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2.1.1 Primary human myofibroblasts

Many of the primary human myofibroblasts used for these studies had been isolated from stomach or oesophagus obtained during surgery for cancer resection. In these cases, tissue had been taken from the tumour (CAM) and usually from an immediately adjacent, macroscopically normal, region within 10mm of the resection border (ATMs).

In addition, some myofibroblasts (NTM) were obtained from normal stomach or oesophagus of transplant donors, and some from the antrum of patients with pernicious anaemia undergoing antrectomy for treatment of ECL cell neuroendocrine tumours. A list of all the cell lines used is provided in Table 2.1. The cultures had been initially prepared by either Dr Peter Hegyi (First Department of Medicine, University of Szeged, Hungary) or by Dr Islay Steele within this research group. In all cases, the method used to prepare myofibroblasts was that described by (Mahida et al., 1997; Wu et al., 1999). Ethical Committee approval was provided by both the University of Szeged and the Royal Liverpool University Hospital; written consent was obtained. The cells had in each case been previously characterised in the laboratory by immunocytochemistry showing positive staining for α-SMA and vimentin, and negative for cytokeratin and desmin.

1 gastric ATM 72 M >59 antrum, corpus,

border medullar N0

intestinal metaplasia, atrophy CAM

2 gastric ATM 82 M 3 antrum intestinal N2 intestinal metaplasia, chronic gastritis CAM

3 gastric ATM 66 F 5 antrum, corpus,

border mixed N4 chronic gastritis CAM

4 gastric ATM 50 F 22 antrum, corpus,

border diffuse N1 chronic gastritis CAM

5 gastric ATM 76 M 25 antrum intestinal N0 intestinal metaplasia, chronic gastritis CAM

6 gastric ATM 77 M >34 antrum intestinal N0 intestinal metaplasia, chronic gastritis CAM 7 gastric ATM 76 M 15 antrum intestinal N2 intestinal metaplasia, atrophy, chronic gastritis CAM

8 gastric ATM 72 M >34 corpus mixed N1 chronic gastritis

CAM

9 gastric ATM 84 F >31 antrum, corpus,

border intestinal N0 chronic gastritis CAM

10 gastric ATM 59 F 17 antrum and corpus diffuse N2 chronic gastritis CAM

11 gastric ATM 51 M 9 antrum, corpus,

border mixed N3 chronic gastritis CAM

12 gastric ATM 67 M >31 antrum intestinal N1 chronic gastritis CAM

13 gastric CAM 39 F >43 antrum, corpus,

15 gastric ATM 61 M n.a. n.a. n.a. n.a. Normal

16 gastric ATM 65 M n.a. n.a. n.a. n.a. intestinal metaplasia,

chronic gastritis

17

gastric corpus

NTM 44 F n.a. n.a. n.a. n.a. n.a.

gastric antrum oesophagus

18

gastric corpus

NTM 45 M n.a. n.a. n.a. n.a. n.a.

gastric antrum oesophagus

19

gastric corpus

NTM 52 F n.a. n.a. n.a. n.a. n.a.

gastric antrum oesophagus

20

gastric corpus

NTM 60 M n.a. n.a. n.a. n.a. n.a.

gastric antrum oesophagus

21

gastric corpus

NTM 52 F n.a. n.a. n.a. n.a. n.a.

gastric antrum oesophagus

22

gastric corpus

NTM 41 M n.a. n.a. n.a. n.a. n.a.

gastric antrum oesophagus

27 oesophagus

ATM

72 M 18 oesophagus adenocarcinoma n.a. Barrett’s oesophagus CAM

28 cardia ATM 70 F >36 cardia adenocarcinoma n.a. chronic gastritis CAM

29 cardia

ATM

63 M 19 cardia adenocarcinoma n.a. chronic gastritis, intestinal metaplasia CAM

30 gastric antrum PA 49 F n.a corpus ECL cell carcinoids n.a. n.a 31 gastric antrum PA 43 M n.a corpus ECL cell carcinoids n.a. n.a

60 32 gastric antrum PA 71 F n.a corpus ECL cell carcinoids n.a. n.a

33 bone marrow MSC 19 M n.a. n.a. n.a. n.a. n.a.

