Anti-mucin 4 fluorescent antibody brightly targets colon cancer in patient-derived orthotopic xenograft mouse models: A proof-of-concept study for future clinical applications


      • In this study, MUC4 was overexpressed in CRC primary and metastatic liver tumors compared to normal colon.
      • Anti-MUC4 antibodies conjugated to NIR dye can improve visualization of primary CRC.
      • Anti-MUC4 antibodies conjugated to NIR can also label CRLM in the liver bed.



      There is a high rate of positive surgical margins with resection of liver metastases in colorectal cancer (CRC). The present study reports using a fluorescent anti-mucin 4 (MUC4) antibodies to label primary CRC and liver metastases to better visualize tumor margins in mouse models.


      Western blotting for MUC4 protein expression of normal colon and CRC tumor lysates was performed. Orthotopic primary and liver metastatic CRC mouse models received anti-MUC4 antibody conjugated to IR800 (MUC4-IR800). Mice were sacrificed and imaged after 48 hours.


      Western blotting demonstrated increased MUC4 expression in a human CRC cell line and patient-derived primary and liver-metastatic CRCs. The LS174T orthotopic primary CRC model tumor to background ratio (TBR) was 2.04 (±0.35). The patient-derived orthotopic xenograft (PDOX) primary CRC model TBR was 2.17 (±0.35). The PDOX liver metastasis model TBR was 1.56 (±0.53).


      MUC4-IR800 provided bright labeling of primary and liver tumors in CRC orthotopic mouse models, demonstrating their future clinical potential for margin visualization in fluorescence guided surgery.


      FGS (Fluorescence guided surgery), CRC (Colorectal cancer), MUC4 (Mucin 4), PDOX (Patient derived orthotopic xenograft), TBR (Tumor to background ratio), CRLM (Colorectal liver metastasis), ICG (Indocyanine green), PBS (Phosphate-buffered saline), SDS (sodium dodecyl sulfate), PBST (phosphate-buffered saline with tween 20), UC (Ulcerative colitis), HRP (horseradish peroxidase)
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        • Peeters K.C.M.J.
        • Marijnen C.A.M.
        • Nagtegaal I.D.
        • et al.
        The TME trial after a median follow-up of 6 years: increased local control but no survival benefit in irradiated patients with resectable rectal carcinoma.
        Ann Surg. 2007; 246: 693-701
      1. Mitchell D, Puckett Y, Nguyen QN. Literature review of current management of colorectal liver metastasis. Cureus. 11(1):e3940. doi:10.7759/cureus.3940.

