Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Identification of wild-type TFF2 and fusion TFF2-CTP-Flag protein delivered by Ad- Tff2 and Ad- Tff2 -CTP-Flag in the blood. a TFF2 level assayed by ELISA in the blood from naive wild-type and CD2- Tff2 transgenic mice and mice at time point 6 months after induction of tumorigenesis with AOM/DSS treatment. Dunn’s multiple comparisons test after one-way ANOVA test, ns, non-significant, * p < 0.05, **** p < 0.001. b Amino acid sequence of fusion protein TFF2-CTP-Flag: TFF2 amino acid sequence—bold regular, CTP—regular, S —underlined, original cysteine substituted for serine, additional amino acids residues -bold italic shadow, Flag-underlined regular, Stop—stop codon. c Schematic presentation of fusion construct Tff2 -2CTP-3Flag. d Map of GV314 vector with inserted TFF2 gene. e – j Time course of TFF2 and TFF2-CTP-Flag level after single administration of adenoviruses Ad- Tff2 and Tff2 -CTP-Flag. Tff2 -null mice were injected with 5 × 10 8 pfu of Ad- Tff2 or Ad- Tff2 -CTP-Flag and sacrificed at indicated times post infection. Time-course of Tff2 mRNA ( e ) and Tff2 -CTP-Flag mRNA ( h ) expression in the liver. Total mRNA was isolated from the liver and then Tff2 mRNA was detected by qPCR, fold of change normalized on housekeeper Hprt mRNA. For each time point 3 mice were used, graphed as mean ± SD. f , i Kinetic of TFF2 protein and TFF2-CTP-Flag fusion protein in the blood assessed by ELISA and expressed as mean ± SD at each time point, three animals per group. g , j western blot for TFF2 and TFF2-CTP-Flag in mouse serum. Five microliter of mouse serum were loaded in each lane and western blot was developed with antibody produced against C-end of TFF2 molecule, arrows indicate the position of TFF2 and TFF2-CTP-Flag with calculated sizes 12 and 25–26 kDa accordingly. k Western blot of blood sample (5 µl) from Tff2 -null mouse taken on day 7 after Ad- Tff2 -CTP-Flag administration. Western blot was developed with anti-Flag antibody; arrows indicate the positions of TFF2-CTP-Flag fusion under reduced and non-reduced conditions

Article Snippet: Fusion TFF2-CTP-Flag protein from this diluted soup was purified using anion-exchange chromatography (Capto Q, GE life sciences).

Techniques: Enzyme-linked Immunosorbent Assay, Transgenic Assay, Sequencing, Construct, Plasmid Preparation, Injection, Infection, Expressing, Isolation, Western Blot, Produced

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Ad- Tff2 or Ad- Tff2 -CTP-Flag reduces splenomegaly and proportion of myeloid cells in the spleen of wild-type and Tff2 -null mice. a – f Wild-type mice were treated 2% DSS for 5 consecutive days, then on day 12 post treatment with DSS single injection of Ad- Tff2 ( a – c ) or Ad- Tff2 -CTP ( d – f ) and Ad-Fc or Ad-Fc-CTP-Flag as controls. Dose 5 × 10 8 pfu was administered in tail vein. Mice were sacrificed on day 19. Spleen appearance ( a , d ), spleen size mass ( b , e ) and proportion of CD11b + Gr-1 + cells ( c, f ) in spleen, unpaired t -test, * p < 0.05. Two experiments with three mice in each group have been done. g – i Ad- Tff2- CTP-Flag reduces splenomegaly ( g , h ) and proportion of CD11b + Gr-1 + cells ( i ) in the spleen of Tff2 -null mice, unpaired t -test, * p < 0.05. One experiment with three mice in each group has been done. j Administration of Ad- Tff2 -CTP-Flag decreases granulocytes/macrophages colonies-forming units in the spleen of wild-type mice treated with DSS. Data obtained from mice treated with 2% DSS for 5 consecutive days and sacrificed on day 19, unpaired t -test, * p < 0.05. k Immunostaining for Gr-1 in the red pulp area in spleens of Tff2 -null mice treated DSS water and received Ad-Fc-CTP (upper raw) or Ad- Tff2 -CTP (lower raw), day 19. Bar size is 100 µm

Article Snippet: Fusion TFF2-CTP-Flag protein from this diluted soup was purified using anion-exchange chromatography (Capto Q, GE life sciences).

Techniques: Injection, Immunostaining

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Validation of Ad- Tff2 -CTP-Flag activity in AOM/DSS-induced colon cancer model. a , b Ad- Tff2 suppresses colon tumorigenesis in wild-type mice treated with AOM/DSS. a Colon appearance (left), tumor number (right), b number of splenic CD11b + Gr-1 + cells. Two experiments have been done with 3–4 mice in each group. c , d Ad Tff2 -CTP-Flag suppresses colon tumorigenesis in AOM/DSS-induced colon cancer model. c Colon appearance and tumor number, d proportion CD11b + Gr-1 + cells in the spleen from wild-type mice treated with Ad- Tff2 -CTP versus Ad-Fc-CTP, unpaired t -test, * p < 0.05. Two experiments have been done with 3–4 mice in each group. e Overlapping staining for Flag and Gr-1 in the spleen of Tff2 -null mice injected with Ad- Tff2 -CTP-Flag. Mice were given 2% DSS for 5 consecutive days, then seven days later adenovirus was administrated via tail vein and mice were sacrificed in one week after adenovirus administration. Bar size is 50 µm. f BrdU incorporation in splenic CD11b + Gr-1 + cells of wild-type mice injected with Ad-Fc-CTP-Flag compare with Ad- Tff2 -CTP-Flag, unpaired t-test. Wild-type mice were treated 2% DSS for 5 consecutive days, then on day 7 post treatment with DSS single injection of Ad- Tff2 -CTP or Ad-Fc-CTP-Flag (both 5 × 10 8 pfu) was administered via tail vein injection. Mice were sacrificed on day 19 after start of DSS treatment, BrdU was injected three hours before sacrifice

Article Snippet: Fusion TFF2-CTP-Flag protein from this diluted soup was purified using anion-exchange chromatography (Capto Q, GE life sciences).

Techniques: Activity Assay, Staining, Injection, BrdU Incorporation Assay

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Clearance of recombinant mouse TFF2 and fusion TFF2-CTP-Flag from the blood. a – c Tff2- null mice were tail vein injected with equal molar amount of purified recombinant TFF2 and TFF2-CTP-Flag per kg of mouse weight. At indicated time points blood was taken and assayed for TFF2 and TFF2-CTP-Flag by western blot with antibodies produced against C-terminus of TFF2 molecule ( a , b ) and ELISA ( c ). Excretion of TFF2 with the urine was analyzed by western blot ( a ). ELISA data are plotted as a mean of value and standard deviation at each time point. d Dose-dependent downregulation of CCND1 mRNA in CD11b + Gr-1 + cells upon administration of recombinant wild-type TFF2 and fusion TFF2-CTP-Flag protein. Tff2 -null mice were given 2.5% DSS for 5 days, then CD11b + Gr-1 + were sorted and cultured with recombinant TFF2 and TFF2-CTP-Flag in indicated concentrations for a 7 days

Article Snippet: Fusion TFF2-CTP-Flag protein from this diluted soup was purified using anion-exchange chromatography (Capto Q, GE life sciences).

Techniques: Recombinant, Injection, Purification, Western Blot, Produced, Enzyme-linked Immunosorbent Assay, Standard Deviation, Cell Culture