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<p>Somatostatin analogs (SSA) are well-established antisecretory drugs in functionally active neuroendocrine tumors (NET). Two placebo-controlled trials have recently demonstrated significant improvement of progression-free survival under SSA treatment. Furthermore, somatostatin receptor (SSTR) overexpression in NET has also been utilized for diagnostic imaging and peptide receptor radionuclide therapy (PRRT). However, PRRT in NET is associated mostly with partial and minor remission, while other radionuclide therapies reach complete remissions in up to 75% of cases. This study assessed a potential radiosensitizing effect of SSA treatment in five established NET cell line models: BON, QGP-1, LCC-18, H727, and UMC-11. Irradiation was found to significantly inhibit proliferation, while no additional effect by octreotide treatment was observed. Intriguingly, no impact of SSA treatment alone was found in any of these NET cell lines when systematically analyzing cell viability, proliferation, and cell cycle distribution. Investigation of the causes for this octreotide resistance led to demonstration of low octreotide binding and scarce SSTR, specifically SSTR2 expression as compared to levels found in human NETs. The resistance toward SSA treatment in viability and proliferation assays could not be overcome by re-expression of SSTR2 in two of the cell lines. These results provide systematic evidence for a lack of authentic, tumor-like SSTR expression, and function in five frequently used NET cell line models and point to the need for more physiologic tumor model systems.</p

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<p>Somatostatin analogs (SSA) are well-established antisecretory drugs in functionally active neuroendocrine tumors (NET). Two placebo-controlled trials have recently demonstrated significant improvement of progression-free survival under SSA treatment. Furthermore, somatostatin receptor (SSTR) overexpression in NET has also been utilized for diagnostic imaging and peptide receptor radionuclide therapy (PRRT). However, PRRT in NET is associated mostly with partial and minor remission, while other radionuclide therapies reach complete remissions in up to 75% of cases. This study assessed a potential radiosensitizing effect of SSA treatment in five established NET cell line models: BON, QGP-1, LCC-18, H727, and UMC-11. Irradiation was found to significantly inhibit proliferation, while no additional effect by octreotide treatment was observed. Intriguingly, no impact of SSA treatment alone was found in any of these NET cell lines when systematically analyzing cell viability, proliferation, and cell cycle distribution. Investigation of the causes for this octreotide resistance led to demonstration of low octreotide binding and scarce SSTR, specifically SSTR2 expression as compared to levels found in human NETs. The resistance toward SSA treatment in viability and proliferation assays could not be overcome by re-expression of SSTR2 in two of the cell lines. These results provide systematic evidence for a lack of authentic, tumor-like SSTR expression, and function in five frequently used NET cell line models and point to the need for more physiologic tumor model systems.</p

Identification of Galectin-4 expression.

<p>(A) Gal-4 expression was detected in cryosections of the GI tract of healthy volunteers. The results are representative for four individuals (original magnifications: ×100). (B) PCR analysis of Gal-4 mRNA expression in resting and anti-CD3/CD28 stimulated PBT. (C) Flow cytometric analysis of intra- and extracellular Gal-4 content in resting and anti-CD3/CD28 stimulated PBT. Data are representative of three individual experiments.</p

Galectin-4 ameliorates experimental colitis by inducing apoptosis and reduction of pro-inflammatory cytokine secretion.

<p>(A) Disease activity index in acute DSS-induced colitis, treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. *p≤0.01 for change vs. control. (B) Detection of apoptotic cells by TUNEL staining in cryosections of colonic tissue from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. (C) Apoptotic cells after TUNEL staining were counted in three power fields on four slices of different animals by an investigator blinded to the protocol. *p≤0.05 for change vs. control. (D) Detection of proliferating cells by BrdU staining in cryosections of colonic tissue from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. (E) BrdU positive cells were counted in three power fields on four slices of different animals by an investigator blinded to the protocol. *p≤0.01 for change vs. control. (F) Cytokine secretion was determined by CBA analysis from colonic cultures from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. All data represent mean±SEM of ten individual experiments. *p≤0.05 for change vs. control.</p

Galectin-4 and its domains distinctively reduce secretion of pro-inflammatory cytokines.

<p>(A) Cytokine release was tested in PBT stimulated with anti-CD3/CD28 for 72 hours. Data represent mean±SEM of six individual experiments. *p≤0.01 for decrease vs. baseline. (B) IL-17 secretion of LPT and PBT was determined by an IL-17 specific ELISA. LPT and PBT were activated by anti-CD2 or -CD3/CD28 mAb, respectively and cultured in the presence or absence of 100 µg/ml Gal-4. Data represent mean±SEM of three individual experiments.</p

Galectin-4 induces T cell apoptosis caspase-independently via calpain-mediated pathways.

