ALCAM (CD166) Human

What is the molecular structure of ALCAM (CD166) Human?

ALCAM is a 583 amino acid type I membrane glycoprotein consisting of:

• 27 amino acid signal peptide

• 500 amino acid extracellular domain containing 5 Ig-like domains

• 24 amino acid transmembrane domain

• 32 amino acid cytoplasmic domain

The extracellular domain's structure enables both homotypic (ALCAM-ALCAM) and heterotypic interactions with binding partners, which are essential for its biological functions.

Where is ALCAM expressed in normal human tissues?

ALCAM was initially identified on activated leukocytes but is now known to be expressed more ubiquitously. Primary expression sites include:

• Thymic epithelial cells

• Activated B and T cells

• Monocytes

• Various epithelial cell types

While initially thought to be primarily involved in leukocyte interactions, ALCAM is now recognized as being present in almost all cell types throughout the body .

What are the primary binding partners of ALCAM and their functional significance?

ALCAM engages in multiple binding interactions that mediate diverse physiological processes:

• Homotypic binding: ALCAM-ALCAM interactions

• CD6: Critical for T cell development and regulation

• NgCAM: Important for functions in the nervous system

• L1CAM: Facilitates tumor-endothelial interactions in cancer progression

• 14-3-3ζ and 14-3-3σ: Identified as interaction partners in oral squamous cell carcinoma

These interactions mediate cell adhesion, migration, and signaling pathways relevant to both normal physiology and disease states.

What methodologies can be used to detect and quantify ALCAM expression in research samples?

Researchers can employ multiple complementary approaches:

• Flow cytometry: Ideal for detecting ALCAM on cell surfaces, particularly using fluorophore-conjugated antibodies (e.g., Alexa Fluor® 647-conjugated antibodies)

• Immunohistochemistry (IHC): For visualizing ALCAM expression patterns (membrane vs. cytoplasmic) in tissue sections

• ELISA: For quantifying soluble ALCAM in serum, ascites, and other biological fluids

• Western blotting: For protein detection in cell or tissue lysates

• qPCR/gene microarray: For analyzing ALCAM mRNA expression levels

Each method has specific applications, with flow cytometry being particularly useful for characterizing ALCAM expression on immune cell populations, as demonstrated in studies with human blood monocytes .

How can I design experiments to validate antibody specificity for ALCAM research?

Antibody validation is critical for reliable ALCAM research and should include:

• Positive controls: Test the antibody on cell lines known to express ALCAM (e.g., activated T cells, certain cancer cell lines)

• Negative controls: Include isotype control antibodies (e.g., IC002R when using FAB6561R)

• Knockdown validation: Compare staining between wild-type cells and those with ALCAM knockdown via siRNA or CRISPR

• Blocking experiments: Pre-incubate antibodies with recombinant ALCAM to confirm binding specificity

• Functional validation: Test antibody effects on known ALCAM functions, such as cell adhesion (e.g., Jurkat cell binding to immobilized CD6 Fc Chimera)

For maximum reliability, researchers should follow standardized protocols, such as those available for staining membrane-associated proteins .

What are the optimal conditions for studying ALCAM-mediated cell adhesion in vitro?

For robust ALCAM adhesion assays:

• Cell selection: Choose cell lines with verified ALCAM expression (e.g., Jurkat cells)

• Substrate preparation:

  • For homophilic interactions: Coat surfaces with recombinant ALCAM

  • For heterophilic interactions: Use recombinant CD6 Fc Chimera (10 μg/mL)

• Blocking controls: Include anti-ALCAM antibodies (25 μg/mL can achieve 80-100% inhibition of adhesion)

• Experimental readouts:

  • Adherent cell quantification

  • Migration assays to assess functional consequences

  • Live-cell imaging to observe dynamic interactions

When optimizing these assays, researchers should consider physiologically relevant conditions, including divalent cation concentrations that may affect adhesion strength.

How does ALCAM expression vary across different cancer types, and what are the prognostic implications?

