TM4SF1 Antibody

What is TM4SF1 and why is it a significant target for antibody development?

TM4SF1 is a small plasma membrane glycoprotein belonging to the tetraspanin family. It has four transmembrane domains and two extracellular loops (EL1 and EL2). TM4SF1 has gained significant research attention because it is highly expressed on the plasma membranes of many human cancer cells and on the vascular endothelium of several human cancers, while showing minimal expression in normal cells. Immunohistochemistry staining of 16 types of cancers and adjacent normal tissues demonstrated that TM4SF1 is highly expressed on cancer cell membranes but undetectably expressed on normal cells . TM4SF1 functions as a cell membrane marker of cancer stem cells (CSCs) and regulates cell motility, proliferation, and angiogenesis . Importantly, TM4SF1 is internalized upon interaction with antibodies, making it an ideal target for antibody-drug conjugates .

What is the structure of TM4SF1 and which domains are targeted by antibodies?

TM4SF1 has a tetraspanin-like structure consisting of:

• Four transmembrane domains

• Two extracellular loops: EL1 (smaller) and EL2 (larger)

• Intracellular N-terminal and C-terminal domains

The extracellular loop 1 (ECL1) of TM4SF1 has been identified as the interaction site with discoidin domain receptor tyrosine kinase 1 (DDR1) . Coimmunoprecipitation assays confirmed this interaction domain. Using ECL1 as an antigen, researchers have screened thousands of monoclonal antibodies and obtained functional antibodies that block the interaction between TM4SF1 and DDR1 . Among these, FC17-7 demonstrated the best blocking activity with dose-dependent binding capacity . Many antibodies also target the larger EL2 domain, as evidenced by the development of 13 antibodies that specifically reacted with this extracellular, lumen-facing domain .

How do I validate the specificity of a TM4SF1 antibody?

To validate TM4SF1 antibody specificity, follow these methodological steps:

• Cell line testing:

  • Test reactivity with cells known to highly express TM4SF1 (e.g., human umbilical vein endothelial cells - HUVEC)

  • Compare with TM4SF1-overexpressing cells (e.g., human dermal fibroblasts transduced to overexpress TM4SF1)

  • Use negative controls expressing extremely low levels of TM4SF1 (e.g., native human dermal fibroblasts with ~5 mRNA copies/cell)

• Immunohistochemistry validation:

  • Test antibody on multiple cancer tissues and adjacent normal tissues

  • Verify membrane-specific staining pattern

  • Use human lung tissue lysate as a positive control

• Specificity confirmation:

  • Use blocking peptides (e.g., PEP-1233 for PA5-21119)

  • Perform fluorescence-activated cell sorting (FACS) analysis to demonstrate dose-dependent binding

• Functional validation:

  • Verify antibody's ability to block TM4SF1-DDR1 interactions if targeting the ECL1 domain

  • Confirm internalization properties following antibody binding

How can TM4SF1 antibodies be used to study cancer stem cells?

TM4SF1 antibodies provide valuable tools for studying cancer stem cells through these approaches:

• Isolation and characterization:

  • Use antibodies to identify and sort TM4SF1high cell populations by flow cytometry

  • Compare TM4SF1high cells with established CSC markers (e.g., CD44high/CD24low in breast cancer)

  • Research has shown there are more CD44high/CD24low cells among TM4SF1high MDA-MB-231 human breast cancer cells than among TM4SF1low cells

• Functional analysis:

  • Assess sphere formation capacity of TM4SF1high vs. TM4SF1low cells

  • TM4SF1high cells from various cancer cell lines form more tumor spheres upon serial passage

  • Analyze expression of pluripotency factors (SOX2, NANOG, POU5F1) which are upregulated in TM4SF1high cells

• In vivo studies:

  • Compare tumor-initiating capacity of TM4SF1high vs. TM4SF1low cells

  • Assess tumor growth rates, latency periods, and metastatic potential

  • Perform serial transplantation assays to evaluate CSC self-renewal

  • Studies show TM4SF1high cells exhibit more rapid tumor growth, higher tumor-initiating cell frequency, and shorter latency periods in mice

How does TM4SF1 signaling contribute to cancer stem cell maintenance?

