Colorectal cancer (CRC) is a type of gastrointestinal cancer and one of the most studied malignancies in oncology. The search for reliable biomarkers continues to shape how we detect, monitor, and understand this disease. Gastrointestinal cancer includes malignancies of the digestive tract, with colorectal cancer being a major subtype. A wide range of colorectal cancer markers have been identified, each offering unique insights into tumor biology, progression, and potential therapeutic targets.

Tumor markers are vital in the diagnosis, prognosis, and monitoring of colon cancer and colorectal cancer patients. Among these, carcinoembryonic antigen (CEA), also called carcinoembryonic antigen CEA, and carbohydrate antigen (CA19-9) are widely used. The combined assessment of CEA CA19-9 is particularly valuable for evaluating tumor marker levels in colorectal dis. Measuring serum CEA in ng/mL, especially preoperative serum levels, provides important prognostic information and helps monitor colorectal cancer progression and treatment response. According to the National Cancer Institute and NCCN Clinical Practice Guidelines, these tumor markers are integral to the clinical management of colorectal cancer.

Among the emerging markers, NOTUM has gained attention for its role in Wnt signaling modulation, particularly in early-stage CRC. Calretinin, a calcium-binding protein, is being explored for its diagnostic value in distinguishing between different tumor types. MUC2, a mucin protein, is typically downregulated in CRC, reflecting changes in epithelial differentiation.

Inflammation-related markers like COX-2 are frequently overexpressed in colorectal tumors and are associated with tumor growth and angiogenesis. LI cadherin, a cell adhesion molecule, is another marker linked to epithelial integrity and tumor progression.

GPA33 is a cell surface antigen expressed in over 95% of colorectal cancers, making it a promising target for antibody-based therapies. Similarly, EGFR and VEGF are well-known for their roles in cell proliferation and angiogenesis, and they are already targets of approved therapies in metastatic CRC.

Tumor suppressor p53 and proliferation marker Ki67 are commonly used in pathology to assess tumor aggressiveness. IGFBP2 and PKM2 are metabolic markers that reflect the altered energy metabolism in cancer cells, often referred to as the Warburg effect.

Transcription factor CDX2 is a reliable marker for intestinal differentiation and is often used to confirm colorectal origin in metastatic tumors. Ep-CAM, Villin, CK7, and CK20 are epithelial markers that help in tumor classification and origin identification. Notably, CK20 is frequently positive in CRC, while CK7 is typically negative, aiding differential diagnosis.

Colorectal cancer is a significant contributor to cancer-related deaths worldwide, with most cancer deaths resulting from metastasis and disease progression. These colorectal cancer markers are not only valuable for diagnosis but also for guiding treatment decisions and monitoring disease progression. As research advances, combining multiple markers may enhance diagnostic accuracy and personalize patient care. Colorectal cancer screening, including the use of the fecal occult blood test as a noninvasive early detection tool, plays a crucial role in identifying cancer at an early stage and improving patient outcomes.

Understanding the expression patterns and biological roles of these markers helps researchers and clinicians navigate the complex landscape of CRC. Early detection through screening and the use of multiple biomarkers can significantly improve therapeutic outcomes for colorectal cancer. Whether you’re developing a cell-based assay or exploring new therapeutic targets, these biomarkers offer a foundation for innovation and discovery.

Here, we look at some of the most common primary IHC markers for colorectal cancer (CRC) and cell-type-specific biomarkers. We also recommend specific antibodies for each biomarker for use in key applications such as IHC.

NOTUM

NOTUM is a secreted enzyme that modifies WNT signaling by removing lipid groups from WNT ligands, limiting their ability to bind to Frizzled receptors. In colorectal cancer, especially in APC-mutant intestinal stem cells, NOTUM expression is often elevated. This shift disrupts normal stem cell dynamics, giving mutant cells a competitive edge. Studies suggest that targeting NOTUM may help rebalance intestinal cell populations and reduce adenoma formation. As research progresses, NOTUM is gaining attention as a biomarker for early detection and a possible therapeutic target in colorectal cancer.

Figure 1. Western blot - Anti-NOTUM antibody [EPR27981-76] - BSA and Azide free (ab315896).

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Calretinin

Calretinin is a calcium-binding protein that plays a role in message targeting and intracellular calcium buffering. While absent in normal colonocytes, it is expressed in most poorly differentiated colon carcinomas. Strongly positive calretinin staining in colon medullary carcinoma, as well as correlations between the expression of calretinin and the degree of differentiation in human colorectal adenocarcinomas, have been reported, emphasizing the potential of calretinin as an IHC biomarker for bowel cancer.

