PET Imaging of VLA-4 in a New BRAF^V600E Mouse Model of Melanoma

Abstract

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 BRAF^V600E 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 (K_d = 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 towards its use for therapeutic patient care.

Introduction

Over the last decade, immune checkpoint blockade has drastically altered the prognosis and medical management of patients with metastatic melanoma. Response rates to dual checkpoint blockade are as high as 50-70% in treatment-naïve melanoma patients, with approximately 25% of patients achieving durable complete responses [1–3]. In addition to checkpoint blockade, small molecule therapies targeting activating mutations in the BRAF kinase (i.e. BRAF^V600E, identified in approximately 50% of cases) have been commonly applied [4]. Combining BRAF inhibitors (BRAFi) (vemurafenib, encorafenib) with inhibitors to MEK (binimetinib, trametinib), another kinase of the MAPK signaling cascade, substantially improves response rates in treatment-naïve patients [5–7]. Despite these advances in immunotherapies and targeted therapies for the treatment of metastatic melanoma, tumor escape, drug resistance, and treatment-associated toxicities remain problematic for many patients [8, 9]. Notably, patients with BRAFi-resistant melanomas display low response rates to immune checkpoint blockade [10, 11] and there remains a major unmet clinical need to improve outcomes in this patient population.

Very late antigen-4 (VLA-4, integrin α₄β₁) is a surface adhesion protein that facilitates the growth and metastasis of melanomas and other malignancies by enhancing tumor angiogenesis and the extravasation of circulating tumor cells into pre-metastatic niches [12, 13]. In melanoma patients, upregulated tumor expression of VLA-4 is a prognostic marker for metastasis [14, 15]. As VLA-4 is also involved in lymphocyte differentiation, trafficking, and homing (via binding to VCAM-1 expressed on the vascular endothelium [16]) in addition to its immunomodulatory properties [17, 18], VLA-4 is a particularly attractive target for imaging and therapeutic intervention.

We previously demonstrated that progressively-growing murine BRAF^wt VLA-4⁺ B16F10 melanomas readily take up the VLA-4 specific peptidomimetic LLP2A (IC₅₀: 2 pM [19]) labeled with radioisotopes Ga-68 and Cu-64 for positron emission tomography imaging (PET) and that LLP2A labeled with the β⁻-emitter Lu-177 mediates anti-tumor efficacy as a therapeutic agent in this model [20–22]. However, B16F10 does not exhibit common oncogenic mutations/deletions associated with the development of most human melanoma [23]. For example, these cells express wild-type PTEN, whose loss from chromosome 10 is implicated in human melanomagenesis [24]. Hence, to test LLP2A-based therapeutic agents in translational models that more faithfully recapitulate human disease, we have now developed BRAF^V600EPTEN^−/−, BRAFi-resistant melanoma models with or without substantial expression of the target antigen VLA-4 (i.e. BPR and BPRα). These BRAFi-resistant melanoma models are expected to support translational research advances and the development of novel imaging and targeted radiotherapeutic approaches for more effective treatment and monitoring of patients with advanced-stage disease.

Materials and Methods

Cell Lines

BPR cells were selected from the BRAF^V600E PTEN^−/− BP melanoma line [25] kindly provided by Dr. Jennifer Wargo (University of Texas M.D. Anderson Cancer Center, Houston, TX), that acquired stable resistance to the BRAF inhibitor (BRAFi) dabrafenib (20 μM) after 1 month in vitro culture [26]. As flow cytometry analyses indicated that BPR cells were deficient in VLA-4 expression (BPR cells only express CD29, the b₁ subunit), they were transduced to express high levels of the α₄ subunit (CD49d) via infection with a recombinant lentiviral vector (Itga4 (NM_010576), CAT#: MR211489L1V, OriGene Technologies, Rockville, MD), generating the BPRa cell line. The cell lines were authenticated by IDEXX BioAnalytics (Columbia, MO) and verified as Mycoplasma-free by in-house PCR. Cells were maintained in DMEM culture medium supplemented with 10% FBS with (BPR, BPRα) or without (B16F10) additional 1% L-glutamine. BPRα cells were selected for purity by flow sorting based on expression of CD49d using a BD FACSAria IIu (BD Biosciences, San Jose, CA).

