HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment methods
Aim. Evidence-backed execution summary for HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment methods from HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Regulation of MDSC function by hypoxia
reagent used in the protocol.
- Use
- Using the hypoxia-sensitive compound pimonidazole, we evaluated distribution of F4/80 + and Gr-1 + cells in more and less hypoxic areas of solid s.c. EL-4 tumor. More hypoxic areas of tumors contained significantly higher numbers of F4/80 + MΦ than less hypoxic areas (P < 0.05; ). These results are consistent w...
Effect of the tumor microenvironment on MDSC function and differentiation
reagent used in the protocol.
- Use
- Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2 + EL-4 tumor-bearing recipients. CD45.1 + donor cells were recovered using magnetic beads 4 h after cell transfer. For...
IFN-γ production.
reagent used in the protocol.
- Use
- The number of IFN-γ-producing cells in response to cognate antigens or CD3/CD28 antibodies were evaluated in an ELISPOT assay as previously described ( ). Each well contained 10 5 splenocytes. The number of spots was counted in triplicates and calculated by an automatic ELISPOT counter (Cellular Technology).
Regulation of MDSC function by hypoxia
reagent used in the protocol.
- Use
- Our data demonstrates that the tumor microenvironment rapidly changes the function of MDSCs and promotes their differentiation to TAM. We investigated the possible mechanism of this effect. Hypoxia is one of the major characteristics of the tumor microenvironment. Therefore, we tested the effect of hypoxia on MDSC....
Regulation of MDSC function by hypoxia
reagent used in the protocol.
- Use
- Regulation of MDSC function by hypoxia. MDSCs were isolated from spleens of CT26 tumor-bearing mice and cultured in medium containing 10 ng/ml GM-CSF and 25% CT26 tumor cell conditioned medium (TCCM) under normoxic and hypoxic (1% O 2 ) conditions using a hypoxic chamber. (A) Expression of NOX subunits was evaluated...
Regulation of MDSC function by hypoxia
reagent used in the protocol.
- Use
- To assess the effect of hypoxia on mouse MDSC differentiation, Gr-1 + CD11b + cells from spleens of tumor-bearing mice were cultured for 5 d in normoxia or hypoxia in the presence of GM-CSF. Hypoxia caused accumulation of cells with macrophage morphology ( ). After a 5-d culture under hypoxic conditions, the proport...
HIF-1α regulates MDSC differentiation and function
reagent used in the protocol.
- Use
- Up-regulation of HIF-1α is one of the major effects of hypoxia. We investigated the possible role of HIF-1α in the regulation of MDSC differentiation and function. A 4-h incubation of spleen MDSC under hypoxia resulted in substantial up-regulation of HIF-1α ( ). To test the possible role of this trans...
HIF-1α regulates MDSC differentiation and function
reagent used in the protocol.
- Use
- HIF-1α regulation of MDSC function. (A) MDSCs isolated from spleens of EL-4 tumor-bearing mice were cultured with 10 ng/ml GM-CSF in hypoxia for 4 or 16 h. The level of HIF1-α was measured by Western blotting. A typical example of three independently performed experiments is shown. (B-F) MDSCs were t...
Effect of the tumor microenvironment on MDSC function and differentiation
Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2 + EL-4 tumor-bearing recipients. CD45.1 + donor cells were recovered using magnetic beads 4 h after cell transfer. For...
- Use
- Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2 + EL-4 tumor-bearing recipients. CD45.1 + donor cells were recovered using magnetic beads 4 h after cell transfer. For...
Regulation of MDSC function by hypoxia
Regulation of MDSC function by hypoxia. MDSCs were isolated from spleens of CT26 tumor-bearing mice and cultured in medium containing 10 ng/ml GM-CSF and 25% CT26 tumor cell conditioned medium (TCCM) under normoxic and hypoxic (1% O 2 ) conditions using a hypoxic chamber. (A) Expression of NOX subunits was evaluated...
- Use
- Regulation of MDSC function by hypoxia. MDSCs were isolated from spleens of CT26 tumor-bearing mice and cultured in medium containing 10 ng/ml GM-CSF and 25% CT26 tumor cell conditioned medium (TCCM) under normoxic and hypoxic (1% O 2 ) conditions using a hypoxic chamber. (A) Expression of NOX subunits was evaluated...
