A Real-Time Near-Infrared Fluorescence Imaging Method for the Detection of Oral Cancers in Mice Using an Indocyanine Green–Labeled Podoplanin Antibody

Ito, Akihiro; Ohta, Mitsuhiko; Kato, Yoshinori; Inada, Shunko A.; Katô, Toshio; Nakata, Susumu; Yatabe, Yasushi; Goto, Mitsuo; Kaneda, Norio; Kurita, K.; Nakanishi, Hayao; Yoshida, Kenji

doi:10.1177/1533033818767936

Cited by 27 publications

(16 citation statements)

References 34 publications

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“…CD44v3-10 ORF was obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. CD44s and CD44v3-10 cDNAs were subcloned into pCAG-Ble-ssPA16 vector possessing signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [ 19 , 49 , 50 , 51 , 52 ], which is detected by NZ-1 [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. CHO/CD44s and CHO/CD44v3-10 were established by transfecting pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas

Ejima

Suzuki

Tanaka

et al. 2023

IJMS

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CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

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Section: Methodsmentioning

confidence: 99%

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas

Ejima

Suzuki

Tanaka

et al. 2023

IJMS

Self Cite

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“…CD44s cDNA was amplified using Hot-Star HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) using LN229 (a glioblastoma cell line) cDNA as a template. CD44v3-10 and CD44 cDNAs were cloned into pCAG-Ble-ssPA16 vector with signal sequence and N-terminal PA16 tag of 16 amino acids (GLEGGVAMPGAEDDVV) [22,[28][29][30][31], which is detected by NZ-1, which was originally developed against human podoplanin [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47]. The pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 vectors were transfected into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.), which offers an innovative electroporation method that utilizes a proprietary biologically compatible pipette tip chamber to generate a more uniform electric field for a significant increase in transfection efficiency and cell viability.…”

Section: Plasmid Construction and Establishment Of Stable Transfectantsmentioning

confidence: 99%

Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C<sub>44</sub>Mab-3 for Multiple Applications against Pancreatic Carcinomas

Kudo¹,

Suzuki²,

Tanaka³

et al. 2023

Preprint

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Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker, and plays tumor promotion and drug resistance in various cancers. Especially, the splicing variants are overexpressed in many carcinomas, and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells, and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant 5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3–10 and PK-1 was 7.1 × 10−10 M and 1.9 × 10−9 M, respectively. C44Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in western blotting, and stained the formalin-fixed paraffin-embedded pancreatic cancer cells, but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications, and expected for the application of pancreatic cancer diagnosis and therapy.

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“…CD44v3-10 ORF was obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. CD44s and CD44v3-10 cDNAs were subcloned into pCAG-Ble-ssPA16 vector possessing signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [19,[45][46][47][48], which is detected by NZ-1 [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64]. CHO/CD44s and CHO/CD44v3-10 were established by transfecting pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.).…”

Section: Cell Linesmentioning

confidence: 99%

Development of a Novel Anti-CD44 variant 6 Monoclonal Antibody for Multiple Applications against Colorectal Carcinomas

Ejima¹,

Suzuki²,

Tanaka³

et al. 2023

Preprint

View full text Add to dashboard Cite

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent KD of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

show abstract

A Real-Time Near-Infrared Fluorescence Imaging Method for the Detection of Oral Cancers in Mice Using an Indocyanine Green–Labeled Podoplanin Antibody

Cited by 27 publications

References 34 publications

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas

Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C<sub>44</sub>Mab-3 for Multiple Applications against Pancreatic Carcinomas

Development of a Novel Anti-CD44 variant 6 Monoclonal Antibody for Multiple Applications against Colorectal Carcinomas

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