Certain genetic variations in the calretinin gene have also been identified as risk factors for colon cancer. Chronic inflammatory conditions such as inflammatory bowel disease, including ulcerative colitis and Crohn's disease, are also established risk factors for colorectal cancer due to persistent inflammation and mucosal disruption.

Figure 2. Immunocytochemistry/ Immunofluorescence - Anti-Calretinin antibody [EP1798] (ab92341).

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MUC2

MUC2 is part of the mucin family and is characteristically observed in small and large bowel mucosa goblet cells. It is involved with coating the epithelia of the intestines, airways, and other mucus membrane-containing organs. Loss of MUC2 expression has been reported to be a predictor of adverse outcomes, and further prospective studies that evaluate adjuvant chemotherapy in stage II patients and stage III patients of colon cancer should include MUC2 expression analysis for patient stratification, as MUC2 expression may vary according to disease stage and can inform treatment decisions, with particular attention to stage II CRC patients.

Studies have shown the prognostic value of MUC2 expression, specifically in stage II and stage III patients. Stage II CRC patients may benefit from tailored follow-up strategies based on MUC2 status.

Figure 3. Immunohistochemistry (Frozen sections) - Anti-MUC2 antibody [EPR23479-47] (ab272692).

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COX-2

COX-2 is an inducible enzyme that regulates prostaglandin synthesis and is overexpressed in several epithelial cancers. It regulates apoptosis, angiogenesis, and tumor cell invasiveness, which contribute to its effects on tumorigenesis. These processes are key contributors to tumor development in colorectal cancer. Multiple studies have shown that selective COX-2 inhibitors are a form of targeted therapy for colorectal cancer, offering a targeted approach to the chemoprevention of CRC.

Figure 4. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800).

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LI Cadherin

LI Cadherin is one of a group of cadherins that act as calcium-dependent cell adhesion proteins that preferentially interact with themselves in a homophilic manner in connecting cells. High LI Cadherin expression has been associated with liver metastasis, a major cause of death associated with colorectal cancer, and poor survival of patients. LI Cadherin expression has also been studied in relation to lymph node metastasis, which is an important factor in colorectal cancer progression.

Figure 5. Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772).

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GPA33

The GPA33 gene codes for A33 antigen, a transmembrane glycoprotein that is expressed in normal colonic and small bowel epithelium as well as more than 95% of human colon cancers. IHC in CRC tissue has shown strong A33 membrane staining in samples of well-differentiated tumors, and there are also proposals for using antibodies to GPA33 as a potentially potent radioimmunotherapy regimen for GPA-33 positive CRC tumors in humans.

Figure 6. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPA33 antibody [EPR4240] (ab108938).

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EGFR

Epidermal growth factor receptor (EGFR) is usually involved in cell growth and is recognized as an important player in CRC initiation and progression. EGFR is also one of the key molecular markers used in colorectal cancer to guide targeted therapy decisions. In metastatic colorectal cancer, the most commonly used targeted therapies are monoclonal antibodies that prevent EGFR activation. However, future development of anti-EGFR-directed nanoparticles that could inhibit overactive EGFR signals could potentially reduce CRC risk.

Figure 7. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894).

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VEGF

VEGF is a heparin-binding glycoprotein with potent angiogenic activity within endothelial cells. Angiogenesis is crucial for the progression of colorectal carcinoma. VEGF is expressed in around 50% of colorectal cancers and shows very low expression in normal colonic mucosa, making it a promising biomarker for diagnosing CRC from histology samples. Increased VEGF expression is often observed in advanced-stage colorectal cancers and is associated with poorer prognosis. VEGF, along with other biomarkers such as CA-125, CA19-9, and CEA, is considered among the key prognostic factors in colorectal cancer.

Figure 8. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VEGFA antibody [EP1176Y] - C-terminal (ab52917).

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p53

The tumor suppressor p53 is a transcription factor involved in cell cycle arrest and apoptosis under cellular stress. Mutations within p53 are one of the most frequent triggers leading to the progression of CRC and are found in 34% of the proximal colon tumors and 45% of distal colorectal tumors.

Figure 9. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049).