TM4SF1 sustains cancer stem cell traits through specific signaling mechanisms:

• TM4SF1-DDR1-JAK2-STAT3 signaling axis:

  • TM4SF1 couples with discoidin domain receptor tyrosine kinase 1 (DDR1)

  • Under collagen I stimulation, this interaction activates JAK2-STAT3 signaling

  • This noncanonical DDR1 signaling pathway induces expression of pluripotency factors SOX2 and NANOG

  • This pathway drives the manifestation of CSC traits and promotes multiorgan metastases

• Experimental evidence for functional significance:

  • Silencing TM4SF1 in TM4SF1high cells reduces sphere formation, tumor growth, tumor-initiating cell frequency, and metastasis

  • TM4SF1 depletion prolongs latency periods and survival times after orthotopic injection

  • Conversely, overexpressing TM4SF1 in TM4SF1low cells enhances stemness properties (sphere formation, tumor growth, tumor-initiating cell frequency, metastasis) and shortens latency periods and survival times

  • TM4SF1high cells maintain their expression level in primary tumors, indicating stable TM4SF1 expression in CSCs

• Long-term maintenance of stemness:

  • Secondary and tertiary transplantation assays demonstrate that TM4SF1high cells maintain their enhanced tumor-forming capacity over multiple generations

  • FACS analysis confirms stable TM4SF1 expression in secondary and tertiary tumors

What approaches can develop effective antibody-drug conjugates targeting TM4SF1?

Developing effective anti-TM4SF1 ADCs requires a methodical approach:

• Antibody selection and engineering:

  • Choose humanized monoclonal antibodies with high specificity and affinity for TM4SF1

  • Target antibodies to extracellular domains that induce internalization

  • Example: v1.10 humanized anti-human TM4SF1 monoclonal antibody

• Payload selection and conjugation:

  • Select potent cytotoxic agents appropriate for the target cells

  • Example: Auristatin cytotoxic agent LP2 (chemical name mc-3377)

  • Ensure proper drug-to-antibody ratio and conjugation chemistry

• Validation in preclinical models:

  • Test selective killing of TM4SF1-expressing cancer cell lines and endothelial cells in vitro

  • Evaluate efficacy in tumor xenograft models

  • Research shows v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts including non-small cell lung cancer, pancreas, prostate, and colon cancers

• Addressing species cross-reactivity limitations:

  • Develop species-specific antibodies when needed

  • Example: When v1.10 did not react with mouse TM4SF1, researchers generated a surrogate anti-mouse TM4SF1 antibody (2A7A) and conjugated it to LP2

  • Combined therapy with both human-targeting and mouse-targeting ADCs proved more effective than either ADC alone, demonstrating the importance of targeting both tumor cells and tumor vasculature

How do TM4SF1 antibodies impact tumor angiogenesis?

TM4SF1 antibodies affect tumor angiogenesis through several mechanisms:

• Expression pattern in tumor vasculature:

  • TM4SF1 is highly expressed on the vascular endothelium of several human cancers

  • A specific endothelial subpopulation (endothelial arterial type 2 [EA2]) is marked by Tm4sf1

  • This subpopulation has a distinct transcriptomic signature enriched for angiogenesis and CXCL12 signaling

  • Trajectory analysis suggests EA2 has a less differentiated state compared to other endothelial subpopulations

• Functional significance in angiogenesis:

  • TM4SF1 regulates endothelial cell functions critical for angiogenesis

  • Knockdown experiments demonstrated that TM4SF1 depletion prevented filopodia formation, inhibited cell mobility, blocked cytokinesis, and inhibited maturation of VEGF-A-induced angiogenesis

  • TM4SF1+CD31+ rat lung endothelial cells were visualized in distal pulmonary arteries and formed tubules in coculture with lung fibroblasts

• Therapeutic targeting approaches:

  • Anti-TM4SF1 antibody-drug conjugates can target both tumor cells and tumor vasculature

  • Combination therapy with antibodies targeting TM4SF1 on both tumor cells and tumor vasculature shows enhanced efficacy

  • This dual action represents a promising therapeutic approach with potential for higher efficacy than strategies targeting only tumor cells

How do I design experiments to study TM4SF1 in species where commercial antibodies show limited cross-reactivity?

When facing limited cross-reactivity of TM4SF1 antibodies across species, consider these methodological approaches:

• Custom antibody development:

  • Generate species-specific antibodies targeting conserved epitopes

  • Example: The development of 2A7A antibody for mouse TM4SF1 when human-targeting antibodies failed to cross-react

• Engineered model systems:

  • Utilize Matrigel models with human endothelial cells to study human TM4SF1 in mice

  • In this approach, endothelial colony-forming cells (ECFC) cultured on collagen-I-coated dishes are mixed with human mesenchymal stem cells (MSC) in a ratio of 2:3

  • The cell mixture is incorporated into Matrigel plugs implanted subcutaneously in nude mice to generate human vessels

• Combined targeting approaches:

  • For therapeutic studies, use both human-specific and mouse-specific antibodies

  • This combined approach targets both the human tumor cells and mouse tumor vasculature in xenograft models

  • Research shows combination therapy with v1.10-LP2 (human-targeting) and 2A7A-LP2 (mouse-targeting) was more effective than either ADC alone

• Alternative detection methods:

  • Use RNA-based approaches like qRT-PCR or RNA-seq

  • Analyze TM4SF1 expression patterns across different datasets

  • Multiple studies have shown TM4SF1 dysregulation in both human and animal disease models, though with some contradictions in expression patterns

What is the optimal protocol for immunohistochemical detection of TM4SF1 in clinical samples?