        • Garden O.J.
        • Rees M.
        • Poston G.J.
        • et al.
        Guidelines for resection of colorectal cancer liver metastases.
        Gut. 2006; 55 (iii1-iii8)
        • Adam R.
        • Kitano Y.
        Multidisciplinary approach of liver metastases from colorectal cancer.
        Ann Gastroenterol Surg. 2019; 3: 50-56
        • Jonas S.
        • Thelen A.
        • Benckert C.
        • et al.
        Extended resections of liver metastases from colorectal cancer.
        World J Surg. 2007; 31: 511-521
        • Shanmugam C.
        • Jhala N.C.
        • Katkoori V.R.
        • et al.
        Prognostic value of MUC4 expression in colorectal adenocarcinomas.
        Cancer. 2010; 116: 3577-3586
        • Lu S.
        • Catalano C.
        • Huhn S.
        • et al.
        Single nucleotide polymorphisms within MUC4 are associated with colorectal cancer survival.
        PLoS One. 2019; 14e0216666
        • Xia P.
        • Choi A.H.
        • Deng Z.
        • et al.
        Cell membrane-anchored MUC4 promotes tumorigenicity in epithelial carcinomas.
        Oncotarget. 2016; 8: 14147-14157
        • Jonckheere N.
        • Skrypek N.
        • Van Seuningen I.
        Mucins and pancreatic cancer.
        Cancers. 2010; 2: 1794-1812
        • Tashiro Y.
        • Aoki T.
        • Hirai T.
        • et al.
        Indocyanine green labeling of tumors in the liver recurring after radiofrequency ablation enables complete resection by fluorescence-guided surgery.
        Anticancer Res. 2022; 42: 1345-1350
        • Fu X.Y.
        • Besterman J.M.
        • Monosov A.
        • Hoffman R.M.
        Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens.
        Proc Natl Acad Sci Unit States Am. 1991; 88: 9345-9349
        • Nishino H.
        • Hollandsworth H.M.
        • Sugisawa N.
        • et al.
        Sutureless surgical orthotopic implantation technique of primary and metastatic cancer in the liver of mouse models.
        Vivo. 2020; 34: 3153-3157
        • Kuo T.H.
        • Kubota T.
        • Watanabe M.
        • et al.
        Liver colonization competence governs colon cancer metastasis.
        Proc Natl Acad Sci Unit States Am. 1995; 92: 12085-12089
      2. Hoffman R.M. Patient-Derived Mouse Models of Cancer : Patient-Derived Orthotopic Xenografts (PDOX). Humana Press, 2017
        • Hollandsworth H.M.
        • Amirfakhri S.
        • Filemoni F.
        • et al.
        Anti-carcinoembryonic antigen-related cell adhesion molecule antibody for fluorescence visualization of primary colon cancer and metastases in patient-derived orthotopic xenograft mouse models.
        Oncotarget. 2020; 11: 429-439
        • Hollandsworth H.M.
        • Amirfakhri S.
        • Filemoni F.
        • et al.
        Humanized anti-tumor-associated glycoprotein-72 for submillimeter near-infrared detection of colon cancer in metastatic mouse models.
        J Surg Res. 2020; 252: 16-21
        • Singh A.P.
        • Chaturvedi P.
        • Batra S.K.
        Emerging roles of MUC4 in cancer: a novel target for diagnosis and therapy: figure 1.
        Cancer Res. 2007; 67: 433-436
        • Chauhan S.C.
        • Singh A.P.
        • Ruiz F.
        • et al.
        Aberrant expression of MUC4 in ovarian carcinoma: diagnostic significance alone and in combination with MUC1 and MUC16 (CA125).
        Mod Pathol. 2006; 19: 1386-1394
        • Mukhopadhyay P.
        • Chakraborty S.
        • Ponnusamy M.P.
        • Lakshmanan I.
        • Jain M.
        • Batra S.K.
        Mucins in the pathogenesis of breast cancer: implications in diagnosis, prognosis and therapy.
        Biochim Biophys Acta. 2011; 1815: 224-240
        • Singh A.P.
        • Chauhan S.C.
        • Bafna S.
        • et al.
        Aberrant expression of transmembrane mucins, MUC1 and MUC4, in human prostate carcinomas.
        Prostate. 2006; 66: 421-429
        • Miyahara N.
        • Shoda J.
        • Ishige K.
        • et al.
        MUC4 interacts with ErbB2 in human gallbladder carcinoma: potential pathobiological implications.
        Eur J Cancer Oxf Engl. 1990; 44 (2008): 1048-1056
        • Tamada S.
        • Shibahara H.
        • Higashi M.
        • et al.
        MUC4 is a novel prognostic factor of extrahepatic bile duct carcinoma.
        Clin Cancer Res. 2006; 12: 4257-4264
        • Das S.
        • Rachagani S.
        • Sheinin Y.
        • et al.
        Mice deficient in Muc4 are resistant to experimental colitis and colitis-associated colorectal cancer.
        Oncogene. 2016; 35: 2645-2654
        • Biemer-Hüttmann A.E.
        • Walsh M.D.
        • McGuckin M.A.
        • et al.
        Immunohistochemical staining patterns of MUC1, MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum.
        J Histochem Cytochem. 1999; 47: 1039-1048
        • Ogata S.
        • Uehara H.
        • Chen A.
        • Itzkowitz S.H.
        Mucin gene expression in colonic tissues and cell lines.
        Cancer Res. 1992; 52: 5971-5978
        • Myerscough N.
        • Warren B.
        • Gough M.
        • Corfield A.
        Expression of mucin genes in ulcerative colitis.
        Biochem Soc Trans. 1995; 23 (536S-536S)
        • Turner M.A.
        • Lwin T.M.
        • Amirfakhri S.
        • et al.
        The use of fluorescent anti-CEA antibodies to label, resect and treat cancers: a review.
        Biomolecules. 2021; 11: 1819
        • Lwin T.M.
        • Hoffman R.M.
        • Bouvet M.
        The development of fluorescence guided surgery for pancreatic cancer: from bench to clinic.
        Expert Rev Anticancer Ther. 2018; 18: 651-662
        • Lwin T.M.
        • Turner M.A.
        • Amirfakhri S.
        • Nishino H.
        • Hoffman R.M.
        • Bouvet M.
        Fluorescence molecular targeting of colon cancer to visualize the invisible.
        Cells. 2022; 11: 249
        • Barth C.W.
        • Gibbs S.L.
        Fluorescence image-guided surgery – a perspective on contrast agent development.
        Proc SPIE-Int Soc Opt Eng. 2020; 11222: 112220J
        • Hoffman R.M.
        Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts.
        Nat Rev Cancer. 2015; 15: 451-452