIF-1α, the master regulator of hypoxia. Images show a gradient of staining intensity from wild-type (WT) to terminally ill mice (Figure 1B). To confirm this observation, we injected mice with PIM, a compound that forms stable adducts with cellular macro- molecules under low oxygen tension. Triple staining with anti-B220, anti-CD3 and anti-PIM antibodies confirmed that the spleen of terminally ill mice was intermixed with B and T cells that were positive for PIM (Figure 1C). Consistently, staining purified B cells from the spleen, peripheral blood and bone marrow with an anti-PIM anti- body demonstrated a marked increase of PIM+ cells in leukemic compared to normal B lymphocytes (Figure 1D).
Upregulation of the adenosine-producing capacity of leukemic cells during leukemia progression
Our previous data indicate that hypoxia acts, at least using in vitro culture systems, through the upregulation of an extracellular enzymatic system that dismantles ATP and ADP, generating adenosine.14 We then sought to deter- mine expression levels and activity of the adenosinergic axis during disease progression.
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