Patients
14 patients (47-78 yr old) with resectable T3 or T4 and N2b stage of HNC were enrolled in the study after signing University of South Florida IRB-approved consent. Patients did not receive radiation or chemotherapy for at least 3 mo before sample collection. Peripheral blood and tumor tissues were collected at...
- Use
- 14 patients (47-78 yr old) with resectable T3 or T4 and N2b stage of HNC were enrolled in the study after signing University of South Florida IRB-approved consent. Patients did not receive radiation or chemotherapy for at least 3 mo before sample collection. Peripheral blood and tumor tissues were collected at...
Reagents
Arginase inhibitor NW-hydroxyl-nor- l -arginine (nor-NOHA) and iNOS inhibitor NG-monomethyl- l -arginine (L-NMMA) were obtained from EMD. 2C-specific (H-2K b, SIYRYYGL) and control (H-2K b RAHYNIVTF) peptides were obtained from American Peptide Company. DCFDA was purchased from Invitrogen. Antibodies against p47 ph...
- Use
- Arginase inhibitor NW-hydroxyl-nor- l -arginine (nor-NOHA) and iNOS inhibitor NG-monomethyl- l -arginine (L-NMMA) were obtained from EMD. 2C-specific (H-2K b, SIYRYYGL) and control (H-2K b RAHYNIVTF) peptides were obtained from American Peptide Company. DCFDA was purchased from Invitrogen. Antibodies against p47 ph...
Cell culture and hypoxic conditions
MDSCs were cultured in complete RPMI media containing 10 ng/ml GM-CSF. A hypoxic environment (1% O 2 with 5% CO 2 ) was created and maintained using a C-Chamber Hypoxic Incubator Chamber (BioSpherix).
- Use
- MDSCs were cultured in complete RPMI media containing 10 ng/ml GM-CSF. A hypoxic environment (1% O 2 with 5% CO 2 ) was created and maintained using a C-Chamber Hypoxic Incubator Chamber (BioSpherix).
ROS detection, arginase activity and NO production
The oxidation-sensitive dye DCFDA was used to measure ROS production by MDSC. Cells were incubated at 37°C in RPMI in the presence of 2.5 µM DCFDA for 30 min. For PMA-induced activation, cells were simultaneously cultured, along with DCFDA, with 30 ng/ml PMA (Sigma-Aldrich). Cells were then labeled with an...
- Use
- The oxidation-sensitive dye DCFDA was used to measure ROS production by MDSC. Cells were incubated at 37°C in RPMI in the presence of 2.5 µM DCFDA for 30 min. For PMA-induced activation, cells were simultaneously cultured, along with DCFDA, with 30 ng/ml PMA (Sigma-Aldrich). Cells were then labeled with an...
ROS detection, arginase activity and NO production
To detect nitrites, equal volumes of culture supernatants (100 µl) were mixed with Greiss reagent. After a 10-min incubation at room temperature, the absorbance at 550 nm was measured using a microplate plate reader (Bio-Rad Laboratories). Nitrite concentrations were determined by comparing the absorbance value...
- Use
- To detect nitrites, equal volumes of culture supernatants (100 µl) were mixed with Greiss reagent. After a 10-min incubation at room temperature, the absorbance at 550 nm was measured using a microplate plate reader (Bio-Rad Laboratories). Nitrite concentrations were determined by comparing the absorbance value...
Statistics
Statistical analysis was performed using a two-tailed Mann-Whitney U or Wilcoxon nonparametric test and Prism 5 software (GraphPad Software, Inc.), with significance determined at P < 0.05.
- Use
- Statistical analysis was performed using a two-tailed Mann-Whitney U or Wilcoxon nonparametric test and Prism 5 software (GraphPad Software, Inc.), with significance determined at P < 0.05.
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analysis was performed using a two-tailed Mann-Whitney U or Wilcoxon nonparametric test and Prism 5 software (GraphPad Software, Inc.), with significance determined at P < 0.05.
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Effect of the tumor microenvironment on MDSC function and differentiation
Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2 + EL-4 tumor-bearing recipients. CD45.1 + donor cells were recovered using magnetic beads 4 h after cell transfer. For controls, CD45.1 + MDSC were transferred i.v. into EL-4 tumor-bearing recipients or naive recipients and recovered from spleens 4 h after cell transfer. (A) After adoptive transfer, CD45.1 + MDSCs were cultured with 2C spleen responder cells (Resp.; 1:4 ratio) stimulated with control and specific peptides. IFN-γ production was measured by ELISPOT assay. The number of spots per 10 5 splenocytes is shown. Values in cells stimulated with control peptide were subtracted. Each experiment was performed in triplicates and repeated twice. *, statistically significant differenc...
