A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers
Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invas...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-04-01
|
| Series: | Current Issues in Molecular Biology |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1467-3045/45/4/238 |
| _version_ | 1797605945993330688 |
|---|---|
| author | Mayuki Tawara Hiroyuki Suzuki Nohara Goto Tomohiro Tanaka Mika K. Kaneko Yukinari Kato |
| author_facet | Mayuki Tawara Hiroyuki Suzuki Nohara Goto Tomohiro Tanaka Mika K. Kaneko Yukinari Kato |
| author_sort | Mayuki Tawara |
| collection | DOAJ |
| description | Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3–10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C<sub>44</sub>Mab-1 (IgG<sub>1</sub>, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C<sub>44</sub>Mab-1 recognizes CD44v9. C<sub>44</sub>Mab-1 could recognize CHO/CD44v3–10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (<i>K</i><sub>D</sub>) of C<sub>44</sub>Mab-1 for CHO/CD44v3–10, COLO201, and COLO205 was 2.5 × 10<sup>−8</sup> M, 3.3 × 10<sup>−8</sup> M, and 6.5 × 10<sup>−8</sup> M, respectively. Furthermore, C<sub>44</sub>Mab-1 was able to detect the CD44v3–10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C<sub>44</sub>Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers. |
| first_indexed | 2024-03-11T05:08:19Z |
| format | Article |
| id | doaj.art-b243de68e8f643f6849c3075db270083 |
| institution | Directory Open Access Journal |
| issn | 1467-3037 1467-3045 |
| language | English |
| last_indexed | 2024-03-11T05:08:19Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Current Issues in Molecular Biology |
| spelling | doaj.art-b243de68e8f643f6849c3075db2700832023-11-17T18:48:13ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452023-04-014543658367310.3390/cimb45040238A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal CancersMayuki Tawara0Hiroyuki Suzuki1Nohara Goto2Tomohiro Tanaka3Mika K. Kaneko4Yukinari Kato5Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, JapanDepartment of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, JapanDepartment of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, JapanDepartment of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, JapanDepartment of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, JapanDepartment of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, JapanCluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3–10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C<sub>44</sub>Mab-1 (IgG<sub>1</sub>, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C<sub>44</sub>Mab-1 recognizes CD44v9. C<sub>44</sub>Mab-1 could recognize CHO/CD44v3–10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (<i>K</i><sub>D</sub>) of C<sub>44</sub>Mab-1 for CHO/CD44v3–10, COLO201, and COLO205 was 2.5 × 10<sup>−8</sup> M, 3.3 × 10<sup>−8</sup> M, and 6.5 × 10<sup>−8</sup> M, respectively. Furthermore, C<sub>44</sub>Mab-1 was able to detect the CD44v3–10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C<sub>44</sub>Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.https://www.mdpi.com/1467-3045/45/4/238CD44CD44v9monoclonal antibodycolorectal cancer |
| spellingShingle | Mayuki Tawara Hiroyuki Suzuki Nohara Goto Tomohiro Tanaka Mika K. Kaneko Yukinari Kato A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers Current Issues in Molecular Biology CD44 CD44v9 monoclonal antibody colorectal cancer |
| title | A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers |
| title_full | A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers |
| title_fullStr | A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers |
| title_full_unstemmed | A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers |
| title_short | A Novel Anti-CD44 Variant 9 Monoclonal Antibody C<sub>44</sub>Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers |
| title_sort | novel anti cd44 variant 9 monoclonal antibody c sub 44 sub mab 1 was developed for immunohistochemical analyses against colorectal cancers |
| topic | CD44 CD44v9 monoclonal antibody colorectal cancer |
| url | https://www.mdpi.com/1467-3045/45/4/238 |
| work_keys_str_mv | AT mayukitawara anovelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT hiroyukisuzuki anovelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT noharagoto anovelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT tomohirotanaka anovelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT mikakkaneko anovelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT yukinarikato anovelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT mayukitawara novelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT hiroyukisuzuki novelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT noharagoto novelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT tomohirotanaka novelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT mikakkaneko novelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers AT yukinarikato novelanticd44variant9monoclonalantibodycsub44submab1wasdevelopedforimmunohistochemicalanalysesagainstcolorectalcancers |