Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2024 Oct 29;21(12):6311–6322. doi: 10.1021/acs.molpharmaceut.4c00777

[64Cu]Cu-NOTA-Trastuzumab and [89Zr]Zr-DFO-Trastuzumab in Xenografts with Varied HER2 Expression

Cristina Simó 1, Shayla Shmuel 1, Alex Vanover 1, Patrícia M R Pereira 1,*
PMCID: PMC11611601  NIHMSID: NIHMS2035922  PMID: 39471823

Abstract

graphic file with name mp4c00777_0007.jpg

Positron emission tomography (PET) has potential as a complementary technique to biomarker analysis, especially for human epidermal growth factor receptor 2 (HER2)-expressing tumors characterized by high heterogeneity. In this study, zirconium-89 (89Zr) and copper-64 (64Cu) labeled trastuzumab were employed to monitor varying levels of tumoral HER2 expression. Additionally, we studied the use of the cholesterol-depleting lovastatin as a pharmacological approach to enhance cell-surface HER2 expression in tumors with moderate to low HER2 levels, aiming to increase antibody accumulation in these tumor types. Both 89Zr- and 64Cu-labeled trastuzumab effectively monitor HER2 expression levels in xenografts exhibiting varying HER2 expression. No significant difference in tumor uptake was observed between 89Zr- or 64Cu-labeled trastuzumab, and tumor uptake for both radioimmunoconjugates positively correlated with HER2 protein levels. These findings underscore the potential of PET to monitor HER2 protein levels across heterogeneous tumors. Furthermore, our results suggest that further optimization of statin dosing and timing could offer a promising strategy to enhance trastuzumab accumulation in HER2-high, HER2-moderate, and HER2-low tumors.

Keywords: Immuno-PET, HER2, 89Zr-labeled trastuzumab, 64Cu-labeled trastuzumab, statin

Introduction

Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor of the epidermal growth factor (EGF) family, which is overexpressed and dysregulated in several tumor types.1 Overexpression of HER2 contributes to tumor progression and poor survival.1,2 In recent years, efforts have been focused on the development of HER2-targeted agents,3 including monoclonal therapeutic antibodies and antibody-drug conjugates.4 For the selection of patients for HER2-targeted therapy, tumors undergo evaluation for HER2 expression through biopsy. However, HER2 expression between primary tumors and metastatic lesions may present discordance,5 along with possible variation of HER2 expression during the progression of the disease6 or treatments,7 requiring continuous HER2 assessments. Positron emission tomography (PET), a noninvasive nuclear imaging technique, has potential as a complementary approach to assess tumor HER2 expression over time.

Trastuzumab is a humanized monoclonal antibody that targets the extracellular domain IV of HER2. PET-labeled trastuzumab has been used to diagnose and monitor tumor response to HER2-targeted therapies.8−13 Particularly, the positron-emitter zirconium-89 (89Zr) has been extensively used for radiolabeling trastuzumab because of its favorable characteristics for immuno-PET.10,11,13,14 However, the long half-life of 89Zr-labeled trastuzumab could result in significant radiation exposure in patients.15 As an alternative, copper-64 (64Cu), with a half-life of 12.7 h, could potentially achieve similar tumor-tissue ratios. In this context, 64Cu-labeled trastuzumab has been applied in different clinical studies to detect primary HER2-positive breast cancer and metastatic lesions.12,15,16

The efficacy of trastuzumab in binding to cancer cells relies on the density and availability of HER2 on the cancer cell membrane.17,18 Previous studies have indicated that drugs disrupting the stability and membrane localization of HER2 may impact the tumor’s response to anti-HER2 therapy.19−21 Cholesterol-depleting drugs — statins —temporally elevate cell-surface HER2 and enhance antibody uptake,17,18,22−24 suggesting a potential combined approach to improve therapeutic outcomes and patient prognosis. However, existing studies have mainly focused on enhancing trastuzumab uptake in HER2-high tumors, leading to uncertainty about the applicability of this pharmacological approach for enhancing cell-surface HER2 in HER2-moderate and HER2-low tumors.

This study aims to explore two objectives. First, we used 89Zr-labeled trastuzumab and 64Cu-labeled trastuzumab for imaging HER2 expression in preclinical models of varying HER2 protein levels. Second, we used immuno-PET imaging to track variations in HER2 expression following intratumoral administration of statin in HER2-high, HER2-moderate, and HER2-low tumors.