HIF-1α regulates MDSC differentiation and function
To directly test this possibility, we used mice with conditional HIF-1α deletion. HIF-1α flox mice were crossed with Mx-Cre mice and HIF-1α deletion was induced by repeated poly:IC administration ( ). Poly:IC is a strong inducer of type I IFN and so would dramatically affect MDSC function. Therefore, to exclude this possibility and to make sure that HIF-1α deletion is confined only to hematopoietic cells, we used BM reconstitution of WT recipients. BM cells from CD45.2 + HIF-1α-deficient (HIF-1α flox Cre +/- ) or control (HIF-1α flox Cre -/- ) mice were used to reconstitute lethally irradiated CD45.1 + congeneic naive mice. BM progenitors from HIF-1α-deficient and WT mice showed similar engraftment potential ( ). 2 wk after BM transfer, mice were inoculated s.c. with EL-4 tumor cells. No significant differences in t...
MATERIALS AND METHODS
All experiments with mice were approved by University of South Florida Animal Care and Use Committee. C57BL/6 and BALB/c female mice (6-8 wk of age) were obtained from the National Cancer Institute. Mice were kept in pathogen-free conditions. CD45.1 + congenic mice (B6.SJL-PtrcaPep3b/BoyJ), gp91 phox-/- (B6.129S6-Cybbtm1Din), HIF-1α flox/flox (B6.129-Hif1atm3Rsjo/JE), and Mx1-Cre +/- (C57BL/6J-Tg(Mx1-cre)1Cgn/J) were purchased from The Jackson Laboratory. 2C TCR transgenic mice have been described previously ( ). EL4 thymoma was obtained from American Type Culture Collection. To establish s.c. tumors, 5 × 10 5 EL-4 tumor cells were injected into C57BL/6 mice. This number of cells formed a tumor with a 1.5-cm diameter within 2-3 wk of injection. EL-4 ascitic tumor was generated by injecting 3 × 10 5 tumor cells i.p. into C57BL/6 mice. To har...
Patients
14 patients (47-78 yr old) with resectable T3 or T4 and N2b stage of HNC were enrolled in the study after signing University of South Florida IRB-approved consent. Patients did not receive radiation or chemotherapy for at least 3 mo before sample collection. Peripheral blood and tumor tissues were collected at the time of surgery from all patients. To obtain single cell suspensions from tumors, solid tissue was subjected to 1 h of enzymatic digestion using 0.1 mg/ml hyaluronidase (Sigma-Aldrich), 2 mg/ml collagenase (Sigma-Aldrich), 600 U/ml DNase (Sigma-Aldrich), and 0.2 mg/ml protease (Sigma-Aldrich) in RPMI 1640. The digested tissue was passed through a 70-µm mesh, and erythrocytes were removed by hypotonic lysis and washed thoroughly to remove debris. Mononuclear cell suspensions were obtained from whole blood using density gradient centrifugation. All cell samples were...
Cell isolation
To collect splenocytes, single cell suspensions were prepared from spleens, and red cells were removed using ammonium chloride lysis buffer. MDSCs were isolated by cell sorting on a FACSAria (BD) after cell staining with APC-conjugated anti-Gr-1 antibody and PE-conjugated anti-CD11b antibodies. To harvest peritoneal macrophages, mice were injected i.p. with 1 ml thioglycollate (BD). 3 d later, peritoneal cells were obtained by peritoneal lavage with 10 ml of ice-cold PBS. Peritoneal macrophages were harvested using magnetic beads and biotinylated anti-F4/80 antibody.
MDSC isolation from solid tumors
Tumors were dissected and digested with 0.7 mg/ml collagenase XI (Sigma-Aldrich) and 30 mg/ml of type IV bovine pancreatic DNase (Sigma-Aldrich) for 45 min at 37°C. Remaining red cells were lysed by ACK and dead cells were removed by centrifugation with Lympholyte M. Gr-1 + cells were isolated by using biotinylated anti-Gr-1 antibody and streptavidin microbeads with MiniMACS columns (Miltenyi Biotec).
