Multiplex detection of surface molecules on colorectal cancers

Area of Science:

• Oncology
• Immunology
• Biotechnology

Background:

• Colorectal cancer (CRC) analysis often requires distinguishing rare cancer cells from normal tissue.
• Accurate immunophenotyping of both cancer cells and tumor-infiltrating lymphocytes (TILs) is crucial for understanding tumor microenvironment and patient prognosis.

Purpose of the Study:

• To develop and validate a fluorescence multiplexing technique for analyzing the plasma membrane proteome of colorectal cancer cells.
• To enable the detection and immunophenotyping of cancer cells within mixed cell populations from surgically resected specimens.
• To profile differential antigen expression on CRC cells and TILs.

Main Methods:

• Preparation of single-cell suspensions from colorectal tumor specimens.
• Capture of mixed cell populations on a CD antibody microarray.
• Detection of cancer cells using fluorescently tagged antibodies for carcinoembryonic antigen (CEA) and epithelial cell adhesion marker (EpCAM).
• Multiplexed labeling with fluorescent antibodies for immunophenotyping of cancer cells and tumor-infiltrating T lymphocytes (e.g., CD3).

Main Results:

• Distinct dot patterns were observed between colorectal cancers and adjacent normal tissue using fluorescence multiplexing.
• Differential expression of specific antigens (CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b, CEA) was identified in cancer cells compared to normal tissue.
• Immunophenotyping of TILs revealed differential expression of various markers including HLA-DR, TCR alpha/beta, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38, and CD71.

Conclusions:

• Fluorescence multiplexing on a single antibody microarray is effective for expression profiling of multiple cell subpopulations within tumor samples.
• This technique allows for the detection and characterization of rare cancer cells and TILs in colorectal cancer specimens.
• The method provides valuable insights into the tumor proteome and immune infiltrate, aiding in cancer research and diagnostics.