Materials and Methods

Cell Culture and Treatments

The human gastric NCIN87, pancreatic MIAPaCa-2, and bladder UMUC3 cancer cell lines were purchased from the American Type Culture Collection. NCIN87 was cultured in RPMI-1640 growth medium supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 100 units/mL penicillin, and 100 μg/mL streptomycin. MIAPaCa-2 was cultured in Dulbecco’s Modified Eagle’s Medium with 4.5 g/L glucose, 4 mM l-glutamine, 1 mM sodium pyruvate, and 3.7 g/L sodium bicarbonate, supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. UMUC3 was cultured in Eagle’s Minimum Essential medium supplemented with 10% (v/v) FBS, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37 °C in an atmosphere with 5% CO2.

Western Blot Analyses

Total protein extracts from NCIN87, MIAPaCa-2 and UMUC3 cancer cells were prepared after cell scrapping in radioimmunoprecipitation assay buffer (RIPA) containing 150 mM sodium chloride (NaCl2), 50 mM Tris hydrochloride (Tris-HCl) pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% (v/v) Tritron X-100, 0.5% (w/v) sodium deoxycholate (DOC), and 0.1% (w/v) sodium dodecyl sulfate (SDS), 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD), and 1 × protease inhibitor cocktail (Roche), and incubating 30 min at 4 °C with vortex every 5 min. After centrifugation of cell lysates at 18,000g for 16 min at 4 °C, supernatants were collected and the amount of total protein was quantified using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific), followed by denaturation of the samples with Laemmli buffer (Thermo Fisher Scientific). The denatured samples underwent electrophoresis and transfer to polyvinylidene difluoride membranes (Bio-Rad). The membranes were incubated in 5% (w/v) milk (Bio-Rad) in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST, EZ BioResearch), followed by incubation with primary antibodies: mouse anti-β-actin (1:10,000, A1978, Sigma), rabbit anti-HER2 (1:800, ab131490, Abcam), and rabbit anti-CAV-1 (1:500, ab2910, Abcam). After incubation with primary antibodies, membranes were washed three times with TBST buffer and incubated with secondary antibodies: goat anti-mouse IgG (heavy- and light-chain) conjugated with Alexa Fluor Plus 800 (1:10,000, Invitrogen) or goat anti-rabbit IgG (heavy- and light-chain) conjugated with Alexa Fluor Plus 680 (1:10,000, Invitrogen) for 1 h at room temperature. Membranes were imaged on an Odyssey infrared imaging system (LI-COR Biosciences), followed by densiometric analyses performed by ImageJ/FIJI (NIH) software.

Biotinylation of Cell-Surface Proteins

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) containing 0.5 mM magnesium chloride (MgCl2) and 1 mM calcium chloride (CaCl2). Cells were incubated with EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) for 30 min at 4 °C with gentle agitation and protected from light. The reaction was stopped by washing the cells twice with PBS containing 0.5 mM MgCl2, 1 mM CaCl2, and 100 mM glycine. Cell scrapping was performed with RIPA buffer and centrifuged at 18000g for 16 min at 4 °C. Supernatants were collected and quantified using the Pierce BCA Protein Assay Kit. A volume of 150 μL of RIPA buffer containing an equal amount of protein was incubated with 20 μL NeutrAvidin Agarose Resins (Thermo Fisher Scientific) overnight at 4 °C with gentle rotation. After incubation, three washes were performed with RIPA buffer (3000g, 2 min, 4 °C) before suspension in Laemmli buffer. Western blot analyses were performed as described above.

Immunofluorescence

NCIN87, MIAPaCa-2, and UMUC3 cells (1.67 million cells/coverslip) were grown on coverslips (1.5 mm, Thermo Fisher Scientific) pretreated with poly-l-lysine (Sigma-Aldrich) for 24 h. Cells were then treated with the active form of lovastatin (Millipore) for 4 h and as the same time that the addition of 100 nM of trastuzumab conjugated with Alexa Fluor 488 (Thermo Fisher Scientific) in 0.1% (w/v) bovine serum albumin (BSA) in PBS for 30 min at 4 °C, followed by incubation for 90 min at 37 °C. Cancer cells were then fixed with 4% (v/v) paraformaldehyde (PFA) for 20 min before incubation with 4′,6-diamidino-2-phenylindole (DAPI, 1:5000, Santa Cruz Biotechnology) for 5 min. Fluorescence images were acquired using a 60X oil immersion objective on the EVOS M5000 Imaging System (Thermo Fisher Scientific). Quantification of the images was performed by ImageJ/FIJI (NIH) software.