ROS detection, arginase activity and NO production
The oxidation-sensitive dye DCFDA was used to measure ROS production by MDSC. Cells were incubated at 37°C in RPMI in the presence of 2.5 µM DCFDA for 30 min. For PMA-induced activation, cells were simultaneously cultured, along with DCFDA, with 30 ng/ml PMA (Sigma-Aldrich). Cells were then labeled with anti-Gr-1 and anti-CD11b antibodies on ice and evaluated by flow cytometry.
ROS detection, arginase activity and NO production
Arginase activity was measured in cell lysates, as previously described ( ). In brief, cells were lysed for 30 min with 0.1% Triton X-100. To 100 µl of protein lysate (25 µg/ml), 100 µl of 25 mM Tris-HCl and 10 µl of 10 mM MnCl 2 were added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was conducted by incubating the lysate with 100 µl of 0.5 M L-arginine, pH 9.7, at 37°C for 120 min. The reaction was stopped with 900 µl H 2 SO 4 (96%)/H 3 PO 4 (85%)/H 2 O (1/3/7, vol/vol/vol). The urea concentration was measured at 540 nm after addition of 40 µl β-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 30 min. One unit of enzyme activity is defined as the amount of enzyme that catalyzed the formation of 1 µmol urea per min.
Measurement outputs
What raw and processed outputs should exist?
MDSCs are a heterogeneous group of cells. Therefore, despite similarities in the morphology and phenotype of MDSC isolated from tumor sites and spleens, it is difficult to forma...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred in...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
It is known that MDSC can differentiate into mature myeloid cells. We therefore investigated the fate of these cells after transfer into the tumor site. CD45.1 + MDSCs isolated...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Because tumors in this model were established in peritoneum, we investigated the possible effect of a tumor-free peritoneum microenvironment on MDSC differentiation using CD45.1...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Effect of the tumor microenvironment on MDSC function and differentiation.
from paperScoring or quantification
Quantify the primary readouts for this experiment: MDSCs are a heterogeneous group of cells. Therefore, despite similarities in the morphology and phenotype of MDSC isolated from tumor sites and spleens, it is difficult to forma...; Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred in...; It is known that MDSC can differentiate into mature myeloid cells. We therefore investigated the fate of these cells after transfer into the tumor site. CD45.1 + MDSCs isolated...; Because tumors in this model were established in peritoneum, we investigated the possible effect of a tumor-free peritoneum microenvironment on MDSC differentiation using CD45.1....
from paperStatistical comparison
Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred in...; Regulation of MDSC function by hypoxia. MDSCs were isolated from spleens of CT26 tumor-bearing mice and cultured in medium containing 10 ng/ml GM-CSF and 25% CT26 tumor cell con...; Up-regulation of HIF-1α is one of the major effects of hypoxia. We investigated the possible role of HIF-1α in the regulation of MDSC differentiation and function. A 4...; HIF-1α regulation of MDSC function. (A) MDSCs isolated from spleens of EL-4 tumor-bearing mice were cultured with 10 ng/ml GM-CSF in hypoxia for 4 or 16 h. The level of HIF...
from paperReporting output
Report representative outputs alongside summary comparisons for MDSCs are a heterogeneous group of cells. Therefore, despite similarities in the morphology and phenotype of MDSC isolated from tumor sites and spleens, it is difficult to forma..., Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred in..., It is known that MDSC can differentiate into mature myeloid cells. We therefore investigated the fate of these cells after transfer into the tumor site. CD45.1 + MDSCs isolated..., Because tumors in this model were established in peritoneum, we investigated the possible effect of a tumor-free peritoneum microenvironment on MDSC differentiation using CD45.1....
inferred from protocolStructured statistical methods
Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred in...; Regulation of MDSC function by hypoxia. MDSCs were isolated from spleens of CT26 tumor-bearing mice and cultured in medium containing 10 ng/ml GM-CSF and 25% CT26 tumor cell con...; Up-regulation of HIF-1α is one of the major effects of hypoxia. We investigated the possible role of HIF-1α in the regulation of MDSC differentiation and function. A 4...; HIF-1α regulation of MDSC function. (A) MDSCs isolated from spleens of EL-4 tumor-bearing mice were cultured with 10 ng/ml GM-CSF in hypoxia for 4 or 16 h. The level of HIF...