ALCAM expression exhibits remarkable cancer type-specific patterns with varying prognostic significance:

These variations highlight the importance of cancer-specific interpretation of ALCAM expression data in both research and clinical contexts.

What is the significance of soluble ALCAM (sALCAM) in oncology research?

Soluble ALCAM represents a truncated form found in circulation with significant research implications:

• Prognostic biomarker potential:

  • In ovarian cancer: Elevated in aggressive tumors and high stages, correlates with CA125/MUC16

  • In thyroid cancer: Increased in patients with lymph node metastases

  • In prostate cancer: Higher in patients with metastasis, nodal-positive disease, and those who died from prostate cancer

  • In breast cancer: Associated with shorter disease-free survival

• Mechanistic insights:

  • Studies show a lack of correlation between serum ALCAM and tissue ALCAM protein/mRNA levels in breast cancer, suggesting non-transcriptional and non-translational contribution to raised serum levels

  • Evidence points to protease cleavage as the likely mechanism for generating soluble ALCAM

• Technical advancements:

  • Recent development of SiNW-on-a-chip biosensor technology allows for rapid detection (30 min) of ALCAM in serum

How does ALCAM contribute to the "seed and soil" theory of cancer metastasis?

ALCAM plays a crucial role in the metastatic cascade through multiple mechanisms:

• Receptor function: ALCAM acts as a "soil sensor receptor" for S100A8/A9/S100P, which serve as "soil signals" that guide metastasizing cancer cells to specific organs

• Cell-cell interaction mediator:

  • L1CAM on breast cancer cells interacts with ALCAM on endothelial cells, promoting tumor-endothelial interactions essential for metastasis

  • ALCAM's extracellular domain facilitates multiple protein-protein interactions that influence cancer cell behavior

• Dynamic regulation:

  • ALCAM undergoes endocytosis for recycling, requiring endophilin (particularly the A3 isoform)

  • This process is driven by galectin-8 of extracellular origin

  • Blocking endophilin-A3 increases cell surface ALCAM and enhances ALCAM-mediated cell adhesiveness

These mechanisms collectively contribute to the complex process by which cancer cells identify and colonize distant sites, representing a significant area for therapeutic intervention.

What are the emerging therapeutic strategies targeting ALCAM in cancer?

Several promising therapeutic approaches targeting ALCAM are under investigation:

• Antibody-based strategies:

  • Single-chain antibody scFv173 has demonstrated ability to reduce ALCAM-mediated cell adhesion and inhibit breast tumor growth in vivo

  • Blocking antibodies against CD166-ILT3 interactions have shown reduced proliferation of leukemia cells and increased survival in tumor-bearing mice

• Targeted imaging approaches:

  • Iron oxide microparticles conjugated to anti-ALCAM antibodies have been used as MRI contrast agents to detect brain metastases with 86% specificity and 79% sensitivity

  • PET imaging approaches using anti-ALCAM immunoglobulin conjugates with various linker designs

• Indirect targeting strategies:

  • NFκB inhibitors have shown effectiveness in suppressing CD166 expression in lung stem-like cancer cells that co-express CD44 and EpCAM

  • Targeting CD6-CD166 interactions by blocking CD6, an established binding partner for CD166

Each approach requires careful consideration of the cancer-specific context and potential off-target effects due to ALCAM's expression in normal tissues.

How can I design experiments to determine whether ALCAM plays a causal role in cancer progression versus being a passive biomarker?

Distinguishing causal involvement from correlation requires systematic experimental approaches:

• Genetic manipulation studies:

  • CRISPR/Cas9 knockout of ALCAM in cancer cell lines

  • Inducible shRNA systems for temporal control of ALCAM expression

  • Domain-specific mutations to identify functionally critical regions

• Functional phenotype assessment:

  • Proliferation, migration, and invasion assays in vitro

  • Colony formation and sphere-formation assays for stem-like properties

  • Xenograft models with modified ALCAM expression

  • Metastasis models to assess effects on dissemination

• Mechanistic investigation:

  • Analysis of binding interactions with partners like CD6 and L1CAM

  • Examination of downstream signaling pathways

  • Investigation of ALCAM trafficking and recycling

• Rescue experiments:

  • Re-expression of ALCAM in knockout models

  • Introduction of mutant ALCAM variants to identify essential domains

These approaches, when used in combination, can provide compelling evidence for ALCAM's functional role in cancer progression.

