Figure Legend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

Techniques Used: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

Figure Legend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

Techniques Used: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

Figure Legend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

Techniques Used: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " title="... a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

Techniques Used: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control

Figure Legend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

Techniques Used: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

Figure Legend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

Techniques Used: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

Figure Legend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

Techniques Used: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " title="... a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

Techniques Used: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control