Conjugation and Radiolabeling of Trastuzumab

Alexa Fluor 488-trastuzumab

For immunofluorescence studies, trastuzumab was conjugated with Alexa Fluor 488 at a molar ratio of 1:3. Briefly, trastuzumab in PBS at pH 8.8–9 was incubated with Alexa Fluor 488 for 1 h at 37 °C with gentle agitation (450 rpm). Purification of Alexa Fluor 488-trastuzumab was performed by size exclusion chromatography (PD-10, preconditioned with PBS pH 7.4; GE Healthcare), followed by sample concentration using 50 kDa amicon filters (Millipore). The concentration of the resulting antibody conjugate was measured by measuring the absorption at 280 nm with a UV–vis spectrophotometer (Nanodrop, Thermo Fisher Scientific).

[89Zr]Zr-DFO-trastuzumab

[89Zr]Zr-oxalate in 0.1 M sodium oxalate solution was obtained from the Washington University School of Medicine Cyclotron Facility. Trastuzumab was first conjugated with p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO; Macrocyclics) in a molar ratio of 1:5, respectively, in 10 mM PBS (≤1% v/v DMSO) at pH 8.8–9 for 1 h at 37 °C with gentle agitation. DFO-trastuzumab was purified using size-exclusion chromatography (PD-10; preconditioned with PBS pH 7.4) and concentrated with a 50 kDa amicon filter. [89Zr]Zr-DFO-Trastuzumab was obtained after incubation of DFO-trastuzumab with [89Zr]Zr-oxalate at pH 7.4 for 1 h at 37 °C. Radiochemical purity was ≥95% as determined by instant thin-layer chromatography (i-TLC) using 50 mM ethylenediaminetetraacetic acid (EDTA) as the mobile phase, and the molar activity was 17.32 ± 0.35 MBq/nmol.

[64Cu]Cu-NOTA-trastuzumab

[64Cu]CuCl2 in 0.1 M ammonium acetate solution pH 6.0 was obtained from the Washington University School of Medicine Cyclotron Facility. Trastuzumab was buffer-exchanged in 0.1 M HEPES buffer pH 8.5 and conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA; Macrocyclics) in a 20-fold molar excess at 4 °C overnight with gentle agitation. The resulting NOTA-trastuzumab was purified by PD10 (preconditioned with 0.1 M ammonium acetate pH 6.0) and concentrated on a 50 kDa amicon filter. [64Cu]Cu-NOTA-trastuzumab was obtained after incubation of NOTA-trastuzumab with [64Cu]CuCl2 in 0.1 M ammonium acetate at pH 6.0 for 1 h at 37 °C. Radiochemical purity was ≥95% as determined by i-TLC using a mixture of 0.1 M ammonium acetate buffer pH 6.0 and 50 mM EDTA (1:1) as mobile phase, and the molar activity was 33.03 ± 6.82 MBq/nmol.

Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry of trastuzumab, DFO-trastuzumab, and NOTA-trastuzumab was performed to determine the number of conjugates per antibody at the Alberta Proteomics and Mass Spectrometry Facility at the University of Alberta in Canada.

Binding Assays

For the total binding study, 1 million of MIAPaCa-2 cells were incubated in 100 μL of PBS containing 1% (w/v) of BSA with 0.037 MBq of [89Zr]Zr-DFO-trastuzumab (1 μCi) in the presence or absence of the active form of lovastatin for 90 min at room temperature with gentle rotation. Media containing noncell bound radiotracer was removed by centrifugation (600g, 2 min, 4 °C), and the cells were washed twice with cold PBS. Radioactivity bounded to the cells was measured on a γ counter calibrated for 89Zr. Results are expressed as percentage binding calculated by dividing the radioactivity of the cells by the total radioactivity obtained from the cells and washes.

To determine membrane-bound radiolabeled trastuzumab, NCIN87, MIAPaCa-2 or UMUC3 cells (2 million cells) were incubated with either 0.037 MBq of [89Zr]Zr-DFO-trastuzumab (1 μCi) or [64Cu]Cu-NOTA-trastuzumab (1 μCi) in PBS containing 0.1% (w/v) of BSA for 10 min at 4 °C, followed by an incubation of 90 min at 37 °C. Media containing noncell bound radiotracer was removed, and the cells were washed twice with cold PBS. The membrane-bound radiotracer was collected by incubation of cells with 50 mM glycine buffer containing 150 mM NaCl at pH 2.8 for 15 min at 4 °C. Internalized fraction was obtained after cell lysis with 1 M of sodium hydroxide (NaOH). Radioactivity bounded to the cell surface was measured on a γ counter calibrated for 89Zr or 64Cu. Results are expressed as a percentage of membrane-bound activity obtained by dividing the radioactivity of the membrane by the total radioactivity obtained from the cells and washes.