Peptide Synthesis and Radiolabeling

Cy3-LLP2A synthesis and CB-TE1A1P-PEG₄-LLP2A radiolabeling with Cu-64 ([⁶⁴Cu]-LLP2A) were conducted as reported previously [27]. Cy3-LLP2A was synthesized by the Molecular Interactions Core at the University of Missouri.

Flow Cytometry Validation of Cy3-LLP2A

Cells in culture were harvested with trypsin and large clumps were excluded via 70 μm cell strainers (Fisher Scientific, Waltham, MA). BPR and BPRα cells (1 x 10⁵) in 0.1 mL ice-cold flow buffer (1x PBS, 0.1% BSA) received either 1 μg Cy3-LLP2A, 0.25 μg anti-CD49d-PE, or isotype control (both BioLegend, San Diego, CA) in 50 μL staining buffer (1x PBS, 1% BSA, 1 mM Mn²⁺). Cells were incubated on ice for 30 min and washed in flow buffer twice prior to analysis.

Cell Binding Assays

To discern differences in the binding affinity of [⁶⁴Cu]-LLP2A to BPR and BPRα cells, cell binding assays were performed as previously described [20], with modifications. Briefly, cells were seeded in 12-well plates (1 x 10⁵ per well) for 24h after the plates were coated with 0.1% gelatin to allow cell adherence to the plate. Multiple wells coated with 0.1% gelatin without cells were included as background for protein quantification. Subsequently, the cells were washed twice with 1 mL HBSS, followed by addition of 0.5 mL of binding medium (HBSS, 0.1% BSA , 1 mM Mn²⁺) per well. For both cell lines, 15 μg LLP2A-PEG₄ was used as a cold block to estimate in vitro nonspecific binding, and [⁶⁴Cu]-LLP2A was added in increasing concentrations (1 nM - 100 nM). The plates were incubated on ice for 2h and the radioactive medium was removed. Cell pellets were dissolved in 0.5% SDS solution after rinsing twice with 1 mL of ice-cold binding buffer. Bound radioactivity following incubation was measured by γ-counting (PerkinElmer 2470 WIZARD², Waltham, MA). The protein content of the cell lysates was determined by BCA assays (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, Waltham, MA) and cell protein content was calculated by subtracting the protein content of 0.1% gelatin in the absence of the cells from the protein content in the presence of the cells and 0.1% gelatin. Lysate radioactivity was normalized to the amount of cellular protein present (fmol/mg). Experiments were conducted in triplicate in different 12-well plates. The dissociation constant K_d and total receptor number B_max were determined with GraphPad Prism 9 software (San Diego, CA).

[⁶⁴Cu]-LLP2A Internalization Assays

Internalization assays were performed to compare the uptake of [⁶⁴Cu]-LLP2A in VLA-4⁻ BPR and VLA-4⁺ BPRα and B16F10 cells. For each cell line, 1 x 10⁵ cells were seeded per well in 12-well plates and incubated 24h prior to the experiment. Cells were first washed twice with 1 mL DMEM and then received 1 mL assay media (DMEM, 1% BSA, 1 mM Mn²⁺). To quantify non-specific internalization, triplicate wells per cell line were blocked with excess (10 μg) LLP2A-PEG₄ and incubated for 15 min at 37°C. Cells received 0.44 MBq (12 μCi; 3.43 MBq/nmol) [⁶⁴Cu]-LLP2A per well prior to incubation at 37°C. At each time point (15 min, 2h, 4h), the assay media was removed, and cells were washed twice with DMEM. The surface-bound fraction was removed by the addition of DMEM containing 20 mM sodium acetate (pH 4.0) followed by incubation of the plates for 10 min at 37°C. Total cellular protein was harvested for analysis by dissolution in 1 mL 0.5% SDS. Sample radioactivity per cell pellet was determined by γ counting (PerkinElmer 2470 WIZARD²). Internalized activity was corrected for background, activity in the corresponding blocked sample, and time-dependent decay. The corrected activity was then normalized to total cellular protein collected (fmol/mg protein) after quantification of the cellular lysate protein via BCA assay.

