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The metastasis-associated gene C4.4A encodes a glycolipid-anchored membraneprotein expressed in several human malignancies. The present study aimed to performa detailed assessment of C4.4A expression in colorectal cancer tissues, in termsof intra-cellular localization, intra-tumoral location and difference in molecularweight. To advance this goal, we developed three new antibodies against the C4.4Aprotein (two polyclonal Abs: C4.4A-119 and C4.4A-277 and one monoclonal Ab: C4.4AGPI-M) to use in addition to the two previously produced polyclonal Abs (C4.4A-81,C4.4A GPI-P). Antibody specificities were confirmed by absorption tests. Westernblot analysis and immunohistochemistry showed that the C4.4A-119 and C4.4-277Abs detected 70-kDa C4.4A, mainly in the cytoplasm, irrespective of intra-tumorallocation. The C4.4A GPI-P and C4.4A GPI-M Abs reacted with the membranous ~40-kDaC4.4A, exclusively at the tumor invasive front, and each detected an identicaltumor cell population. The tested antibodies showed varied C4.4A detection ratesin 33 CRC tissues. The C4.4A-277 Ab yielded the highest positive rate in 29 of33 CRC tissues (87.9%), while the C4.4A GPI-P and C4.4A GPI-M Abs each only showed33.3% positivity. The present findings suggest that the GPI anchor signaling sequencemay be essential for detecting membranous C4.4A at the invasive front of CRC tissues.

Distinct expression of C4.4A in colorectal cancer detected by different antibodies