For optimal immunohistochemical detection of TM4SF1:

• Sample preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • Human lung tissue is recommended as a positive control

  • Include both tumor samples and adjacent normal tissue for comparison

• Antibody selection:

  • Choose antibodies validated for immunohistochemistry applications

  • Consider using antibodies targeting different epitopes of TM4SF1

  • For polyclonal antibodies like PA5-21119, use with corresponding blocking peptide (e.g., PEP-1233) to confirm specificity

• Staining protocol optimization:

  • Optimize antigen retrieval method (heat-induced epitope retrieval)

  • Determine optimal antibody concentration through titration

  • Include appropriate negative controls (isotype control, blocking peptide)

• Interpretation guidelines:

  • Assess membrane-specific staining pattern

  • Compare expression between tumor cells and tumor vasculature

  • TM4SF1 is typically highly expressed on cancer cell membranes but undetectably expressed on normal cells

  • Document both staining intensity and percentage of positive cells

How do I optimize flow cytometry protocols for TM4SF1 detection and cell sorting?

Optimizing flow cytometry for TM4SF1:

• Sample preparation:

  • Prepare single-cell suspensions from cell cultures or tissue samples

  • Minimize cell clumping and optimize viability

  • For clinical samples, consider gentle enzymatic digestion methods

• Antibody selection and staining:

  • Choose antibodies targeting extracellular domains of TM4SF1

  • Optimize antibody concentration through titration experiments

  • Include appropriate fluorochrome selection based on instrument configuration

  • Consider co-staining with other markers (e.g., CD44/CD24 for breast cancer CSCs)

• Gating strategy:

  • Exclude dead cells and doublets

  • Use fluorescence minus one (FMO) controls to set gates

  • Define TM4SF1high and TM4SF1low populations based on fluorescence intensity

  • Studies have successfully used this approach to isolate TM4SF1high populations with enhanced CSC properties

• Validation of sorted populations:

  • Verify TM4SF1 expression levels by qRT-PCR or western blot

  • Assess functional properties through sphere formation assays

  • Evaluate expression of stemness markers (SOX2, NANOG, POU5F1)

  • Test tumor-initiating capacity in animal models

What functional assays best demonstrate the effect of TM4SF1 antibodies on cancer stem cells?

The following functional assays effectively demonstrate antibody effects on TM4SF1-expressing CSCs:

• In vitro sphere formation assay:

  • Culture cells in low-attachment conditions with or without antibody treatment

  • Quantify sphere number and size after 7-14 days

  • Assess serial sphere-forming capacity over multiple passages

  • Studies show TM4SF1high cells form more tumor spheres upon serial passage, a property that can be targeted with antibodies

• Cell migration and invasion assays:

  • Use transwell migration/invasion chambers

  • Assess the impact of TM4SF1-blocking antibodies on cell mobility

  • TM4SF1 regulates cell motility, and knockdown experiments have shown inhibition of cell mobility

• Protein interaction studies:

  • Perform co-immunoprecipitation assays to evaluate antibody blocking of TM4SF1-DDR1 interaction

  • Measure downstream signaling (JAK2-STAT3 pathway activation) using phosphorylation-specific antibodies

  • Assess expression of stemness factors (SOX2, NANOG) following antibody treatment

• In vivo limiting dilution assays:

  • Inject decreasing numbers of antibody-treated cells into immunodeficient mice

  • Calculate tumor-initiating cell frequency

  • Monitor tumor growth rates and latency periods

  • Studies show TM4SF1high cells exhibit higher tumor-initiating cell frequency and shorter latency periods

• Cytotoxicity assays for antibody-drug conjugates:

  • Measure selective killing of TM4SF1-expressing cells by ADCs

  • Compare effects on TM4SF1high versus TM4SF1low populations

  • Research demonstrates v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells expressing TM4SF1

How do TM4SF1 expression patterns differ across various cancer types and what implications does this have for antibody therapy?