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Evidence quotes (8)
Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2 + EL-4 tumor-bearing recipients. CD45.1 + donor cells were recovered using magnetic beads 4 h after cell transfer. For controls, CD45.1 + MDSC were transferred i.v. into EL-4 tumor-bearing recipients or naive recipients and recovered from spleens 4 h after cell transfer. (A) After adoptive transfer, CD45.1 + MDSCs were cultured with 2C spleen responder cells (Resp.; 1:4 ratio) stimulated with control and specific peptides. IFN-γ production was measured by ELISPOT assay. The number of spots per 10 5 splenocytes is shown. Values in cells stimulated with control peptide were subtracted. Each experiment was performed in triplicates and repeated twice. *, statistically significant differences (P < 0.05) from the values in splenocytes cultured alone. Error bars show mean and SD. (B) Similar experiments performed using stimulation with anti-CD3/CD28 antibodies. Error bars show mean and SD. (C) Evaluation of gene expression of argI, iNOS, and p47 phox in MDSC postadoptive transfer. Eac...
To directly test this possibility, we used mice with conditional HIF-1α deletion. HIF-1α flox mice were crossed with Mx-Cre mice and HIF-1α deletion was induced by repeated poly:IC administration ( ). Poly:IC is a strong inducer of type I IFN and so would dramatically affect MDSC function. Therefore, to exclude this possibility and to make sure that HIF-1α deletion is confined only to hematopoietic cells, we used BM reconstitution of WT recipients. BM cells from CD45.2 + HIF-1α-deficient (HIF-1α flox Cre +/- ) or control (HIF-1α flox Cre -/- ) mice were used to reconstitute lethally irradiated CD45.1 + congeneic naive mice. BM progenitors from HIF-1α-deficient and WT mice showed similar engraftment potential ( ). 2 wk after BM transfer, mice were inoculated s.c. with EL-4 tumor cells. No significant differences in tumor growth between the groups were seen (unpublished data). Populations of myeloid cells were evaluated when the tumor reached 1.5 cm in diameter (3 wk after tumor injection). Gr-1 + CD11b + MDSC were expanded in spleens of tumor-bearing mice. To assess the effect of the tumor microenvironment on H...
All experiments with mice were approved by University of South Florida Animal Care and Use Committee. C57BL/6 and BALB/c female mice (6-8 wk of age) were obtained from the National Cancer Institute. Mice were kept in pathogen-free conditions. CD45.1 + congenic mice (B6.SJL-PtrcaPep3b/BoyJ), gp91 phox-/- (B6.129S6-Cybbtm1Din), HIF-1α flox/flox (B6.129-Hif1atm3Rsjo/JE), and Mx1-Cre +/- (C57BL/6J-Tg(Mx1-cre)1Cgn/J) were purchased from The Jackson Laboratory. 2C TCR transgenic mice have been described previously ( ). EL4 thymoma was obtained from American Type Culture Collection. To establish s.c. tumors, 5 × 10 5 EL-4 tumor cells were injected into C57BL/6 mice. This number of cells formed a tumor with a 1.5-cm diameter within 2-3 wk of injection. EL-4 ascitic tumor was generated by injecting 3 × 10 5 tumor cells i.p. into C57BL/6 mice. To harvest cells from ascitic tumors, mice were sacrificed and the peritoneum was washed with 10 ml of ice-cold PBS. Cells were then aspirated and placed on ice immediately. The CT26 colon carcinoma model used in some in vitro experiments was established by injecting 5 × 10 5 CT26 tumor cells s.c. in...
14 patients (47-78 yr old) with resectable T3 or T4 and N2b stage of HNC were enrolled in the study after signing University of South Florida IRB-approved consent. Patients did not receive radiation or chemotherapy for at least 3 mo before sample collection. Peripheral blood and tumor tissues were collected at the time of surgery from all patients. To obtain single cell suspensions from tumors, solid tissue was subjected to 1 h of enzymatic digestion using 0.1 mg/ml hyaluronidase (Sigma-Aldrich), 2 mg/ml collagenase (Sigma-Aldrich), 600 U/ml DNase (Sigma-Aldrich), and 0.2 mg/ml protease (Sigma-Aldrich) in RPMI 1640. The digested tissue was passed through a 70-µm mesh, and erythrocytes were removed by hypotonic lysis and washed thoroughly to remove debris. Mononuclear cell suspensions were obtained from whole blood using density gradient centrifugation. All cell samples were analyzed within 3 h after collection. Cells were loaded with DCFDA. To identify live MDSC, mononuclear cells were first labeled with PerCP-Cy5.5-conjugated anti-CD14, APC-conjugated anti-CD11b, and PE-Cy-7-conjugated anti-CD33. Antibody-labeled cells were then finally resuspended in DAP...