Animal Studies

Four- to six-week-old male and female C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All applicable institutional and/or national guidelines for the care and use of animals were followed. For PET/CT imaging and biodistribution experiments, mice were inoculated subcutaneously (s.c.) in the right flank with 5 x 10⁵ BPR or BPRα cells mixed with an equal volume of Matrigel (Corning Life Sciences, Tewksbury, MA).

PET/CT Imaging and Analysis

C57BL/6 mice bearing s.c. BPR or BPRα tumors (n=9 each, 3m/6f) were injected intravenously (lateral tail vein) with [⁶⁴Cu]-LLP2A (7.4 MBq [200 μCi]; 57.2 MBq/nmol). At 4 and 24h post-injection, mice were anesthetized with 2% isoflurane, and small animal PET/CT images were acquired as static images collected over 10 min with an Inveon PET/CT scanner (Siemens Medical Solutions, Knoxville, TN). PET and CT images were co-registered and PET images were reconstructed with an Ordered Subsets Expectation Maximizing 3D algorithm (OSEM-3D) with Inveon Research Workstation (IRW) software (Siemens Medical Solutions, Knoxville, TN). Regions of interest (ROI) were drawn from CT images, and the corresponding PET activities were calculated using IRW software. Standard uptake values (SUVs) at time t were calculated via the formula: SUV(t) = image-derived activity concentration (Bq/mL) x [animal weight (g)/injected dose (Bq)].

Biodistribution Experiments

Immediately following the 24h PET/CT imaging time point, BPR and BPRα tumor-bearing mice (n=6; 3m/3f) were euthanized. To observe the time course of the tracer biodistribution in BPRα, BPRα tumor-bearing mice were euthanized at 4h (n=3) and 48h (n=4) post-tracer injection at an equivalent dose in separate experiments. Blood, muscle (gastrocnemius of uninvolved leg), bone, heart, lungs, spleen, kidney, liver, intestines, thymus, and tumor were harvested, weighed, and the activity counted with a γ counter (PerkinElmer 2470 WIZARD²). The percent injected activity per gram of tissue (%IA/g) was calculated by decay correction of each sample normalized to a standard of known weight containing activity corresponding to the injected dose.

Statistics

Statistical analyses were performed using GraphPad Prism 9 software (San Diego, CA). Groups were compared using two-tailed student t-test or one-way ANOVA with Tukey’s HSD for multiple comparisons. A p-value <0.05 was considered statistically significant. Outliers as determined by two-sided Grubbs’ test (α = 0.05) [28] were removed from analysis.

Results

Generation of BPRα and Flow Cytometry Validation of Cy3-LLP2A

BPR cells only express the β₁ subunit (CD29) of VLA-4, so to simulate a more aggressive variant of these BRAFi-resistant melanomas, the cells were transduced with the α₄ subunit (CD49d) using recombinant lentiviral particles to express high levels of VLA-4 (BPRα, Fig. 1a). To verify α₄ induction and to isolate transduced clones, flow sorting was conducted (Suppl. Fig. 1, see Electronic Supplementary Material). Approximately 70% of the resulting cells stained positively for both CD49d (α₄) and CD29 (β₁) and were collected for further propagation as the stable cell line designated BPRα. LLP2A selectivity to the parental and transfected lines was assessed using Cy3-LLP2A (Suppl. Fig. 2a) as a flow cytometry probe on BPRα vs. BPR cells. Although some staining of the probe was observed for VLA-4⁻ BPR cells, the median fluorescence intensity of staining on BPRα cells was significantly greater (p<0.0001, Fig. 1b).

