Fig 2: Secretory clusterin promotes survival of gastric cancer cells after starvation- and chemotherapy-induced stress.(A) Quantification of basal and gastrin-induced migration of AGS-GR cells after 18 hours in the presence of either non-immunized IgG (8 µg/ml) or anti-clusterin (anti-CLU) (8 µg/ml). Relative migration ratio was estimated using data from 5 independent experiments (2–4 technical replicates in each experiment), normalizing the data to the median cell index of untreated cells with non-immunized IgG in each independent experiment. (B-D) Apoptosis was induced by serum-starvation for 48 hours and caspase 3/7 activity measured in: (B) AGS-GR cells grown in the presence of either non-immunized IgG (8 µg/ml) or anti-CLU (8 µg/ml) with or without gastrin (10 nM). Relative caspase activity was estimated using data from 2–5 independent experiments (2–6 technical replicates in each experiment), normalizing the data to the median intensity of cells with only non-immunized IgG present in each independent experiment. (C, D) AGS-GR cells grown in (C) the absence or presence of gastrin (10 nM), sCLU (200 nM), or both; (D) the absence or presence of cisplatin (10 µM) combined with either anti-CLU (8 µg/ml) or non-immunized IgG (8 µg/ml). Relative caspase activity was estimated using data from 3–4 independent experiments (6 technical replicates in each experiment), normalizing the data to the median intensity of untreated cells in each independent experiment. Data is presented as mean with error bars representing 95% confidence intervals. *Students t-test with Bonferroni-adjusted p value < 0.05.

Fig 3: Clusterin and TFF2 expression in oxyntic mucosa of Mongolian gerbils increase after H. pylori infection and normalize when antagonizing the CCK2R.(A, B) Number per oxyntic area (0.44 mm2) of (A) CLU-positive/VMAT2(neuroendocrine (NE))-positive cells and CLU-positive/VMAT2(NE)-negative cells and (B) dual CLU-positive/Ki67-positive cells from uninfected control (n = 4), H. pylori-infected (H. pylori) (n = 7), and CCK2R-antagonized H. pylori-infected gerbils (H. pylori+CCK2Ra) (n = 4). (C, D) Oxyntic expression of CLU (brown) and dual CLU-positive (green)/VMAT2-positive (red) ECL cells in (C) uninfected control (n = 4) and (D) CCK2R-antagonized H. pylori-infected gerbils (n = 4). (E) ISH and IHC showing expression of clusterin in oxyntic mucosa of H. pylori-infected gerbils (n = 7). (F) Double immunofluorescence staining showing co-expression of CLU (green) and TFF2 (red, upper figure) and CLU (green) and Ki67 (red, lower figure) in oxyntic mucosa of H. pylori-infected gerbils. (G) IHC showing TFF2 expression (brown) in oxyntic mucosa of uninfected controls and H. pylori-infected gerbils untreated and treated with a CCK2R antagonist. Data presented as means ± SEM. *ANOVA with Tukey-adjusted p value < 0.05. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 µm from the gland bottom) is highlighted with a dotted line. Scale bars = (C, D left column, E left column, G) 200 µm; (E right column, F upper figure) 100 µm; (D right column, F lower figure) 50 µm.

Fig 4: Human gastric adenocarcinoma cells express and secrete clusterin in response to gastrin and cisplatin.(A) In-house gene expression dataset (ILMN_1815184 Illumina HumanHT-12) showing higher expression of CLU mRNA in diffuse tumors containing signet ring cells (SRC tumors) compared with tumors without SRCs (diffuse tumors). Each dot represents individual mRNA expression levels (log2 intensities) and the black lines mark mean. *corrected p value < 0.0001. (B) IHC of CLU protein (brown) and ISH of CLU mRNA (brown) showing localization in tumor cells in human diffuse SRC tumors. (C) Western blot showing different isoforms of CLU expressed in the gastric cancer cell lines AGS-GR, MKN-45 and KATO-III. (D) Western blot showing that gastrin and/or cisplatin for 48 hours stimulated increased expression and secretion (lower panel) of CLU in AGS-GR cells. Similar results were found after 24 hours (S3 Fig). The image is representative of 3 independent experiments. ß-tubulin was used as loading control. pre-sCLU = precursor of secretory CLU; iCLU = intracellular CLU; sCLU = secretory CLU. (E) Immunocytochemical staining of AGS-GR cells showing cytoplasmic vesicular expression of CLU (green) in unstimulated cells or cells treated with gastrin for 24 hours. Actin is stained with Rhodamine Phalloidin (red) and nuclear DNA with DAPI (blue). Scale bars = (B left column) 50 µm; (B right column, E) 20 µm.

Fig 5: Clusterin expression is regulated by CCK2R signaling and suppression of gastric acid secretion.(A, B, C) HDC protein level (A) and CLU protein level (B) in whole mucosa lysates by targeted mass spectrometry and plasma (C) by ELISA from control (Ctr) (n = 9), PPI-induced hypergastrinemic (PPI) (n = 6), CCK2R-antagonized (CCK2Ra) (n = 5–8) and CCK2R-antagonized PPI-induced hypergastrinemic (PPI+CCK2Ra) (n = 6–8) rats. Each dot represents an individual animal and the black lines mark median. (D) ISH (n = 4) and IHC (n = 8) of oxyntic mucosa from CCK2R-antagonized rats showing expression of clusterin (brown). (E) ISH (n = 5) and IHC (n = 8) of oxyntic mucosa from CCK2R-antagonized hypergastrinemic PPI-rats showing expression of clusterin (brown) (compare (D) and (E) to control and hypergastrinemic PPI-rats in Fig 2A). (F-H) Number per oxyntic gland of (F) CLU-positive cells in IHC-stained sections (n = 5–8 rats per group), (G) dual CLU-positive/PGA5-positive cells (n = 4 rats per group), and (H) dual CLU-positive/PCNA-positive cells (n = 4 rats per group). Data presented as means ± SEM. *ANOVA with Tukey-adjusted p value < 0.05. Nuclei were counterstained with hematoxylin (blue). The basal zone (~100 µm from the gland bottom) is highlighted with a dotted line. Scale bars = 100 µm.