To collect splenocytes, single cell suspensions were prepared from spleens, and red cells were removed using ammonium chloride lysis buffer. MDSCs were isolated by cell sorting on a FACSAria (BD) after cell staining with APC-conjugated anti-Gr-1 antibody and PE-conjugated anti-CD11b antibodies. To harvest peritoneal macrophages, mice were injected i.p. with 1 ml thioglycollate (BD). 3 d later, peritoneal cells were obtained by peritoneal lavage with 10 ml of ice-cold PBS. Peritoneal macrophages were harvested using magnetic beads and biotinylated anti-F4/80 antibody.
Tumors were dissected and digested with 0.7 mg/ml collagenase XI (Sigma-Aldrich) and 30 mg/ml of type IV bovine pancreatic DNase (Sigma-Aldrich) for 45 min at 37°C. Remaining red cells were lysed by ACK and dead cells were removed by centrifugation with Lympholyte M. Gr-1 + cells were isolated by using biotinylated anti-Gr-1 antibody and streptavidin microbeads with MiniMACS columns (Miltenyi Biotec).
The oxidation-sensitive dye DCFDA was used to measure ROS production by MDSC. Cells were incubated at 37°C in RPMI in the presence of 2.5 µM DCFDA for 30 min. For PMA-induced activation, cells were simultaneously cultured, along with DCFDA, with 30 ng/ml PMA (Sigma-Aldrich). Cells were then labeled with anti-Gr-1 and anti-CD11b antibodies on ice and evaluated by flow cytometry.
Arginase activity was measured in cell lysates, as previously described ( ). In brief, cells were lysed for 30 min with 0.1% Triton X-100. To 100 µl of protein lysate (25 µg/ml), 100 µl of 25 mM Tris-HCl and 10 µl of 10 mM MnCl 2 were added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was conducted by incubating the lysate with 100 µl of 0.5 M L-arginine, pH 9.7, at 37°C for 120 min. The reaction was stopped with 900 µl H 2 SO 4 (96%)/H 3 PO 4 (85%)/H 2 O (1/3/7, vol/vol/vol). The urea concentration was measured at 540 nm after addition of 40 µl β-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 30 min. One unit of enzyme activity is defined as the amount of enzyme that catalyzed the formation of 1 µmol urea per min.
Machine-readable layer
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"text": "Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1 + mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2 + EL-4 tumor-bearing recipients. CD45.1 + donor cells were recovered using magnetic beads 4 h after cell transfer. For controls, CD45.1 + MDSC were transferred i.v. into EL-4 tumor-bearing recipients or naive recipients and recovered from spleens 4 h after cell transfer. (A) After adoptive transfer, CD45.1 + MDSCs were cultured with 2C spleen responder cells (Resp.; 1:4 ratio) stimulated with control and specific peptides. IFN-γ production was measured by ELISPOT assay. The number of spots per 10 5 splenocytes is shown. Values in cells stimulated with control peptide were subtracted. Each experiment was performed in triplicates and repeated twice. *, statistically significant differenc..."
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"name": "HIF-1α regulates MDSC differentiation and function",
"text": "To directly test this possibility, we used mice with conditional HIF-1α deletion. HIF-1α flox mice were crossed with Mx-Cre mice and HIF-1α deletion was induced by repeated poly:IC administration ( ). Poly:IC is a strong inducer of type I IFN and so would dramatically affect MDSC function. Therefore, to exclude this possibility and to make sure that HIF-1α deletion is confined only to hematopoietic cells, we used BM reconstitution of WT recipients. BM cells from CD45.2 + HIF-1α-deficient (HIF-1α flox Cre +/- ) or control (HIF-1α flox Cre -/- ) mice were used to reconstitute lethally irradiated CD45.1 + congeneic naive mice. BM progenitors from HIF-1α-deficient and WT mice showed similar engraftment potential ( ). 2 wk after BM transfer, mice were inoculated s.c. with EL-4 tumor cells. No significant differences in t..."