TM4SF1 expression across cancer types:

• Expression pattern analysis:

  • Immunohistochemistry staining of 16 types of cancers showed TM4SF1 is highly expressed on cancer cell membranes but undetectably expressed on normal cells

  • This widespread expression across multiple cancer types suggests broad therapeutic potential

• Cancer stem cell populations:

  • TM4SF1high populations have been identified in various human cancer cell lines:

    • Breast cancer (MDA-MB-231, MDA-MB-453)

    • Melanoma (A375, A2058)

    • Lung cancer (H460, H2030, H1975)

  • In all these cell lines, TM4SF1high cells exhibited enhanced stemness properties

• Therapeutic implications:

  • The broad expression across multiple cancer types suggests antibody therapies could have wide applications

  • Anti-TM4SF1 ADCs have shown efficacy against multiple cancer types in xenograft models:

    • Non-small cell lung cancer

    • Pancreatic cancer

    • Prostate cancer

    • Colon cancer

  • Cancer-specific optimization of antibody properties or conjugates may be required for maximum efficacy

• Dual-targeting potential:

  • TM4SF1 expression on both tumor cells and tumor vasculature enables dual-targeting strategies

  • This approach could be particularly effective for highly vascularized tumors

  • Combined targeting of both compartments has shown enhanced efficacy in preclinical models

What explains the apparently contradictory TM4SF1 expression patterns observed in different disease models?

Understanding contradictory TM4SF1 expression patterns:

• Cancer versus non-cancer contexts:

  • In cancer: TM4SF1 is consistently upregulated in cancer cells and tumor vasculature

  • In pulmonary arterial hypertension (PAH): More complex pattern observed

• Tissue-specific versus cell-specific expression in PAH:

  • Whole tissue level: TM4SF1 is upregulated in rat models of PAH (MCT and SuHx rats) by bulk RNA-seq

  • Human lung tissue: TM4SF1 is downregulated in human PAH lungs

  • Circulating cells: TM4SF1 is significantly upregulated in peripheral blood mononuclear cells of PAH patients

• Disease severity correlation:

  • Higher TM4SF1 expression in blood is associated with worse World Health Organization functional class in PAH patients

  • This suggests potential utility as a biomarker despite contradictory tissue expression

• Methodological considerations:

  • Different detection methods (bulk RNA-seq vs. scRNA-seq vs. protein detection)

  • Species differences (human vs. rat models)

  • Cell type heterogeneity within tissues

  • Disease stage and progression

These contradictions highlight the importance of context-specific analysis of TM4SF1 expression and function when developing targeted therapies.

How can TM4SF1 antibodies be combined with other therapeutic approaches for maximum efficacy?

Combination strategies with TM4SF1 antibodies:

• Multi-targeting antibody combinations:

  • Combine antibodies targeting different epitopes of TM4SF1

  • Use species-specific antibodies to target both tumor cells and tumor vasculature

  • Research demonstrates combination therapy with v1.10-LP2 (targeting human tumor cells) and 2A7A-LP2 (targeting mouse tumor vasculature) was more effective than either ADC alone

• Pathway-based combinations:

  • Combine TM4SF1 antibodies with inhibitors of downstream pathways

  • Target the JAK2-STAT3 pathway that is activated by TM4SF1-DDR1 interaction

  • Consider combinations with DDR1 inhibitors

• Cancer stem cell-directed therapies:

  • Combine with other CSC-targeting approaches

  • Target multiple CSC markers simultaneously (TM4SF1 plus CD44/CD24 or CD133)

  • Integrate with therapies targeting the tumor microenvironment that supports CSCs

• Conventional therapy enhancement:

  • Use TM4SF1 antibodies to sensitize tumors to chemotherapy or radiation

  • Target the CSC population that often survives conventional treatments

  • Exploit the dual-targeting of tumor cells and tumor vasculature to enhance drug delivery

• Immunotherapy combinations:

  • Explore combinations with immune checkpoint inhibitors

  • Investigate whether targeting tumor vasculature through TM4SF1 might enhance immune cell infiltration

  • Consider development of TM4SF1-targeted bispecific antibodies engaging immune effector cells

What quality control measures are essential when working with TM4SF1 antibodies?

Essential quality control measures for TM4SF1 antibodies:

• Antibody characterization:

  • Validate antibody specificity using positive and negative control cells

  • Confirm target epitope (ECL1, ECL2, or other domains)

  • Determine antibody affinity (e.g., 8G4 antibody has Kd ~1 nM for human TM4SF1)

  • Verify species cross-reactivity or lack thereof

• Application-specific validation:

  • For immunohistochemistry: Use human lung tissue as positive control

  • For flow cytometry: Perform titration to determine optimal concentration

  • For blocking experiments: Confirm ability to inhibit TM4SF1-DDR1 interaction

  • For antibody-drug conjugates: Verify selective killing of TM4SF1-expressing cells

• Reproducibility measures:

  • Test multiple antibody lots for consistency

  • Use blocking peptides (e.g., PEP-1233 for PA5-21119) to confirm specificity

  • Include appropriate controls in each experiment

• Functional validation:

  • Confirm predicted biological effects (e.g., inhibition of sphere formation)

  • Verify downstream signaling pathway modulation

  • Test for expected outcomes in vivo (tumor growth inhibition, metastasis reduction)

These quality control measures ensure reliable and reproducible results when working with TM4SF1 antibodies in research applications.