Recombinant Rabbit CD166 antigen (ALCAM)

What is CD166/ALCAM and what cellular functions does it regulate?

CD166/ALCAM is a member of a subfamily of immunoglobulin receptors with five immunoglobulin-like domains (VVC2C2C2) in the extracellular domain. It functions as a cell adhesion molecule that binds to T-cell differentiation antigen CD6 and is implicated in cell adhesion and migration processes . Functionally, ALCAM may act as a cell surface sensor to register local growth saturation and regulate cellular signaling and dynamic responses. The ALCAM-CD6 interaction is required for optimal activation of T-cells, suggesting ALCAM involvement in immunologic responses to tumor cells .

ALCAM expression is transcriptionally regulated and differentially expressed across various cell types, with highest expression typically found in stem cell populations. Research indicates that ALCAM may favor interactions between tumor and endothelial cells, potentially contributing to cancer progression .

How is CD166/ALCAM differentially expressed across tissue types and developmental stages?

CD166/ALCAM shows distinct expression patterns across different tissues and developmental stages. In hematopoietic cell populations, ALCAM is differentially regulated and most highly expressed in long-term hematopoietic stem cells (LT-HSCs) . Studies have demonstrated that ALCAM expression progressively increases with age in LT-HSCs, with significant upregulation observed in 12-month-old mice compared to 2-month-old mice .

Analysis of ALCAM mRNA levels in sorted hematopoietic stem and progenitor cell (HSPC) subsets by qRT-PCR reveals a differential expression pattern that mirrors cell surface protein levels. This indicates transcriptional regulation of ALCAM expression. Across age groups, ALCAM maintains preferential expression in LT-HSCs, with significant age-associated upregulation showing approximately 2-fold and 5-fold increases at 12 months and 16 months, respectively .

What are the primary applications for CD166/ALCAM antibodies in research?

Recombinant rabbit monoclonal antibodies against CD166/ALCAM are valuable tools in various research applications:

For optimal results, researchers should validate antibodies in their specific experimental systems as performance may vary based on tissue type and preparation method .

How can I optimize CD166/ALCAM detection in Western blot experiments?

For optimal Western blot detection of CD166/ALCAM, consider the following methodological approach:

• Sample preparation: Include protease inhibitors in lysis buffer to prevent degradation.

• Protein loading: Load 20-40 μg of total protein per lane.

• Gel selection: Use 8-10% SDS-PAGE gels for optimal separation.

• Transfer conditions: Transfer to PVDF membranes at 100V for 60-90 minutes.

• Blocking: Block with 5% non-fat milk or BSA in TBS-T for 1 hour at room temperature.

• Antibody selection: Use validated recombinant rabbit monoclonal antibodies (e.g., ARC1720) at manufacturer-recommended dilutions .

• Incubation time: Incubate with primary antibody overnight at 4°C for best results.

• Controls: Include positive controls such as THP-1 or SH-SY5Y cell lysates .

Note that CD166/ALCAM is often detected as a band of approximately 65 kDa, though this may vary due to glycosylation. Deglycosylation treatments can be used to confirm specificity if multiple bands are observed.

What are the best approaches for quantifying CD166/ALCAM expression in clinical samples?

Quantitative measurement of CD166/ALCAM in clinical samples requires careful consideration of methodology. ELISA assays have been developed with high sensitivity and specificity for CD166/ALCAM detection in serum samples:

• ELISA development considerations:

  • Ensure recovery of 90-100% when recombinant human ALCAM protein is added to control diluent and serum samples.

  • Verify negligible cross-reactivity with other adhesion molecules of the Ig superfamily.

  • Test assay reproducibility and linearity across the dynamic range.

  • Evaluate sample stability under various storage conditions .

• Clinical performance metrics:
  In breast cancer studies, ALCAM outperformed traditional markers with an area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] compared to CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63) .

• Alternative methods:

  • Flow cytometry for cellular expression profiling

  • Immunohistochemistry for spatial distribution in tissues

  • qRT-PCR for transcriptional analysis

Each method has specific advantages and limitations that should be considered based on the research question and available sample types.

How does CD166/ALCAM contribute to hematopoietic stem cell function?

CD166/ALCAM plays a crucial role in hematopoietic stem cell (HSC) function, particularly in maintaining long-term repopulating capacity and engraftment potential. Studies using Alcam knockout (Alcam−/−) mouse models have revealed several key aspects of ALCAM function in HSCs:

• Long-term repopulation: Alcam−/− HSCs demonstrate reduced long-term replating capacity in vitro and diminished long-term engraftment potential upon transplantation .

• Frequency of long-term repopulating cells: Alcam−/− bone marrow contains a markedly lower frequency of long-term repopulating cells compared to wild type (WT) .

• Age-associated regulation: ALCAM levels progressively increase with age in LT-HSCs, suggesting a role in age-related stem cell regulation. There is a significant (p=0.0159) elevation in ALCAM surface expression in 12-month-old LT-HSCs compared to 2-month-old LT-HSCs .

• Differentiation capacity: Despite engraftment deficiencies, Alcam−/− HSCs retain normal differentiation potential. In single-cell in vitro differentiation assays, Alcam−/− (n=273) and WT (n=267) HSCs gave rise to similar phenotypes and numbers of all colony types, including megakaryocyte-erythroid, granulocyte-macrophage, granulocyte, and mixed colonies .

These findings indicate that ALCAM is particularly important for HSC self-renewal and long-term engraftment functions rather than differentiation capacity.

Can CD166/ALCAM be used as a marker to isolate and enrich stem cell populations?

