Fig 1: ENTPD2 is a direct transcriptional target of HIF-1a. a Representative IHC staining images of ENTPD2 and two hypoxia markers, CA9 and GLUT1, in human HCC. Overlapping staining pattern of ENTPD2, CA9, and GLUT1 was observed. b ENTPD2 mRNA expression in MHCC97L-EV, -shHIF-1a, and shHIF-2a clones. Cells were exposed to 20 and 1% O2 for 24 h and ENTPD2 mRNA expression was quantitated by qRT-PCR. Values were normalized to 20% O2 EV or WT (n = 3). c ENTPD2 mRNA (left) and protein (right) expression in MHCC97L- HIF-1a-WT (parental) and HIF-1a-knockout (HIF-1a-/-) clones. Cells were exposed to 20 and 1% O2 for 24 h. The mRNA and protein expressions of ENTPD2 were quantitated by qRT-PCR and flow cytometry, respectively. All values were normalized to 20% O2 WT (n = 3). d Three putative hypoxia-responsive elements (HREs), HIF binding consensus sequence, 5'-A/GCGTG-3' in the promoter region of ENTPD2 were located by in silico analysis. e ChIP assay was performed in MHCC97L cells exposed to 20 and 1% O2 with IgG, HIF-1a, and HIF-1ß antibodies. Fold of enrichment was normalized to the according IgG controls (n = 3). Data are presented as mean ± s.d. (Student’s t-test, ***P < 0.001)
Fig 2: Combined treatment with ENTPD2 inhibitor and anti-PD-1/anti-CTLA-4 immune checkpoint inhibitors in C57BL/6 HCC-bearing mice. a C57BL/6 mice were orthotopically implanted with 3 × 106 Hepa1-6. On day 3, mice were administered with vehicle, 3 mg kg-1 ARL67156 or 10 mg kg-1 POM-1 through i.p. injection for 8 consecutive days (n = 5 for each group). Images of tumors collected from mice and tumor size was measured with caliper. b C57BL/6 mice were orthotopically implanted with 3 × 106 Hepa1-6. On day 3, mice were administered with vehicle or 10 mg kg-1 POM-1 i.p. injection for 8 consecutive days, whereas on day 5 and day 9, mice were administered with 4 mg kg-1 control IgG antibody or 2 mg kg-1 anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors (CTLA-4/PD-1 Ab) through i.p. injection (n = 6 for each group). Images of tumors collected from mice and tumor size was measured with caliper. c Tumors collected at Day 11 were dissociated and the percentages of CD11b+Ly6C+ M-MDSCs and CD11b+Ly6G+ PMN-MDSCs in CD45+ cell population and the total number of MDSCs were determined by flow cytometry. d Tumors collected at Day 18 were sectioned and stained with hematoxylin and eosin for histological analysis (n = 5 for each group). The representative pictures of tumor boundaries (front) and tumor cores (core) of vehicle and combined treatment group were shown. Black arrows indicated the tumor cells. White arrows indicated the immune cells. e Survival test of chemically-induced HCC mice. Two-week-old C57BL/6 mice were administered with single dose of 1 mg kg-1 DEN through i.p. injection (Sigma-Aldrich) and after 8 weeks, with 1.25 ml kg-1 30% CCl4 (Sigma-Aldrich) through i.p. injection twice weekly for 8 weeks. Twenty-six-week-old mice were with administered 10 mg kg-1 POM-1 through i.p. injection thrice weekly or 2 mg kg-1 PD-1and CTLA-4 monoclonal antibodies through i.p. injection twice weekly (n = 5 for each group). Data are presented as mean ± s.d. (Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001)
Fig 3: Schematic representation of hypoxia-induced MDSC accumulation. Hypoxia stabilizes HIF-1a in cancer cells. HIF-1a binds to the HRE in the promoter of ENTPD2 to initiate transcription in cancer cells. ENTPD2 facilitates the dephosphorylation of extracellular ATP into 5'-AMP. Increase in extracellular 5'-AMP prevents M-MDSCs from maturation at tumor sites, thereby promoting tumor-mediated immune escape. Inhibition of ENTPD2 reduces the number of tumor-infiltrating MDSCs and increase the efficacy of PD-1/CTLA-4 immune checkpoint inhibitors
Fig 4: ENTPD2 hydrolyzes ATP to 5'-AMP that contributes to MDSC maintenance. a The effect of ENTPD2 on MDSC maintenance. Splenic MDSCs isolated from C57BL/6 mice were cultured in conditioned media collected from MHCC97L-EV and -ENTPD2-overexpressing (OE) clones. The percentages of CD11b+Gr1+ cells after 4-day culturing were analyzed (n = 3). b Liquid chromatography-mass spectrometry (LC-MS) analysis of the rate of ATP hydrolysis in conditioned media (n = 3). c Liquid chromatography-mass spectrometry (LC-MS) analysis of adenosine monophosphate (5'-AMP) level in conditioned media. MHCC97L and Hepa1-6 cells were pre-exposed to 20% O2 and 1% O2 for 48 h. Cells were trypsinized and 2 × 106 cells were re-suspended in serum-free DMEM medium supplemented with 100 µM ATP and incubated at 37 °C for 1 h. Conditioned media were collected and subjected to LC-MS analysis (n = 3). d–f The effect of 5'-AMP on MDSC maintenance. Splenic CD11b+Gr1+ cells isolated from C57BL/6 mice were cultured in the presence of d different concentrations of 5'-AMP and e 100 µM 5'-AMP with or without NT5E inhibitor, APCP. The percentages of CD11b+Gr1+ cells after 4-day culturing were analyzed (n = 3). f The effect of 5'-AMP on MDSC induction from bone marrow progenitors. Bone marrow progenitors collected from C57BL/6 mice were cultured with GM-CSF and IL4 with or without 5'-AMP. The percentages of CD11b+Gr1+ cells induced after 4-day culturing were analyzed (n = 3). Data are presented as mean ± s.d. (Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001)
Fig 5: ENTPD2 expression in HCC. a Real-time quantitative PCR analysis revealed the expression of ENTPD family members and NT5E in three HCC cell lines, MHCC97L, PLC/PRF/5, and Hep3B, exposed to 20 and 1% O2. Gene expression level was normalized to 20% O2 (n = 3). b TCGA database revealed the expression of ENTPD family members and NT5E in 49 cases of HCC tissues and their corresponding non-tumorous liver (NT) tissues. Gene expression level was normalized to NT. c Waterfall plot showed that ENTPD2 was overexpressed in 58% (36/62) of HCC patients. d Left: ENTPD2 mRNA expression in HCC tissues and their corresponding NT tissues from 62 patients = (Queen Mary Hospital (QMH), the University of Hong Kong (HKU)) was determined by qRT-PCR. Right: ENTPD2 mRNA expression in HCC and NT tissues in 49 HCC patients from the TCGC database. For qRT-PCR, values were normalized with house-keeping gene, HPRT. (Wilcoxon signed-rank test, **P < 0.01, ***P < 0.001) e High expression of ENTPD2 was associated with aggressive clinico-pathological features (n = 62, Student’s t-test). f Data from TCGA showed that high expression (z-score > 1) of ENTPD2 was significantly associated with poorer overall and disease free survival in 368 and 347 HCC patients, respectively (log-rank test, P < 0.01). g Representative IHC staining images of ENTPD2 in human HCC and NT tissues (10 cases). High ENTPD2 staining was only observed in HCC tissues but not in NT tissues
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