Fig 1: Working model of molecular interplay between LEC and CCA during CCA progression. LECs and CCA cells in inflamed TME actively produce several cytokines and chemokines. CXCL5 produced by inflamed LECs interacts with CXCR2 in CCA cells and potentiates EMT, matrix remodeling and alters cellular metabolism. The proinflammatory cytokines and chemokines in the TME in turn induces metabolic alterations in LECs resulting in ATP generation and activation of lymphangiogenesis. This signaling axis potentiates CCA LN metastasis. Thus CXCL5-CXCR2 crosstalk between LECs and CCA cells in inflamed environment results in aiding the lymphatic metastasis of CCA cells. (This schematic was created with BioRender.com accessed on 25 October 2021).
Fig 2: Knockdown of CXCR2 blocked UA-induced monocyte migration in vitro. (A) Monocytes were loaded to the upper chamber, and cardiomyocytes were cultured in the lower chamber. Migrated monocytes in transwell assays were stained by 4',6-diamidino-2-phenylindole (DAPI) at 12 h after addition of CMC-Na-saline or UA to the lower chamber. (B) Quantitation of migrated cell numbers is presented in the right panel. Statistical analysis was performed by two-way ANOVA. ***P < 0.001 vs. CMC-Na-saline + Ad-GFP; ###P < 0.001 vs. UA + Ad-siCXCR2; n = 3/group.
Fig 3: Effects of ZD on the expressions of Cyclin D1, PCNA, COX-2 and CXCR2. ZD increased the immunoreactivities of Cyclin D1, PCNA, and COX-2 in the colon of Apcmin/+ mice. (A) Immunoreactivities of Cyclin D1, PCNA, and COX-2 were detected by immunohistochemistry. (B) The data of relative optical density of immunoreactivities. n=6. (C) ZD increased expressions of CXCR2 and COX-2 in the colon of Apcmin/+ mice. Expressions of CXCR2 and COX-2 were detected by Western blot. GADPH was used to normalize the expression level. (D) The data of relative protein expression of Western blot. N=3. Data are showed as mean ± S.D., *P < 0.05, **P < 0.01 and ***P < 0.001 vs ZS groups.
Fig 4: Inflamed LECs express high levels of CXCL5 and induce upregulation of CXCR2 expression in CCA cells. (A) Cytokines present in conditioned medium (CM) of LECs primed with LPS for 24 h were analyzed using human cytokine array kit (left panel) and the cytokine, CXCL5 (right panel) was quantified by densitometric analysis of the spots using ImageJ. Values are plotted as mean pixel density and represented as mean ± SEM, n = 3; **** p = 0.0001 represents value versus LEC CM (B) CXCL5 levels in LEC and LPS-LEC-CM were quantified by ELISA. ** p = 0.01 value versus control (C) LECs were treated with CCA-CM for 24 h and the expression of inflammatory cytokine/chemokines including CXCL5 were measured by qPCR. Values are represented as mean ± SEM, n = 3. ** p = 0.01, *** p = 0.001 and **** p = 0.0001 value versus control (5% EGM). Expression of CXCR2 was measured by qPCR in (D) livers of control and orthotopic CCA tumor mice, ** p = 0.01 value versus control liver and (E) CCA cells treated with LPS-LEC-CM. Values are represented as mean ± SEM, n = 3. **** p = 0.0001 value versus control (5% EGM). (F) CCA cells were treated with LPS LEC CM and the protein level expression of CXCR2 was measured by Western blot. Values are represented as mean ± SEM, n = 3, ** p = 0.01 value versus control.
Fig 5: Tumor draining liver LNs and tumor liver express high levels of CXCL5 and CXCR2. Representative immunofluorescent images at 40× magnification of (A) CXCL5 expression (green fluorescence) and LYVE1 expression (red fluorescence) in liver LN of control and orthotopic CCA mice. Scale bars are 20 µm. Representative immunofluorescent images at 10× magnification of (B) CXCR2 expression green fluorescence) and CK19 expression (red fluorescence) in control liver and tumor from control and orthotopic CCA mice respectively. Scale bars are 100 µm. At least 3–5 images were taken for all tissue sections.
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