Fig 2: Anti-IGFBP2 neutralizing antibody suppressed the CAF-like cell-induced migration and invasion in three esophageal squamous cell carcinoma (ESCC) cell lines. (A,B) Transwell migration (A) and invasion (B) assays of TE-8, TE-9, and TE-15 ESCC cell lines co-cultured with MSCs or CAF-like cells compared with those of monocultured ESCC cells. Migrating and invading cells on the bottom of the upper chamber were counted in five randomly selected fields. (C,D) Transwell migration (C) and invasion (D) assays of ESCC cell lines co-cultured with CAF-like cells and with the anti-IGFBP2 neutralizing antibody or control goat IgG antibody, compared with those without antibody. Migrating and invading cells on the bottom of the upper chamber were counted in five randomly selected fields. Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, N.S. not significant).

Fig 3: IGFBP2 promoted migration and invasion in ESCC cell lines through Akt, Erk, and NF?B signaling pathways. (A) Transwell migration assay of three ESCC cell lines stimulated with recombinant human IGFBP2 (rhIGFBP2). RhIGFBP2 was added to the lower chamber at a concentration of 100 ng/mL. Migrating cells on the bottom of the upper chamber were counted in five randomly selected fields. (B) Transwell invasion assay of three ESCC cell lines stimulated with rhIGFBP2. RhIGFBP2 was added to the lower chamber at a concentration of 100 ng/mL. Invading cells on the bottom of the upper chamber were counted in five randomly selected fields. (C) Western blotting to confirm the changes in the levels of total and phosphorylated Akt, Erk, and NF?B for 0, 10, 30, and 60 min after stimulation with rhIGFBP2 (100 ng/mL). TE-8, TE-9, and TE-15 cells were seeded on the day before protein extraction and incubated overnight in serum-free RPMI-1640. (D,E) Transwell migration (D) and invasion (E) assay of ESCC cell lines with inhibitors of phosphatidylinositol 3-kinase (PI3K inhibitor; LY294002, 10 µM), MEK1/2 (PD98059, 10 µM), and NF?B (Bay117082, 10 µM), stimulated with rhIGFBP2. Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 4: Gold nanoparticles in combination with dual kinase inhibitor PI-103 exhibit inhibition of tumors in a human xenograft of ovarian cancer. A–C) Reduction in tumor growth by combined delivery of GNPs and PI-103. Tumor harboring mice were treated with DMSO and with intravenous injections of GNPs, PI-103 (daily or twice weekly, intraperitoneally), or GNPs plus PI-103 for 10-days. Tumor volume (A), tumor images (B), and tumor mass (C) at 10 days are shown. Data are expressed as means ± SD and one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. *P = 0.05, **P = 0.01, ***P = 0.001, and ****P = 0.001, n = 8. D) Tumor tissues after completion of Day10 treatments, 8 mice from each group were taken out for IHC staining of tumor xenografts for IGFBP2, pHHB3, TUNEL, CD31 and H&E staining. Scale bar represents 100 µm. E) Quantification of intensity. Slides were immunostained with IGFBP2, pHHB3, TUNEL, and CD31. Positive cells or intensities were counted using ImageJ. The fold changes or counts/filed with respect to the control are graphically shown. Control is DMSO treated control; PI-103 (twice weekly); PI-103 (daily); GNPs (thrice weekly) and GNPs (thrice weekly) +PI-103 (twice weekly). *P < 0.05 compared with the control group by student t-test.

Fig 5: GPC4 is expressed on the astrocyte surface via a GPI-anchorage. A, Diagram of GPC4 sequence, protein, and posttranslational modifications. GPC4 contains an N-terminal signal peptide and GPI-anchor signal peptide for GPI-anchor attachment. Mature GPC4 is subject to furin cleavage, intrachain di-sulfide bonds, heparan sulfate attachment, and GPI-anchorage to the cell surface membrane. B, Western blotting of HA tagged GPC4 construct expressed in HEK293T cells. The ~37-kDa band shows the HA tagged N-terminal GPC4 in the reducing condition. PI-PLC treatment in HEK293T cells drives robust release of GPC4; however, GPC4 is detected in media in the absence of PI-PLC. C, Western blotting of concentrated ACMs for endogenous GPC4, IGFBP2, and TSP1, with and without PI-PLC treatment. PI-PLC treatment strongly facilitates release of endogenous GPC4 from astrocytes. D, Biotinylation of surface GPC4 shows that pretreatment of PI-PLC removes endogenous GPC4 from the cell surface of astrocytes. Extended Data Figure 1-1 supports Figure 1.