<p>(A) PBT and LPT were stimulated with anti-CD3/CD28 or -CD2, respectively, in the presence of 0, 50, 100 or 200 µg/ml Gal-4 and apoptosis was determined by annexin-V staining. Data represent mean±SEM of eight individual experiments. *p≤0.05 for increase vs. baseline. (B) PBT were stimulated in the presence of 0, 25, 50, 100 or 200 µg/ml Gal-4. Apoptosis was detected by annexin V/PI staining, necrosis by PI staining followed by flow cytometry. Data represent mean±SEM of six individual experiments. *p≤0.05 for increase vs. baseline. (C) PBT were transfected with Gal-4 siRNA or scrambled control, activated by anti-CD3/CD28 and cultured in the presence of the TNF-α blocker adalimumab for three days. Apoptosis was detected by annexin V/PI staining followed by flow cytometry. Data are representative for three individual experiments. (D) Caspase-3, -8, and -9 activity in anti-CD3/CD28 stimulated T cells cultured in the presence or absence of 100 µg/ml Gal-4. Data are representative for three individual experiments. (E) PBT were activated by anti-CD3/CD28 and cultured for 24 h in the presence or absence of 100 µg/ml Gal-4 and 50 mM Calpain inhibitor z-LLY-fmk, 4 mM EGTA or 30 mM BAPTA-AM. Data are representative for three individual experiments. (F) Disruption of the mitochondrial membrane potential was detected by rhodamine123 staining followed by flow cytometric analysis. T cells were stimulated with anti-CD3/CD28 and incubated for 3 h in the presence or absence of 100 µg/ml Gal-4. Afterwards cells were analysed by flow cytometry. All data are representative for three individual experiments. (G) Disruption of the mitochondrial membrane potential was detected after 12 h by rhodamine123 staining followed by flow cytometric analysis. All data are representative for three individual experiments.</p

Galectin-4 binds to stimulated, but not resting T cells.

<p>(A) Flow cytometric analysis of Gal-4 binding to resting and anti-CD3/CD28 stimulated T cells using Gal-4 detected by an anti-Gal-4 Ab and PE-labeled secondary Ab. Carbohydrate-dependence of the binding was analysed by addition of 50 mM lactose as a pan-galectin inhibitor and 50 mM sucrose as control. Data are representative of three individual experiments. (B) Immunoprecipitation of Gal-4 binding complexes. BSA served as negative, Gal-1 as positive control. Data are representative for four individual experiments.</p

CD4 T-cells primed by liver-derived antigen display deficient Th1-effector function.

<p>(A) Four million CFSE-labeled OT-II T-cells were transferred intravenously into TF-OVA mice or into B6 control mice. After 68 hours, non-parenchymal cells were purified from liver and spleen and incubated <i>in vitro</i> with PMA/ionomycin. Production of Interferon-γ and IL-2 was analyzed after 4 hours. Representative results are depicted (n = 6). (B) Mice were treated as in (A), but cells were analyzed for CD25 and FoxP3 expression immediately after purification of cells from the indicated organs. Representative results are depicted from n = 6 mice in each group. All plots depict data gated on CD4⁺CFSE⁺ cells. CD25/FoxP3 plots on the right depict the frequency of CD25⁺FoxP3⁺ double positive cells.</p

Priming of CD4 T-cells by endogenous antigen in the inflamed liver.

<p>(A) Eight million OT-I T-cells were transferred intravenously into TF-OVA mice (+ Hepatitis), or mice were left untreated (- Hepatitis). ALT levels were determined at day 6. Values from individual mice and mean ± SEM are depicted (*** p<0.0001 by Mann-Whitney test). (B) Four million CFSE-labeled OT-II T-cells were transferred intravenously into TF-OVA mice at day 6 after transfer of OT-I T-cells (+ Hepatitis) or into untreated TF-OVA mice (- Hepatitis). Non-parenchymal cells were isolated from liver and spleen analyzed for the presence of CFSE⁺ cells. The absolute number of CFSE⁺ cells in liver or spleen was determined 20 hours after transfer of OT-II T-cells. Cumulative results are depicted from n = 6 mice per group (mean ± SEM, ** p<0.005 by Student's t-test). (C) Mice were treated as in (B). Proliferation was analyzed 20 and 44 hours after transfer of OT-II T-cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results are depicted from n = 6 mice in each group. All plots display data gated on CD4⁺Vα2⁺ cells.</p

Hepatitis is amplified by effector but not naive CD4 T-cells.

<p>(A) Four million naïve (open squares) or effector (open circles) OT-II T-cells were transferred alone or together with 4 million naïve OT-I T-cells (filled squares and circles) into TF-OVA mice. Naïve OT-II T-cells were also transferred three days prior to transfer of OT-I T-cells to allow timely redistribution to the liver (half-filled squares). As a control, 4 million naïve OT-I T-cells (filled triangles) were transferred alone. Alanine aminotransferase levels were determined at day 5. Individual values and mean ± SEM are depicted (**p<0.005 by Mann-Whitney test). (B) One million naïve OT-I T-cells were transferred alone (filled triangles) or together with 4 million effector OT-II T-cells (half-filled circles) into TF-OVA mice. Alanine aminotransferase levels were determined at day 5. Individual values and mean ± SEM are depicted (**p<0.005 by Mann-Whitney test). (C) Four million effector OT-II T-cells were transferred alone (CD4) or together with 1 million naïve OT-I T-cells (CD8+CD4) into TF-OVA mice. As controls, 1 million naïve OT-I CD8 T-cells (CD8) were transferred alone or no cells were transferred (control). At day 6, equal numbers of CFSE^high SIINFEKL-pulsed and CFSE^low unpulsed B6 splenocytes were injected. After 5 hours cells from the indicated organs were analyzed for CFSE staining. Histogram blots depict data gated on CFSE-positive cells. (D) Antigen-specific cytolysis was calculated as described in methods. Cumulative results are depicted from n = 6 mice per group (mean ± SEM; ** p<0.005 by Mann-Whitney test).</p