34 bone marrow MSC 43 M n.a. n.a. n.a. n.a. n.a.

35 bone marrow MSC 22 F n.a. n.a. n.a. n.a. n.a.

36 bone marrow MSC 36 M n.a. n.a. n.a. n.a. n.a.

37 bone marrow MSC 21 M n.a. n.a. n.a. n.a. n.a.

38 bone marrow MSC 19 M n.a. n.a. n.a. n.a. n.a.

Table 2.1 Primary human myofibroblasts and MSC with corresponding patient data. Patient No, type of cells generated, origin of cells, age, gender, tumour location, tumour classification, post-operative survival and pathology assessment of tissue taken adjacent to the tumour are presented. A post-operative survival indicates survival at the time of thesis submission. (Key: PA-Pernicious anaemia, ECL cell-Enterochromaffin-like cell, MSC-Mesenchymal stem cell, NTM- Normal tissue myofibroblasts, CAM-Cancer, ATM-Adjacent tissue, N-Regional lymph nodes: N0-No regional lymph node metastasis, N1-Metastasis in 1 to 6 regional lymph nodes, N2-Metastasis in 7 to 15 regional lymph nodes, N3-Metastasis in more than 15 regional lymph nodes).

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2.1.2 Human mesenchymal stem cells

Six human bone-marrow derived mesenchymal stromal (stem) cell (MSC) types were purchased from Lonza, USA and PromoCell, UK (Table 1). These cells had been routinely characterised by the producer as positive for surface antigens CD29, CD44, CD105 and CD166, and negative for CD14, CD34 and CD45, and by functional in vitro assays for differentiation into adipocytes, chondrocytes and osteocytes.

2.1.3 Tissue culture

Primary myofibroblasts were used at passages 4 to 15. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, Poole, Dorset, UK) supplemented with 10% v/v fetal bovine serum (FBS, BioWhittaker, Lonza, Belgium), 1% v/v antibiotic-antimycotic solution (Sigma), 1% v/v penicillin- streptomycin (Sigma) and 1% v/v non-essential amino acids (Sigma). Media was prepared fresh and changed every 48 – 72 h. Human MSCs were cultured in a special media containing Mesenchymal Stem Cell Basal Medium and SingleQuots® kit (MSCBM, Lonza, Belgium). The kit includes mesenchymal cell growth supplement, L-glutamine and gentamicin sulphate/amphotericin-B. Media was prepared and changed every 72 - 96 h. All cells were incubated at 370C in an atmosphere of 5.0% v/v CO2. At about 90% confluence, cells were

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2.1.4 Release assay

Cells were washed twice with 10ml sterile phosphate buffered saline (PBS, Invitrogen, Paisley, Renfrew, UK) and harvested with 0.25% w/v trypsin (Sigma) or trypsin-EDTA in the case of MSCs. Trypsin was inactivated with media containing serum, and cells were counted with a haemocytometer (Weber Scientific International Ltd, Middlx, England). Two 10cm dishes were plated for each condition and were left overnight for cells to attach. Media was removed the following day and cells were washed 3 times with 10ml sterile PBS. Afterwards, cells were starved in 5ml serum-free media for 1 h, which was then replaced. Cells were stimulated, as appropriate, with 100ng/ml rhIGF-II (R&D Systems Inc., Oxfordshire, UK), 50ng/ml rhIGF-I (Calbiochem, Merck4Bioscheinces, Beeston, UK), 2µg/ml rhMMP-7 (Calbiochem) and 1µM ionomycin calcium salt (Sigma-Aldrich). Preincubations with 10µg/ml brefeldin A (Epicentre Biotechnologies, Cambio Ltd, Cambridge, UK), 10µg/ml cycloheximide (Sigma), 2µg/ml actinomycin D (Sigma), 3.2µM AG1024 (Calbiochem) and 5µM marimastat (Calbiochem) were performed in particular experiments. After stimulation for 30 min, media was collected and centrifuged (800g 40C, 7 min) to remove cell debri.