Fig 1: Depletion of MDSC using anti-Gr-1 inhibits low SLC7A2 mediated HCC metastasis.A Tumor growth in mice subcutaneously injected with Hepa1-6-shcontrol or Hepa1-6-shSLC7A2 cells, treated with anti-Gr-1 antibody (400 µg/body) or IgG twice a week from day 1 after tumor inoculation. n = 10. B Flow cytometric images of subcutaneous Hepa1-6-shcontrol or Hepa1-6-shSLC7A2 cells treated with anti-Gr-1 antibody or IgG at day 27. CD8+ T cells (upper) and MDSCs (lower). C Flow cytometric analyses the ratio of CD8+ T cells and MDSCs between groups at day 27 respectively, n = 10. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. D–H In vivo assays shown that MDSCs depletion could suppress loss of SLC7A2-mediated HCC metastasis. D The representative Bioluminescence images were shown in the different groups, treated with anti-Gr-1 antibody or IgG in the C57BL/6 injected with the indicated cells in the liver, n = 10. E The Bioluminescence intensity in the tumors at the indicated time point was presented as the total photon flux. F Incidence of lung metastasis and the number of metastatic lung nodules in lung in the C57BL/6 mice. n = 10. G Overall survival of the mice in each group. H Representative H&E-stained lung metastatic nodules. The scale bars represent 1 mm (upper panel) and 100 µm (lower panel). The data are shown as the mean ± SD; *P < 0.05 and **P < 0.01.
Fig 2: Low SLC7A2 induces CXCL1 secretion and MDSC infiltration.A, B Levels of CXCL1 in human HCC cells by qRT-RCR and ELISA. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05, **P < 0.01. C Levels of CXCL1 by qRT-RCR in Hepa1-6-shSLC7A2 tumor-bearing mice tumors (n = 5). The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. D ELISA of CXCL1 in subcutaneous tumors and in blood from tumor-bearing mice. (n = 5). The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05, ****P < 0.0001. E Labeled CD11b+Gr-1+ cells myeloid MDSCs from Hepa1-6 bearing mice were determined by fluorescence-activated cell sorted (FACS). F Schematic diagram about the migration of MDSCs. G Conditioned media (CM) from Hepa1-6-shSLC7A2 and their controls clones were placed in the lower chambers. Freshly Myeloid MDSCs were put in the upper chambers and invasion for 24 h. Total numbers were counted. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. H Pre-treated with mCXCL1 (0, 10,100 ng/mL, 24 h), the chemotaxis of mouse MDSCs is shown. The data are shown as the mean ± SD from the at least three independent experiments; ****P < 0.0001. I Representative IHC staining images for SLC7A2 and CD11b in human HCC tissues. Scale bars, 200 µm (upper), 50 µm (lower). J Kaplan–Meier analysis of the associations between MDSCs infiltration and overall survival in the HCC cohort. The data are shown as the mean ± SD; **P < 0.01.
Fig 3: Treatment of CXCR2 antagonist suppresses low SLC7A2-mediated HCC metastasis.A, B Conditioned media (CM) from Hepa1-6 clones was placed in the lower chambers with different concentration of SB265610 (0, 100,1000 ng/mL, 24 h). Freshly Myeloid MDSCs were put in the upper chambers and invasion for 24 h. Total numbers were counted. The scale bars represent 100 µm. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05, **P < 0.01. C Pre-treated with mCXCL1 (10 ng/mL, 24 h) and SB265610 (100 ng/mL, 24 h), the chemotaxis index of mouse MDSCs is shown. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. D Tumor growth in mice subcutaneously injected with Hepa1-6-shcontrol or Hepa1-6-shSLC7A2 cells, treated with CXCR2 antagonist (SB265610: 2 mg/kg body weight) or PBS six times a week from day 1 after tumor inoculation. n = 10. E Flow cytometric images of subcutaneous Hepa1-6-shcontrol or Hepa1-6-shSLC7A2 cells treated with SB265610 or PBS at day 27. CD8+ T cells (upper) and MDSCs (lower). Flow cytometric analyses the ratio of CD8+ T cells and MDSCs between groups at day 27 respectively, n = 10. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001. F Immunofluorescent staining of the CD11b, CD8, and granzyme B protein expression patterns in mice tumor cells. G–K In vivo assays shown that the treatment of SB265610 can block knock-down SLC7A2-mediated HCC metastasis. G The representative Bioluminescence images were shown in the different groups, treated with SB265610 or PBS in the C57BL/6 injected with the indicated cells in the liver, n = 10. The Bioluminescence intensity in the tumors at the indicated time point was presented as the total photon flux. H Incidence of lung metastasis I the number of metastatic lung nodules in lung in the C57BL/6 mice. n = 10. J Overall survival of the mice in each group. K Representative H&E-stained lung metastatic nodules. The scale bars represent 1 mm (upper panel) and 100 µm (lower panel). Images shown are representative of at least three independent experiments. *P < 0.05, **P < 0.01.