CD166/ALCAM has proven valuable as a cell surface marker for isolating and enriching various stem cell populations:

• Prostate stem/progenitor cells: CD166 can further enrich sphere-forming activity of Lin−;Sca1+;CD49fhi (LSChi) populations, which are already enriched more than 10-fold for in vitro sphere-forming activity .

• Human prostate cells: CD166 enriches sphere-forming ability of benign primary human prostate cells in vitro and induces the formation of tubule-like structures in vivo .

• Hematopoietic stem cells: ALCAM surface expression correlates with LT-HSC identity and function. Flow cytometry analysis of HSPC subsets based on ALCAM expression can separate populations with differential stem cell properties .

Methodological approach for using CD166/ALCAM in stem cell isolation:

• Use high-quality monoclonal antibodies specific for CD166/ALCAM

• Include appropriate isotype controls

• Combine with other established stem cell markers for higher purity

• Validate isolated populations through functional assays (e.g., sphere formation, transplantation)

The use of CD166 as an enrichment marker is particularly valuable in cancer research, where it can help identify cancer-initiating cell populations with enhanced tumorigenic potential.

How is CD166/ALCAM expression altered in cancer, and what are its diagnostic implications?

CD166/ALCAM expression is significantly altered in various cancer types, with important diagnostic implications:

• Breast cancer: ALCAM is overexpressed in breast cancer tissues and can be detected at elevated levels in patient serum. As a diagnostic biomarker, ALCAM (AUC = 0.78 [95% CI: 0.73, 0.84]) outperforms traditional markers like CA15-3 (AUC = 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC = 0.63) .

• Prostate cancer: CD166 expression is upregulated in human prostate cancers, with particularly high expression in castration-resistant prostate cancer (CRPC) samples .

• Methodology for assessment:

  • Serum ELISA assays can quantify soluble ALCAM levels

  • Immunohistochemistry can assess tissue expression patterns

  • Flow cytometry can measure cellular expression levels

For research purposes, it's important to note that though genetic deletion of murine CD166 in the Pten-null prostate cancer model does not block cancer progression or CRPC development, the presence of CD166 on prostate stem/progenitors and castration-resistant sub-populations suggests potential for targeted therapeutic delivery .

What experimental approaches can be used to study CD166/ALCAM function in cancer progression?

Several experimental approaches are effective for investigating CD166/ALCAM's role in cancer progression:

• Genetic knockdown/knockout studies:

  • siRNA or shRNA-mediated knockdown in cancer cell lines

  • CRISPR/Cas9-mediated knockout in cell lines or animal models

  • Conditional knockout mouse models (e.g., Alcam−/− mice crossed with cancer-prone models)

• Functional assays:

  • Sphere formation assays to assess cancer stem cell properties

  • Migration and invasion assays to evaluate metastatic potential

  • Colony formation assays to measure clonogenic potential

  • Limiting dilution transplantation assays to determine tumor-initiating cell frequency

• Molecular mechanism studies:

  • Co-immunoprecipitation to identify binding partners

  • Signaling pathway analysis using phosphorylation-specific antibodies

  • Transcriptome analysis to identify downstream effectors

For example, in prostate cancer research, the Lin−;Sca1+;CD49fhi;CD166+ (LSChi;CD166+) population showed enhanced sphere-forming ability compared to LSChi;CD166− cells, demonstrating CD166's utility in enriching for cancer-initiating cells .

What are common technical challenges when working with CD166/ALCAM antibodies and how can they be addressed?

Researchers may encounter several technical challenges when working with CD166/ALCAM antibodies:

• Antibody specificity issues:

  • Solution: Validate antibodies using positive and negative controls (e.g., CD166-expressing and non-expressing cell lines)

  • Use recombinant monoclonal antibodies like ARC1720 for improved consistency

  • Confirm specificity with genetic knockdown/knockout samples when possible

• Detection of multiple isoforms:

  • CD166/ALCAM has multiple identified isoforms

  • Solution: Use antibodies targeting conserved epitopes for detecting all isoforms

  • For isoform-specific detection, select antibodies recognizing unique regions

• Post-translational modifications:

  • CD166/ALCAM undergoes glycosylation which can affect antibody binding

  • Solution: Consider deglycosylation treatments for consistent results

  • Use antibodies raised against peptide sequences less affected by modifications

• Low expression levels in certain samples:

  • Solution: Implement signal amplification methods

  • Concentrate samples when appropriate

  • Use more sensitive detection systems (e.g., chemiluminescence for Western blot)

How can contradictory findings about CD166/ALCAM function be reconciled in experimental design?

Contradictory findings regarding CD166/ALCAM function across different studies may arise from several factors that should be considered in experimental design:

• Cell/tissue context differences:

  • Solution: Clearly define and characterize the cellular system being studied

  • Compare results across multiple cell lines or primary tissues

  • Consider microenvironmental factors that may influence ALCAM function

• Methodology variations:

  • Solution: Standardize protocols across experiments

  • Use multiple complementary techniques to validate findings

  • Report detailed methodological information to facilitate reproducibility

• Species differences:

  • Human and murine ALCAM may have functional differences

  • Solution: Be cautious when extrapolating findings between species

  • Validate key findings in both systems when possible

• Differentiating correlation vs. causation:

  • Altered ALCAM expression may be a consequence rather than cause of disease

  • Solution: Use carefully designed gain/loss-of-function studies

  • Implement temporal analysis to establish sequence of events

For example, while Alcam−/− HSCs show compromised long-term repopulating potential, deletion of CD166 in the Pten-null prostate cancer model does not block cancer progression . This apparent contradiction highlights the context-dependent nature of CD166/ALCAM function and necessitates careful experimental design when studying this molecule in different biological systems.