Fig. 1.

Cy3-LLP2A staining of BPR and BPRα cells by flow cytometry. (a) BPR cells natively express only the β₁ subunit (CD29) of VLA-4; however, both α₄ and β₁ subunits are required for LLP2A binding. Therefore, BPR cells were transduced with the α₄ subunit of VLA-4 (CD49d) to generate BPRα (VLA-4/integrin α₄β₁⁺). (b) Flow cytometry indicates that LLP2A requires intact VLA-4 for appreciable binding. Median fluorescence intensity of Cy3-LLP2A-stained and unstained BPR and BPRα. At least 10,000 events were analyzed per replicate, and each point represents an individual sample. ****: p < 0.0001, by unpaired t-test.

Radiolabeling Chemistry

The radiochemical purity of [⁶⁴Cu]-LLP2A was >99% (Suppl. Figs. 2b, 3) and a molar activity of 57.2 MBq/nmol (1 mCi/μg LLP2A) was utilized for all applicable studies. We have previously demonstrated [⁶⁴Cu]-LLP2A stability in human serum, with negligible binding of the conjugate to plasma proteins [20].

[⁶⁴Cu]-LLP2A Cell Binding Assays in BPR and BPRα Cells

To interrogate differences in LLP2A binding affinity to BPR and BPRα cells, saturation binding assays were conducted. [⁶⁴Cu]-LLP2A was competed with PEG₄-LLP2A at 4°C. Representative saturation binding curves of [⁶⁴Cu]-LLP2A to BPR and BPRα cells (Fig. 2) indicate significant binding affinity to the VLA-4⁺ BPRα cells (K_d: 1.4 ± 0.45 nM), whereas in VLA-4⁻ BPR cells, no saturation occurs, as total binding cannot be differentiated from non-specific (blocked) binding. The BPRα B_max was 145 ± 8.8 fmol/mg, while the BPR B_max was not interpretable (although metrics can be calculated, the model fit is poor; R²=0.0002). These observations suggest that α₄ is not present in BPR and that LLP2A binding is specific only to BPRα cells given their expression of intact VLA-4.

Fig. 2.

Saturation binding data of [⁶⁴Cu]--LLP2A in (a,b) BPRα and (c,d) BPR. (a) Overall and (b) specific binding isotherms of BPRα. (c) Saturation is not reached for BPR, as total (non-specific + specific) and non-specific binding alone are essentially indistinguishable. (d) A specific binding isotherm cannot be properly fitted for BPR. In both cell lines, non-specific binding refers to the off-target binding that occurs despite prior blocking of receptors with excess unlabeled LLP2A (PEG₄-LLP2A). Radioligand concentrations were evaluated in triplicate.

Internalization of [⁶⁴Cu]-LLP2A in BPR and BPRα Cells

We next sought to compare the extent of [⁶⁴Cu]-LLP2A internalization in BPR and BPRα cells relative to VLA-4⁺ B16F10 melanoma cells, as LLP2A internalization in B16F10 has previously been demonstrated [20]. The presence of cell surface VLA-4 is likely required for appreciable tracer uptake as internalization significantly increased (p<0.05 - p<0.0001) relative to baseline only for BPRα and B16F10 (Fig. 3a). Furthermore, blocking VLA-4 with excess unlabeled LLP2A prior to introducing [⁶⁴Cu]-LLP2A prevented binding of the radiolabeled agent (Figs. 3b, 3c). The tracer was readily internalized by BPRα and B16F10 within 15 min (10 ± 1.3 fmol/mg and 9.2 ± 1.4 fmol/mg, respectively), increasing to a peak value at 2h of incubation for both cell lines. Beyond this point, uptake plateaued in both cell lines (no statistical difference in B16F10 2h vs. 4h). Greater tracer accumulation occurs in BPRα, which became statistically significant relative to B16F10 at 4h (p=0.008). Only internalized tracer was considered, as a control condition without the stripping of surface-bound material (‘no strip’, wells received PBS) displayed greatly increased activity for BPRα and B16F10 but not BPR (Suppl. Fig. 4).

