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Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand.

Lin TY, Zhang H, Wang S, Xie L, Li B, Rodriguez CO, de Vere White R, Pan CX - Mol. Cancer (2011)

Bottom Line: In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.No significant changes in cell viability or proliferation were observed upon incubation with PLZ4.The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology and oncology, Department of Internal Medicine, University of California-Davis Cancer Center, Sacramento, CA 95817, USA.

ABSTRACT

Objective: To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.

Experimental design: The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.

Results: PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.

Conclusions and clinical relevance: PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.

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PLZ4 binds to Canine TCC cells. A. whole cell binding assay to determine cell binding specificity of PLZ4. Cells were resuspended at 106 cells/ml and incubated with beads coated with PLZ4. If PLZ4 binds to cells in solution, bead surface would be covered with cells and exhibit a rosette pattern under the microscopy examination. This experiment was repeated 3 times for cell lines. The cell binding assay of normal canine bladder urothelial cells was repeated on 3 different dogs. a. 5637 human bladder cancer cell line; b. K9TCC-PU cell line; c. normal canine urothelial cells; d. urothelial cells from a bladder with chronic cystitis. The average diameter of the beads is 90 μm. B. Affinitofluorescence of PLZ4 peptide toward Canine TCC cell lines. Affinitofluorescence staining was performed with all five canine TCC cell lines and normal canine bladder urothelial cells of dogs euthanized for non-bladder diseases. Only fluorescence staining to normal urothelial cells (a) and K9TCC-PU cells (c) was shown. b and d showed the corresponding DAPI nuclear staining of a and c, respectively.
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Figure 1: PLZ4 binds to Canine TCC cells. A. whole cell binding assay to determine cell binding specificity of PLZ4. Cells were resuspended at 106 cells/ml and incubated with beads coated with PLZ4. If PLZ4 binds to cells in solution, bead surface would be covered with cells and exhibit a rosette pattern under the microscopy examination. This experiment was repeated 3 times for cell lines. The cell binding assay of normal canine bladder urothelial cells was repeated on 3 different dogs. a. 5637 human bladder cancer cell line; b. K9TCC-PU cell line; c. normal canine urothelial cells; d. urothelial cells from a bladder with chronic cystitis. The average diameter of the beads is 90 μm. B. Affinitofluorescence of PLZ4 peptide toward Canine TCC cell lines. Affinitofluorescence staining was performed with all five canine TCC cell lines and normal canine bladder urothelial cells of dogs euthanized for non-bladder diseases. Only fluorescence staining to normal urothelial cells (a) and K9TCC-PU cells (c) was shown. b and d showed the corresponding DAPI nuclear staining of a and c, respectively.

Mentions: To determine the binding of PLZ4 to canine invasive TCC cell lines, a whole cell binding assay was performed (Additional File 1). We first synthesized PLZ4 on TentaGel S NH2 resin beads (Rapp Polymere Gmbh, Germany) [1,7], and incubated the PLZ4-coated beads with 106 cells/ml single-cell suspensions of five different canine invasive TCC cell lines including K9TCC-PU, K9TCC-PU-AxA, K9TCC-PU-In, K9TCC-PU-AxC, and K9TCC-PU-Nk (kindly provided by Deborah Knapp at Purdue University, West Lafayette, IN, USA) [3]. Human bladder cancer cell line 5637 cells served as the positive control. Over 95% of the bead surface was covered with 5637 and K9TCC cells (Figure 1A: a and 1A: b, respectively). In contrast, there was no cell binding and a smooth bead surface was observed when the beads were incubated with the normal canine bladder urothelial cells (Figure 1A: c), or bladder cells from a dog with chronic cystitis (Figure 1A: d).


Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand.

Lin TY, Zhang H, Wang S, Xie L, Li B, Rodriguez CO, de Vere White R, Pan CX - Mol. Cancer (2011)

PLZ4 binds to Canine TCC cells. A. whole cell binding assay to determine cell binding specificity of PLZ4. Cells were resuspended at 106 cells/ml and incubated with beads coated with PLZ4. If PLZ4 binds to cells in solution, bead surface would be covered with cells and exhibit a rosette pattern under the microscopy examination. This experiment was repeated 3 times for cell lines. The cell binding assay of normal canine bladder urothelial cells was repeated on 3 different dogs. a. 5637 human bladder cancer cell line; b. K9TCC-PU cell line; c. normal canine urothelial cells; d. urothelial cells from a bladder with chronic cystitis. The average diameter of the beads is 90 μm. B. Affinitofluorescence of PLZ4 peptide toward Canine TCC cell lines. Affinitofluorescence staining was performed with all five canine TCC cell lines and normal canine bladder urothelial cells of dogs euthanized for non-bladder diseases. Only fluorescence staining to normal urothelial cells (a) and K9TCC-PU cells (c) was shown. b and d showed the corresponding DAPI nuclear staining of a and c, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040722&req=5

Figure 1: PLZ4 binds to Canine TCC cells. A. whole cell binding assay to determine cell binding specificity of PLZ4. Cells were resuspended at 106 cells/ml and incubated with beads coated with PLZ4. If PLZ4 binds to cells in solution, bead surface would be covered with cells and exhibit a rosette pattern under the microscopy examination. This experiment was repeated 3 times for cell lines. The cell binding assay of normal canine bladder urothelial cells was repeated on 3 different dogs. a. 5637 human bladder cancer cell line; b. K9TCC-PU cell line; c. normal canine urothelial cells; d. urothelial cells from a bladder with chronic cystitis. The average diameter of the beads is 90 μm. B. Affinitofluorescence of PLZ4 peptide toward Canine TCC cell lines. Affinitofluorescence staining was performed with all five canine TCC cell lines and normal canine bladder urothelial cells of dogs euthanized for non-bladder diseases. Only fluorescence staining to normal urothelial cells (a) and K9TCC-PU cells (c) was shown. b and d showed the corresponding DAPI nuclear staining of a and c, respectively.
Mentions: To determine the binding of PLZ4 to canine invasive TCC cell lines, a whole cell binding assay was performed (Additional File 1). We first synthesized PLZ4 on TentaGel S NH2 resin beads (Rapp Polymere Gmbh, Germany) [1,7], and incubated the PLZ4-coated beads with 106 cells/ml single-cell suspensions of five different canine invasive TCC cell lines including K9TCC-PU, K9TCC-PU-AxA, K9TCC-PU-In, K9TCC-PU-AxC, and K9TCC-PU-Nk (kindly provided by Deborah Knapp at Purdue University, West Lafayette, IN, USA) [3]. Human bladder cancer cell line 5637 cells served as the positive control. Over 95% of the bead surface was covered with 5637 and K9TCC cells (Figure 1A: a and 1A: b, respectively). In contrast, there was no cell binding and a smooth bead surface was observed when the beads were incubated with the normal canine bladder urothelial cells (Figure 1A: c), or bladder cells from a dog with chronic cystitis (Figure 1A: d).

Bottom Line: In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.No significant changes in cell viability or proliferation were observed upon incubation with PLZ4.The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology and oncology, Department of Internal Medicine, University of California-Davis Cancer Center, Sacramento, CA 95817, USA.

ABSTRACT

Objective: To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.

Experimental design: The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.

Results: PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.

Conclusions and clinical relevance: PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.

Show MeSH
Related in: MedlinePlus