What challenges exist in translating ALCAM research findings into clinical applications?

Several significant challenges must be addressed:

• Contradictory prognostic associations:

  • ALCAM shows different prognostic associations across cancer types

  • Membrane versus cytoplasmic expression may have opposite implications

  • Reconciling these differences requires cancer-specific interpretation

• Biomarker standardization issues:

  • High baseline levels in normal populations

  • Presence in multiple non-cancerous conditions

  • Need for standardized detection methods and cutoff values

• Therapeutic targeting concerns:

  • Ubiquitous expression across multiple tissues raises potential for off-target effects

  • Blood-brain barrier considerations for CNS applications

  • Tumor selectivity varies by cancer type (e.g., better detection of breast cancer metastases to brain compared to lung or melanoma)

• Technical limitations:

  • Species differences in antibody recognition (e.g., human vs. murine tissues)

  • Challenges in distinguishing between membrane-bound and soluble forms

Addressing these challenges requires integrated approaches combining basic research, translational studies, and careful clinical validation.

How can single-cell technologies advance our understanding of ALCAM in the tumor microenvironment?

Single-cell approaches offer unprecedented insights into ALCAM biology:

• Single-cell RNA sequencing:

  • Reveals heterogeneity of ALCAM expression within tumors

  • Identifies cell populations where ALCAM may play critical roles

  • Allows correlation with other markers to define functional subsets

• Mass cytometry (CyTOF):

  • Enables simultaneous analysis of ALCAM with dozens of other proteins

  • Can identify rare cell populations with unique ALCAM expression patterns

  • Provides insights into signaling networks associated with ALCAM

• Spatial transcriptomics/proteomics:

  • Preserves spatial context of ALCAM-expressing cells

  • Reveals interactions between ALCAM+ cells and other components of the tumor microenvironment

  • Can identify niches where ALCAM may be particularly important

These technologies can help resolve contradictory findings by revealing context-specific functions of ALCAM that may be obscured in bulk analyses.

What is the relationship between ALCAM expression and cancer stem cell properties?

Emerging evidence suggests important connections between ALCAM and cancer stemness:

• Co-expression patterns:

  • In lung cancer, CD166 is co-expressed with established stem cell markers CD44 and EpCAM in stem-like cancer cells

  • These cells show particular sensitivity to NFκB inhibitors

• Functional relationships:

  • ALCAM may contribute to maintenance of stem-like properties through modulation of adhesion and signaling

  • Interaction with the tumor microenvironment may create niches supporting cancer stem cell maintenance

• Methodological approaches:

  • Sphere formation assays to assess self-renewal capacity

  • Limiting dilution assays to quantify tumor-initiating potential

  • Lineage tracing to track ALCAM+ cells during tumor growth and treatment

Understanding these relationships may identify new therapeutic vulnerabilities, particularly in cancers where conventional treatments fail to eliminate cancer stem cells.

How do post-translational modifications affect ALCAM function in cancer progression?

Post-translational modifications represent an understudied aspect of ALCAM biology:

• Proteolytic processing:

  • Generation of soluble ALCAM through proteolytic cleavage

  • Different proteases may be involved in different cancer contexts

  • The released extracellular domain may have distinct biological activities

• Glycosylation patterns:

  • The extracellular domain contains multiple potential glycosylation sites

  • Cancer-specific alterations in glycosylation may affect binding properties

  • Methods such as lectin arrays and glycoproteomics can characterize these changes

• Phosphorylation of cytoplasmic domain:

  • May regulate ALCAM trafficking and signaling

  • Phosphoproteomic analysis can identify cancer-specific modifications

  • Mutational studies can determine functional significance

These modifications may explain some of the context-dependent functions of ALCAM and represent potential targets for therapeutic intervention.