Production of nerve growth factor by mouse hepatocellular carcinoma cells and expression of TrkA in tumor-associated arteries in mice  Kan Kishibe, Yoshihisa Yamada, Katsuhiro Ogawa  Gastroenterology  Volume 122, Issue 7, Pages 1978-1986 (June 2002) DOI: 10.1053/gast.2002.33581 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of NGF by HCC. (A) cDNA array analysis. αNGF (open arrowheads) is present in an HCC at 15.7 times the normal liver level and βNGF (solid arrowheads) at 4.0 times. (B) RT-PCR analysis of α and βNGF. Both proved detectable in all HCC tissues and βNGF in all HCC cell lines but negative in normal liver. (C) Northern blot analysis of α and βNGF. Expression is apparent in the HCC tissue but not in normal liver. (D) Left, H&E staining, and right, immunohistochemical staining for NGF. A, adenoma; N, normal liver. NGF is positive in the adenoma but negative in the surrounding normal liver. Gastroenterology 2002 122, 1978-1986DOI: (10.1053/gast.2002.33581) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Bioassay of NGF produced by HCC cells using PC12 cells. (A) Although PC12 cells showed only a few neurites without NGF, (B) they showed prominent neurite outgrowth after treatment with NGF or (C) the CM of HCC cells. (D) However, when the CM was treated with the neutralizing anti-NGF antibody, the neurite outgrowth was markedly inhibited. (E) Neurite outgrowth in PC12 cells with the CM of 6 HCC cell lines. Although the activity of pure NGF was almost completely inhibited by the neutralizing anti-NGF antibody, the degree of inhibition was less prominent with the HCC cells. Open columns represent CM without treatment with the anti-NGF antibody, and closed columns represent CM with the antibody treatment. Gastroenterology 2002 122, 1978-1986DOI: (10.1053/gast.2002.33581) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Establishment and properties of HCC cells introduced with antisense NGF. (A) RT-PCR for NGF mRNA. Although the parental (HCC) and sense cells (S) express NGF mRNA, the antisense cells (AS) are negative. (B) The NGF level in the CM of antisense cells (AS) is markedly reduced compared with the parental (HCC) and sense cells (S). WE, Williams' E medium supplemented with 10% FBS. (C) Neurite outgrowth activity of antisense cells (AS) is markedly decreased compared with the parental (HCC) and sense cells (S). (D) Changes in the numbers of viable PC12 cells after treatment with the CM of parental (HCC), antisense (AS), and sense cells (S) relative to the number at the start of cultivation. The number of viable PC12 cells is decreased in the antisense case. (E) Although apoptotic cells are rare in PC12 cells treated with parental HCC cell CM (top), they are frequently seen in the PC12 cells treated with the antisense cell CM (arrows, bottom) Hoechst 33248 staining. (F) Apoptotic index of PC12 cells is increased by cultivation with serum-free RPMI1640 medium (−) compared with the medium supplemented with serum (+) or NGF. However, the apoptotic index is increased when PC12 cells were treated with the antisense cell CM (AS), parental HCC cell CM (HCC), and sense cell CM (S); particularly high with the antisense cell CM (AS). (G) Growth curves of the parental (HCC), antisense (AS), and sense cells (S). Note the lack of any difference between them. Gastroenterology 2002 122, 1978-1986DOI: (10.1053/gast.2002.33581) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 RT-PCR analysis of NGF expression in developing and regenerating livers and cultured hepatocytes. (A) NGF is not detectable in fetal (18 ED) and newborn livers (1D, 2W). (B) Ten-week-old male mice were subjected to a two-thirds hepatectomy. NGF is detectable in regenerating livers from the second day after the operation at least until the 10th day. (C) Primary cultured hepatocytes also express NGF from 2 days after the start of cultivation with a peak on the fourth day. Gastroenterology 2002 122, 1978-1986DOI: (10.1053/gast.2002.33581) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 (A and B) RT-PCR for TrkA and p75NTR and (C and D) immunostaining for TrkA. Neither receptor is evident in (A) HCC tissues and cell lines or (B) regenerating liver, although both are detectable in the brain. (C) TrkA is expressed in the wall of HCC artery (arrow) but not in the wall of portal vein (P) or bile duct (B). A, artery; N; normal liver, H; HCC, *; arterial wall. (D) Higher magnification of C (*). Smooth muscle cells in the arterial wall show the TrkA staining (arrow). Gastroenterology 2002 122, 1978-1986DOI: (10.1053/gast.2002.33581) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 NPY immunostaining in the normal liver and HCC tissue. Brown dots represent NPY-positive nerve fibers (arrows). (A) In the normal liver, only a few nerve fibers are evident in the portal tract. P, portal vein; B, bile duct; A, artery. (B) In HCC (H), nerve fibers are thick and abundant in the vessel walls, especially at arteries. Gastroenterology 2002 122, 1978-1986DOI: (10.1053/gast.2002.33581) Copyright © 2002 American Gastroenterological Association Terms and Conditions