Fig. 3.

VLA-4 expression is required for [⁶⁴Cu]-LLP2A internalization. (a) Time-dependent specific uptake (unblocked minus blocked internalization) of [⁶⁴Cu]-LLP2A in B16F10, BPR, and BPRα cells. BPRα vs. BPR: *: p < 0.05, **: p < 0.01, ***: p < .001. B16F10 vs. BPR: I: p < 0.05, I I: p < 0.01. Total binding and blocking of [⁶⁴Cu]-LLP2A in (b) BPRα, (c) B16F10, and (d) BPR cells. n=3 per time point. φ: p < 0.05, φφ: p < 0.01, φφφ: p < 0.001, by unpaired t-test.

Biodistribution of [⁶⁴Cu]-LLP2A in BPR and BPRα Tumor Models

The biodistribution of [⁶⁴Cu]-LLP2A was determined in BPR and BPRα tumor-bearing mice at 24h post-injection of the tracer (Fig. 4a, Suppl. Table 1). There was notable uptake of the tracer in each tumor type, with significantly increased uptake in BPRα vs. BPR tumors (BPR: 1.7 ± 0.2% IA/g and BPRα: 2.8 ± 0.2% IA/g, p<0.0001). As we have previously observed over both shorter (up to 4h) [20] and longer (up to 96h) [21] timescales in B16F10 melanoma, LLP2A accumulates in organs enriched in VLA-4 given its role in lymphocyte maturation, leukocyte homeostasis, and/or hematopoiesis - namely the thymus (BPR: 3.5 ± 0.9% IA/g and BPRα: 4.2 ± 4.0% IA/g), spleen (BPR: 8.1 ± 4.4% IA/g and BPRα: 6.0 ± 1.9% IA/g), and bone marrow (BPR: 2.2 ± 0.4% IA/g and BPRα: 1.6 ± 0.5% IA/g). Apart from the tumor, there was no significant difference in LLP2A uptake per organ examined in the BPR and BPRα models.

Fig. 4.

BPRα displays improved [⁶⁴Cu]-LLP2A pharmacokinetics and tumor uptake relative to BPR. (a) Biodistribution of [⁶⁴Cu]-LLP2A in mice bearing BPR and BPRα tumors at 24h post-injection (n=6). Inset: comparison of tumor uptake only. Data are presented as % injected activity per gram of tissue. (b) [⁶⁴Cu]-LLP2A PET/CT images at 4h (left) and 24h (right), (c) Tumor mean standard uptake values (SUV), and tumor-to-muscle SUV of BPR and BPRα tumor-bearing mice at 4h and 24h post-injection (n=9). **p<0.01, ***p<0.001, ****p<0.0001 by unpaired t-test.

PET/CT Imaging of BPR and BPRα Tumor Models

At 4h post-injection of [⁶⁴Cu]-LLP2A, there was a significant increase (p=0.0021) in tumor uptake in BPRα relative to BPR (Figs. 4b, 4c), which likely reflects the addition of VLA-4⁺ tumor cells to the VLA-4⁺ immune cell component infiltrating the baseline tumor microenvironment. In both models, we observed marked uptake in the thymus, spleen, and bone marrow, in accordance with the biodistribution results. Notable activity was also seen in the bladder, indicating rapid renal clearance of the tracer. These observations parallel our previous studies using LLP2A in B16F10 melanoma [20, 21]. Unlike BPR tumors, BPRα tumor uptake remained detectable at 24h. The tumor-to-muscle ratio of BPRα was significantly greater than BPR at 4h (p=0.0005) but not at 24h (p=0.2715).