Fig 4: G9a inhibitor, UNC0642, suppresses SLC7A2-mediated HCC immune escape, invasion, and metastasis.A, B MHCC97H and HCCLM3 HCC cells treated with 5-aza (2 µm, 3 days), EPZ-6438 (1 µm, 3 days), UNC0642 (5 µm, 2 days), ITF-2357 (1 µm, 3 days) then qRT-RCR and western blotting was used to test the expression of SLC7A2. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. C ChIP-quantitative real-time PCR analysis to detect H3K9me2 association with SLC7A2 gene after treatment of G9a inhibitor UNC0642. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. D Representative IHC staining images for SLC7A2 and G9a in human HCC tissues. Scale bars, 200 µm (upper), 50 µm (lower). E Bioinformatics analysis of G9a mRNA expression on the TCGA database in human HCC tissues. F Kaplan–Meier analysis of the associations between G9a expression and overall survival in the HCC cohort. G–I Treatment of UNC0642 promoted HCC cell proliferation and metastasis in vitro. G Usage of UNC0642 on HCC cell colony formation. H The effects of UNC0642 on HCC cell proliferation were measured by a CCK-8 assay. I Transwell assay shown the abilities of migration and invasion. The scale bar represents 100 µm. J Western blotting was used to detect the G9a, SLC7A2, and its downstream target genes expression in MHCC97H and HCCLM3 cells treated with UNC0642. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. K, L Tumor growth in mice subcutaneously injected with H22-control or H22-SLC7A2 cells, treated with UNC0642 daily by intraperitoneal injection at 5 mg/kg. The tumor volume was monitored every 3 days. n = 5. M Flow cytometric images of subcutaneous H22-control or H22-SLC7A2 cells treated with UNC0642 or its control at day 27. CD8+ T cells (upper) and MDSCs (lower). Flow cytometric analyses the ratio of CD8+ T cells and MDSCs between groups at day 27, respectively, n = 5. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. N Immunofluorescent staining of the CD11b, CD8, and granzyme B protein expression patterns in mice tumor cells. Scale bars, 20 µm. O–R In vivo assays shown that the treatment of UNC0642 can block knock-down SLC7A2-mediated HCC metastasis. O The representative Bioluminescence images were shown in the different groups, treated with UNC0642 or DMSO in the BALB/C injected with the indicated cells in the liver and Incidence of lung metastasis, n = 10. The Bioluminescence intensity in the tumors at the indicated time point was presented as the total photon flux. P The number of metastatic lung nodules in lung in the BALB/C mice. n = 10. Q Overall survival of the mice in each group. R Representative H&E-stained lung metastatic nodules. The scale bars represent 1 mm (upper panel) and 100 µm (lower panel). *P < 0.05, **P < 0.01. S A schematic diagram of the SLC7A2 signaling in HCC immune evasion. SLC7A2 deficiency upregulated CXCL1 expression through PI3K/Akt/NF-?B pathway. CXCL1 promoted HCC growth and metastasis through recruiting MDSCs to the tumors. Neutralizing or suppressing MDSC infiltration abolished deficient SLC7A2-mediated HCC growth and metastasis. Usage of G9a inhibitor (UNC0642) suppressed loss of SLC7A2-meidated HCC metastasis.
Fig 5: SLC7A2 disruption facilitates HCC proliferation, invasion, and metastasis in vitro and vivo.A, B Relative mRNA and protein expression of SLC7A2 in normal liver tissue and HCC cell lines. C Western blotting analyzed performed the expression of SLC7A2 in Huh7 and MHCC97H cells after lentivirus transfection. D, E Deficient SLC7A2 promoted HCC cell proliferation in vitro. D Effects of SLC7A2 on HCC cell colony formation. E The effects of SLC7A2 on HCC cell proliferation were measured by a CCK-8 assay. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. F–L Deficient SLC7A2 promoted HCC cell growth and migration in vivo. F Growth curves and G weight curves of tumors in C57BL/6 (n = 10 mice per group) in subcutaneously model. H IHC staining for Ki67 in the indicated tumors. I Representative Bioluminescent images and total photon flux were shown. J Incidence of lung metastasis and the number of metastatic nodules in the different group. K Overall survival time in C57BL/6 mice. L Representative images of H&E stained-lung tissues from the different. The scale bars represent 1 mm (upper panel) and 100 µm (lower panel). The data are shown as the mean ± SD; *P < 0.05, **P < 0.01.
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