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"text": "All experiments with mice were approved by University of South Florida Animal Care and Use Committee. C57BL/6 and BALB/c female mice (6-8 wk of age) were obtained from the National Cancer Institute. Mice were kept in pathogen-free conditions. CD45.1 + congenic mice (B6.SJL-PtrcaPep3b/BoyJ), gp91 phox-/- (B6.129S6-Cybbtm1Din), HIF-1α flox/flox (B6.129-Hif1atm3Rsjo/JE), and Mx1-Cre +/- (C57BL/6J-Tg(Mx1-cre)1Cgn/J) were purchased from The Jackson Laboratory. 2C TCR transgenic mice have been described previously ( ). EL4 thymoma was obtained from American Type Culture Collection. To establish s.c. tumors, 5 × 10 5 EL-4 tumor cells were injected into C57BL/6 mice. This number of cells formed a tumor with a 1.5-cm diameter within 2-3 wk of injection. EL-4 ascitic tumor was generated by injecting 3 × 10 5 tumor cells i.p. into C57BL/6 mice. To har..."
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"text": "14 patients (47-78 yr old) with resectable T3 or T4 and N2b stage of HNC were enrolled in the study after signing University of South Florida IRB-approved consent. Patients did not receive radiation or chemotherapy for at least 3 mo before sample collection. Peripheral blood and tumor tissues were collected at the time of surgery from all patients. To obtain single cell suspensions from tumors, solid tissue was subjected to 1 h of enzymatic digestion using 0.1 mg/ml hyaluronidase (Sigma-Aldrich), 2 mg/ml collagenase (Sigma-Aldrich), 600 U/ml DNase (Sigma-Aldrich), and 0.2 mg/ml protease (Sigma-Aldrich) in RPMI 1640. The digested tissue was passed through a 70-µm mesh, and erythrocytes were removed by hypotonic lysis and washed thoroughly to remove debris. Mononuclear cell suspensions were obtained from whole blood using density gradient centrifugation. All cell samples were..."
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"name": "Cell isolation",
"text": "To collect splenocytes, single cell suspensions were prepared from spleens, and red cells were removed using ammonium chloride lysis buffer. MDSCs were isolated by cell sorting on a FACSAria (BD) after cell staining with APC-conjugated anti-Gr-1 antibody and PE-conjugated anti-CD11b antibodies. To harvest peritoneal macrophages, mice were injected i.p. with 1 ml thioglycollate (BD). 3 d later, peritoneal cells were obtained by peritoneal lavage with 10 ml of ice-cold PBS. Peritoneal macrophages were harvested using magnetic beads and biotinylated anti-F4/80 antibody."
},
{
"@type": "HowToStep",
"position": 6,
"name": "MDSC isolation from solid tumors",
"text": "Tumors were dissected and digested with 0.7 mg/ml collagenase XI (Sigma-Aldrich) and 30 mg/ml of type IV bovine pancreatic DNase (Sigma-Aldrich) for 45 min at 37°C. Remaining red cells were lysed by ACK and dead cells were removed by centrifugation with Lympholyte M. Gr-1 + cells were isolated by using biotinylated anti-Gr-1 antibody and streptavidin microbeads with MiniMACS columns (Miltenyi Biotec)."
},
{
"@type": "HowToStep",
"position": 7,
"name": "ROS detection, arginase activity and NO production",
"text": "The oxidation-sensitive dye DCFDA was used to measure ROS production by MDSC. Cells were incubated at 37°C in RPMI in the presence of 2.5 µM DCFDA for 30 min. For PMA-induced activation, cells were simultaneously cultured, along with DCFDA, with 30 ng/ml PMA (Sigma-Aldrich). Cells were then labeled with anti-Gr-1 and anti-CD11b antibodies on ice and evaluated by flow cytometry."
},
{
"@type": "HowToStep",
"position": 8,
"name": "ROS detection, arginase activity and NO production",
"text": "Arginase activity was measured in cell lysates, as previously described ( ). In brief, cells were lysed for 30 min with 0.1% Triton X-100. To 100 µl of protein lysate (25 µg/ml), 100 µl of 25 mM Tris-HCl and 10 µl of 10 mM MnCl 2 were added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was conducted by incubating the lysate with 100 µl of 0.5 M L-arginine, pH 9.7, at 37°C for 120 min. The reaction was stopped with 900 µl H 2 SO 4 (96%)/H 3 PO 4 (85%)/H 2 O (1/3/7, vol/vol/vol). The urea concentration was measured at 540 nm after addition of 40 µl β-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 30 min. One unit of enzyme activity is defined as the amount of enzyme that catalyzed the formation of 1 µmol urea per min."
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"datePublished": "2010",
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