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Ki67

Ki67 is an important protein involved in cell division and is commonly used as a marker of cellular proliferation. Quantifying Ki67 expression by IHC will give you the Ki67 labeling index, which is commonly used in clinical pathology to estimate cancer prognosis. High Ki67 expression is thought to be an indication of a good prognosis for patients with colorectal cancer. Ki67 is one of several biomarkers with established prognostic values in colorectal cancer, helping to predict patient outcomes and guide treatment decisions.

Figure 10. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] (ab16667).

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Insulin-like growth factor binding protein 2 (IGFBP2)

IGFBP-2 interacts with the extracellular matrix, proteoglycans, and integrin receptors within many different cell types. It may also act as a transcription factor to stimulate gene expression. Studies have shown that IGFBP-2 is uniquely distributed at the bottom of human colonic crypts and that increased levels of IGFBP-2 can be seen in many colorectal cancers. IGFBP-2 is often overexpressed in cancer tissue compared to normal tissue, highlighting its potential as a biomarker for tumor activity. IGFBP-2 is one of several serum tumor markers used in the diagnosis and monitoring of colorectal cancer. This expression co-localizes with the phosphorylated p65 subunit of NF-kB, making these good biomarkers for CRC.

Figure 11. Immunohistochemistry (Frozen sections) - Anti-IGFBP2 antibody [EPR18012-257] (ab188200).

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PKM2

The M2 isoform of PK (PKM2) is a glycolytic enzyme involved in aerobic glycolysis and anabolic metabolism in cancer cells. PKM2 also promotes the transcription of Oct-46, HIF-1α7, STAT38, and β-catenin during the progression of various cancers.

Chronic inflammation in the colon can contribute to increased PKM2 expression and promote tumorigenesis, highlighting its role in colorectal cancer development.

Figure 12. Immunocytochemistry/ Immunofluorescence - Anti-PKM antibody [EPR10138(B)] - BSA and Azide free (ab206129).

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CDX2

CDX2 is a transcription factor expressed specifically in the intestine, where it is involved in the maintenance of intestinal cell types. Low CDX2 expression in CRC is associated with advanced stages of cancer progression, vessel invasion, and metastasis. Over 20% of CRC show some reduction of CDX2 protein by histology. Seeing normal or high levels of CDX2 expression is a biomarker for a good prognosis for survival from the disease.

Loss of CDX2 expression has been identified as an independent negative prognostic marker in colorectal cancer. CDX2, along with other biomarkers, is considered among the independent prognostic factors in colorectal cancer, helping to predict survival outcomes regardless of other clinical variables.

Figure 13. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDX2 antibody [EPR2764Y] (ab76541).

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Ep-CAM

Ep-CAM is a common biomarker used in colorectal adenocarcinoma to discriminate cancer cells from mesothelial cells. Being able to distinguish between adenocarcinoma and mesothelial cells by histology is crucial for clinicians to be able to correctly diagnose and assign treatment to patients. Ep-CAM immunohistochemistry is a valuable diagnostic test in the evaluation of colorectal cancer tissue samples.

Ep-CAM is also commonly used to identify and isolate tumor cells in colorectal cancer tissue samples.

Figure 14. Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] (ab223582).

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Villin

Villin is an actin-binding protein expressed in the epithelial cells lining the gut and has been shown to regulate epithelial-mesenchymal transition (EMT) in colorectal cancers. Microsatellite instability (MSI) colorectal cancers have also been shown to have very low levels of Villin expression compared to normal gut epithelial cells. Studies have found a significant difference in Villin expression between normal gut epithelial cells and microsatellite instability colorectal cancers. This loss of Villin leads to poorly differentiated histology of the CRC.

Figure 15. Immunoprecipitation - Anti-Villin antibody [SP145] (ab130751).

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CK7 (Cytokeratin 7) and CK20 (Cytokeratin 20)

CK7 and CK20 are cytokeratins expressed in the gut epithelia. Different CK7 and CK20 IHC expression patterns are commonly used to distinguish colorectal adenocarcinomas. Most colorectal cancers are CK7 negative and CK20 positive, making the combination of these cytokeratins an excellent biomarker combination. Significant differences in CK7 and CK20 expression patterns are observed between colorectal adenocarcinomas and other epithelial neoplasms. Assessment of CK7 and CK20 expression patterns is a standard procedure in routine clinical practice for colorectal cancer diagnosis. CK7 and CK20 expression varies between colorectal carcinomas according to histological grade and tumor location.

Figure 16. Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 7 antibody [EPR1619Y] - Cytoskeleton Marker (ab68459).

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References

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