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Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Farnebo L, Shahangian A, Lee Y, Shin JH, Scheeren FA, Sunwoo JB - Oncotarget (2015)

Bottom Line: Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation.TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice.In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA, USA.

ABSTRACT
Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

No MeSH data available.


Related in: MedlinePlus

TLR2 is expressed on HNSCC cell lines and promotes growth of organoids in a 3D model of in vitro tumor growthA. Representative flow cytometry histograms of TLR2 expression (black line) on HNSCC cell lines and isotype-matched control staining (gray shaded histogram). B. HNSCC cell lines were grown in organoid cultures in eight replicate wells, and treated with 5 μg/ml mIgG1 antibody, 5 μg/ml α-TLR2 antibody, or 10 μg/ml zymosan. Images shown are representative of cultures at day 14. C. Graphs show the mean ± s.e.m. of the total surface area (arbitrary units) of the organoids formed, indicative of cell proliferation, as measured with ImageJ software. Statistical analysis was performed using the Kruskal-Wallis one-way-ANOVA test with Dunn's post-hoc test for multiple comparisons (n = 8, *p < 0.05, **p < 0.005, ns = non-significant).
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Figure 1: TLR2 is expressed on HNSCC cell lines and promotes growth of organoids in a 3D model of in vitro tumor growthA. Representative flow cytometry histograms of TLR2 expression (black line) on HNSCC cell lines and isotype-matched control staining (gray shaded histogram). B. HNSCC cell lines were grown in organoid cultures in eight replicate wells, and treated with 5 μg/ml mIgG1 antibody, 5 μg/ml α-TLR2 antibody, or 10 μg/ml zymosan. Images shown are representative of cultures at day 14. C. Graphs show the mean ± s.e.m. of the total surface area (arbitrary units) of the organoids formed, indicative of cell proliferation, as measured with ImageJ software. Statistical analysis was performed using the Kruskal-Wallis one-way-ANOVA test with Dunn's post-hoc test for multiple comparisons (n = 8, *p < 0.05, **p < 0.005, ns = non-significant).

Mentions: We assessed the expression of TLR2 on established HNSCC cell lines (SCC4, UM-SCC-6, UPCI:SCC103 and PCI-13) by flow cytometry (Fig. 1A), and observed that all four cell lines uniformly expressed the receptor. To determine if TLR2 has a functional role in the growth of HNSCC, we first assessed whether a monoclonal antibody (α-TLR2 mAb, clone T2.5) known to block TLR2 signaling [40], would have any effect on these cell lines in a 3D in vitro organoid model of tumor growth. Addition of the α-TLR2 mAb to the organoid cultures resulted in significant reduction of organoid sizes in all cell lines compared to the isotype control (Fig. 1B and 1C), indicating that constitutive activation of the receptor promotes tumor growth. Furthermore, experimental activation of the receptor with a well-characterized yeast-derived ligand of TLR2, zymosan, resulted in a significant increase in the size of the organoids (Fig. 1B and 1C), again indicating that the receptor is functional and has a growth-promoting effect on these cells. Of note, the absolute number of organoids was not consistently affected by the addition of zymosan (Supplemental Fig. 1A), but the individual and aggregate size of the organoids was significantly increased, suggesting that activation of TLR2 may have a profound effect in vivo.


Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Farnebo L, Shahangian A, Lee Y, Shin JH, Scheeren FA, Sunwoo JB - Oncotarget (2015)

TLR2 is expressed on HNSCC cell lines and promotes growth of organoids in a 3D model of in vitro tumor growthA. Representative flow cytometry histograms of TLR2 expression (black line) on HNSCC cell lines and isotype-matched control staining (gray shaded histogram). B. HNSCC cell lines were grown in organoid cultures in eight replicate wells, and treated with 5 μg/ml mIgG1 antibody, 5 μg/ml α-TLR2 antibody, or 10 μg/ml zymosan. Images shown are representative of cultures at day 14. C. Graphs show the mean ± s.e.m. of the total surface area (arbitrary units) of the organoids formed, indicative of cell proliferation, as measured with ImageJ software. Statistical analysis was performed using the Kruskal-Wallis one-way-ANOVA test with Dunn's post-hoc test for multiple comparisons (n = 8, *p < 0.05, **p < 0.005, ns = non-significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496405&req=5

Figure 1: TLR2 is expressed on HNSCC cell lines and promotes growth of organoids in a 3D model of in vitro tumor growthA. Representative flow cytometry histograms of TLR2 expression (black line) on HNSCC cell lines and isotype-matched control staining (gray shaded histogram). B. HNSCC cell lines were grown in organoid cultures in eight replicate wells, and treated with 5 μg/ml mIgG1 antibody, 5 μg/ml α-TLR2 antibody, or 10 μg/ml zymosan. Images shown are representative of cultures at day 14. C. Graphs show the mean ± s.e.m. of the total surface area (arbitrary units) of the organoids formed, indicative of cell proliferation, as measured with ImageJ software. Statistical analysis was performed using the Kruskal-Wallis one-way-ANOVA test with Dunn's post-hoc test for multiple comparisons (n = 8, *p < 0.05, **p < 0.005, ns = non-significant).
Mentions: We assessed the expression of TLR2 on established HNSCC cell lines (SCC4, UM-SCC-6, UPCI:SCC103 and PCI-13) by flow cytometry (Fig. 1A), and observed that all four cell lines uniformly expressed the receptor. To determine if TLR2 has a functional role in the growth of HNSCC, we first assessed whether a monoclonal antibody (α-TLR2 mAb, clone T2.5) known to block TLR2 signaling [40], would have any effect on these cell lines in a 3D in vitro organoid model of tumor growth. Addition of the α-TLR2 mAb to the organoid cultures resulted in significant reduction of organoid sizes in all cell lines compared to the isotype control (Fig. 1B and 1C), indicating that constitutive activation of the receptor promotes tumor growth. Furthermore, experimental activation of the receptor with a well-characterized yeast-derived ligand of TLR2, zymosan, resulted in a significant increase in the size of the organoids (Fig. 1B and 1C), again indicating that the receptor is functional and has a growth-promoting effect on these cells. Of note, the absolute number of organoids was not consistently affected by the addition of zymosan (Supplemental Fig. 1A), but the individual and aggregate size of the organoids was significantly increased, suggesting that activation of TLR2 may have a profound effect in vivo.

Bottom Line: Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation.TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice.In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA, USA.

ABSTRACT
Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

No MeSH data available.


Related in: MedlinePlus