Discussion

Although immune checkpoint inhibitors and targeted therapy (BRAFi, MEKi) are potent additions to the therapeutic arsenal against metastatic melanoma, not all patients benefit, and the development of treatment resistance is common. As such, new therapeutic targets must be explored. Expression of VLA-4 (integrin α₄β₁), an adhesion molecule that facilitates circulating tumor cell extravasation among other tumor-potentiating functions, is correlated to the development of metastatic disease in patients. Further, upregulated VLA-4 expression in melanoma often portends a more aggressive, metastatic phenotype [14]. As early-stage melanoma is generally well-managed by surgical resection [29], the capability to selectively image and stage disease progression via biomarkers such as VLA-4 may improve treatment outcomes. We have previously demonstrated that [⁶⁴Cu]-TE1A1P-PEG₄-LLP2A ([⁶⁴Cu]-LLP2A) is readily taken up in VLA-4⁺ BRAF^wt B16F10 melanomas in vivo, providing good image contrast [20]. However, to our knowledge LLP2A has not been evaluated in a melanoma model that recapitulates aspects of aggressive patient tumors - namely high VLA-4 expression [14, 15] and the presence of common underlying oncogenic driver mutations that are characteristic of human disease.

Here, we generated and investigated a novel matched pair of drug-resistant BRAF-mutant, PTEN-null cell lines (VLA-4⁻ and VLA-4⁺) for their differential targeting by LLP2A towards establishing their wider use in translational melanoma research. BRAF^V600E BP cells with acquired resistance to dabrafenib (BPR) were successfully transduced with the α₄ subunit of VLA-4 (BPRα). This was verified directly by specific antibody staining (Suppl. Fig. 1) and was also suggested by the substantially increased binding of the Cy3-LLP2A probe to BPRα vs. BPR cells (Fig. 1b). Despite the purported selectivity of LLP2A to intact VLA-4 [19] (BPR cells only express the β₁ subunit paired to other integrins), there remains a detectable degree of staining to BPR cells. This is likely nonspecific, as a relatively high degree of interaction between Cy3 dye and lipid bilayers has been reported [30]. Although LLP2A fails to bind to other heterodimeric integrins containing the β₁ subunit (ex. α₂β₁) or to α₄β₁ with anti-α₄ blocking [19], to our knowledge LLP2A binding to β₁ alone has never been observed.

Saturation binding and internalization assays were performed with [⁶⁴Cu]-LLP2A for both the BPR and BPRα cell lines. As expected, LLP2A displays high binding affinity to BPRα cells (K_d: 1.4 ± 0.45 nM), but its binding to BPR cells is not distinguishable from control (blocked) conditions. Similarly, the number of receptor sites bound by LLP2A was indeterminate in BPR cells - values can be calculated via the least squares method for one-site binding, but the model is very poorly fitted. The binding affinity of [⁶⁴Cu]-LLP2A in BPRα is comparable (1.4 ± 0.45 vs 0.28 ± 0.03 nM) and the B_max roughly half (145 ± 8.8 fmol/mg vs 300 ± 4.5 fmol/mg) that of what we have previously reported for B16F10 melanoma cells (Suppl. Fig. 5) [20]. These results are somewhat surprising given the significantly greater tracer internalization in BPRα vs. B16F10 after 4h (Fig. 3). We hypothesize that these observations may be due to reduced tracer efflux from BPRα. BPRα displays a similar uptake pattern to B16F10, with internalization reaching a peak at 2h, followed by a plateau. Total and specific uptake in BPR are indistinguishable. Blocking of BPRα and B16F10 with excess LLP2A-PEG₄ renders internalization negligible, mirroring total BPR uptake. Taken together, these data indicate that internalization requires VLA-4 and that the low level of LLP2A binding to BPR seen in flow cytometry does not lead to significant uptake.

