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Updated: Oct 15, 2025
Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma
Published on: September 8, 2021
PET Imaging of VLA-4 in a New BRAFV600E Mouse Model of Melanoma
Michael C Bellavia1, Lea Nyiranshuti2,3,4, Joseph D Latoche2
1Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
Purpose:
Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4β1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+).
Procedures:
BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors.
Results:
[64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses.
Conclusions:
[64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.
Insights
The novel PET tracer [64Cu]-LLP2A effectively targets very late antigen-4 (VLA-4) expressing BRAF-mutant melanoma. This imaging agent distinguishes VLA-4 positive from negative tumors, supporting its use in future theranostic applications for melanoma.
Area of Science:
- Oncology
- Radiochemistry
- Molecular Imaging
Background:
- Melanoma patients often progress despite targeted therapy and immune checkpoint inhibitors, necessitating new treatment strategies.
- The peptidomimetic LLP2A targets very late antigen-4 (VLA-4), a protein overexpressed in melanoma and implicated in tumor growth and metastasis.
- Existing murine melanoma models like B16F10 do not fully represent human melanoma biology, particularly lacking common BRAF mutations.
Purpose of the Study:
- To evaluate the Positron Emission Tomography (PET) tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in translational BRAFV600E mutant melanoma models.
- To assess the tracer's ability to differentiate melanoma variants based on VLA-4 expression levels.
Main Methods:
- Developed BRAFV600E mutant melanoma models, BPR (VLA-4 negative) and BPRα (VLA-4 positive) by transducing BPR cells with α4 (CD49d).
- Determined the binding affinity of [64Cu]-LLP2A to BPR and BPRα cells using saturation binding assays.
- Quantified tracer internalization and evaluated biodistribution and PET/CT imaging in mice bearing BPR and BPRα tumors.
Main Results:
- [64Cu]-LLP2A exhibited high binding affinity to VLA-4 positive BPRα cells (Kd = 1.4 nM) but not to VLA-4 negative BPR cells.
- VLA-4 positive BPRα and B16F10 cells showed comparable tracer internalization, while BPR cells showed undetectable internalization.
- PET/CT imaging revealed significantly higher [64Cu]-LLP2A uptake in BPRα tumors compared to BPR tumors in vivo.
Conclusions:
- [64Cu]-LLP2A successfully differentiates VLA-4 positive (BPRα) from VLA-4 negative (BPR) BRAF-mutated melanomas.
- These findings support the translational utility of these models for prospective imaging and theranostic studies.
- The study extends the application of LLP2A to target clinically relevant, therapy-resistant melanoma variants for potential therapeutic use.