The biodistribution results show a strong increase in tumor LLP2A accumulation in BPRα vs. BPR tumors in vivo (p<0.0001), with notably lower uptake in BPRα for organs enriched in VLA-4 (spleen, thymus, bone marrow) at 24h, although none of these comparisons reached statistical significance. Similar observations emerged in a separate study interrogating tracer biodistribution in BPRα tumor mice conducted at 4, 24, and 48h post-injection, with time-dependent clearance (Suppl. Fig. 5, Suppl. Table 2). Notably, kidney activity appears to increase over time, though the extent of this is inflated by an extreme value at 48h. This observation underscores the importance of individual patient dosimetry for the clinical use of LLP2A in radionuclide therapy.

PET/CT demonstrates greater [⁶⁴Cu]-LLP2A uptake and retention in BPRα tumors relative to BPR tumors, despite considerable clearance by 24h. Image quantification analysis corroborates the PET/CT observations, with higher tumor SUV and tumor-to-muscle SUV ratios in BPRα versus BPR at both time points. These differences are statistically significant with the exception of the tumor-to-muscle SUV at 24h, when most of the tracer has cleared. Compared to the dynamics we previously determined for B16F10, BPRα demonstrates reduced tracer uptake by tumor: muscle SUV ratio at 4h, with uptake increasing to be nearly equivalent by 24h (Suppl. Table 3).

B16F10 has been a conventional model in cancer research since the 1970s due to its aggressiveness and tendency to spontaneously metastasize to the lungs [31]. However, B16F10 does not express the common oncogenic driver mutations/genetic deletions found in human melanomas. BP, the parent cell line to BPR and BPRα, was isolated from an immunocompetent BRAF^V600E PTEN^−/− genetically-engineered mouse model after induction with tamoxifen [25]. These lines, which more accurately simulate developing melanomas in humans [32], can be transplanted onto MHC-matched immunocompetent mice, allowing for the testing of interventional treatment protocols for future translation into the clinic.

2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) PET/CT is widely utilized clinically for metastatic melanoma detection, staging, and evaluation of response to treatment [33, 34]. However, ¹⁸F-FDG PET/CT is often unable to detect subclinical lesions less than 1 cm in diameter [35] that appear in preferential metastatic sites (brain, lungs, liver) due to non-disease background [36, 37]. We have previously demonstrated that [⁶⁴Cu]-LLP2A can effectively delineate B16F10 lung metastases with high contrast [20]. Further, as ¹⁸F-FDG signal reflects glucose metabolism, which is elevated in inflamed tissue and increases with immunotherapy, ¹⁸F-FDG may not resolve tumor from regions of immune infiltration [38]. Numerous small molecules (ex. benzamide derivatives [39–41]), antibodies (ex. anti-GD2 [42, 43]), and peptides that circumvent these issues have been generated to image metastatic melanoma, and these typically bind overexpressed surface antigens (ex. MC1R) or melanin [44]. Intriguingly, VLA-4 has been shown to prompt extravasation of circulating tumor cells only when in the activated state [45]. As such, LLP2A may be especially well-suited to indicate the aggressiveness of melanomas, which is not possible with currently employed tracers.

A limitation of the study is that transduction of the α₄ subunit to increase VLA-4 may not recapitulate the VLA-4 cell surface copy number/density observed in clinical melanoma specimens. In this regard, a genetic model of drug-resistant BRAF^V600E VLA-4⁺ melanoma would be ideal; however, these are not available for use. BPRα graft models can serve as faithful approximations of human disease by their genetic and cell surface protein expression profile. Although we would predict that BPRα cells would have greater metastatic potential vs. BPR cells in vivo, this was not formally tested in the current report, but is planned in future studies.

Conclusions

BPRα, a VLA-4^hi variant of a spontaneous BRAF^V600E murine melanoma cell line, shows avid binding and uptake of the VLA-4-targeted PET tracer [⁶⁴Cu]-CB-TE1A1P-PEG₄-LLP2A ([⁶⁴Cu]-LLP2A), providing high image contrast. This was not observed for its VLA-4⁻ parental cell line BPR. These findings support the use of these novel BRAFi-resistant cell lines in translational studies related to on-treatment tumor imaging and VLA-4 targeted therapies.