Subcutaneous Bilateral Tumor Model

All animals were treated according to the guidelines approved by the Research Animal Resource Center and Institutional Care and Use Committee at Washington University School of Medicine in St. Louis. Six- to eight-week-old nu/nu female mice (Charles River Laboratories) were injected subcutaneously with 5 million NCIN87 cells (n = 8), 5 million MIAPaCa-2 cells (n = 8) or 2 million UMUC3 cells (n = 8) in a 100 μL cell suspension of a 1:1 (v/v) mixture of Matrigel (BD Biosciences) on the bilateral dorsal flank regions. The tumor volume (V/mm3) was estimated by external vernier caliper measurements of the longest axis, α/mm, and the axis perpendicular to the longest axis, b/mm. The tumors were assumed to be spheroidal, and the volume was calculated in accordance with the equation V = (4π/3) × (α/2)2 × (b/2).

PET Imaging

PET imaging was performed when tumor volumes reached approximately 100–400 mm3 (see details in Supplementary Table 1). Mice were randomly distributed into two experimental groups: [89Zr]Zr-DFO-trastuzumab (n = 3 per tumor type) and [64Cu]Cu-NOTA-trastuzumab (n = 3 per tumor type). Lovastatin (0.44 mg/kg, 50 μL) was intratumorally injected into the right tumor, while the left tumor was injected with PBS (50 μL) as a control. Lovastatin or saline was administered 4 h before and simultaneously to the tail vein administration of [89Zr]Zr-DFO-trastuzumab (7.2–7.5 MBq, 50 μg protein) or [64Cu]Cu-NOTA-trastuzumab (8.4–8.7 MBq, 50 μg protein). PET imaging studies were conducted on a Mediso nanoScan PET/CT scanner at 24 and 48 h. The mice were anesthetized by inhalation of 2% isoflurane in an oxygen gas mixture 10 min before starting the CT-PET scan. CT scan was recorded for 5 min to obtain anatomical information, followed by a static PET scan for 20 min with the mice under isoflurane anesthesia (2%) in an oxygen gas mixture.

Quantification of PET Imaging

PET/CT images of the same mouse were coregistered and analyzed using 3D Slicer software (version 5.2.2). Regions of interest (ROI) were manually delineated in the tumor and muscle. Activity values were obtained as Bq/cm3 (decay-corrected) and converted as a percentage of injected dose per cm3 of tissue (%ID/cm3). The values represented in the graphs were expressed as %ID/cm3 normalized to tumor volume or tumor/muscle ratio. For the PET images in Figure 4, in which only control tumors are shown, the “mask volume” function on 3D Slicer software was used to blank out image regions of the lovastatin-administered tumor.

Figure 4.

Figure 4

Open in a new tab

89Zr and 64Cu immuno-PET imaging. (A) PET images (maximum intensity projections, sagittal view) of NCIN87, MIAPaCa-2 and UMUC3 tumors obtained at 24 and 48 h postinjection of [89Zr]Zr-DFO-trastuzumab (7.2–7.5 MBq, 50 μg) or [64Cu]Cu-NOTA-trastuzumab (8.4–8.7 MBq, 50 μg). Comparison between NCIN87, MIAPaCa-2, and UMUC3 xenograft (B) and 89Zr versus 64Cu isotopes (C) at 24 and 48 h after intravenous administration of [89Zr]Zr-DFO-trastuzumab or [64Cu]Cu-NOTA-trastuzumab. Results are expressed as %ID/cm3 (mean ± S.E.M, n = 3 per group). Statistical analyses were performed with two-way ANOVA for cell line comparison and unpaired t-test for 89Zr versus 64Cu-labeled trastuzumab.

Blocking Biodistribution Study

An additional biodistribution study was performed to evaluate the specificity of [89Zr]Zr-DFO-trastuzumab in the HER2-moderate MIAPaCa-2 cell line. Six- to eight-week-old nu/nu female mice (Charles River Laboratories) were injected subcutaneously with 5 million MIAPaCa-2 cells (n = 8) and randomly distributed into two experimental groups: blocking and non-blocking (n = 4 per group). Biodistribution studies were conducted at 48 h postinjection of the radiolabeled antibody (0.37 MBq, 5 μg protein). For the blocking group, mice were administered intravenously with unlabeled DFO-trastuzumab (25X, 125 μg protein) 30 min before [89Zr]Zr-DFO-trastuzumab injection. Following mice euthanasia by controlled carbon dioxide overdose followed by cervical dislocation, organs were collected, and radioactivity was assessed using a γ counter (2480 Wizard, PerkinElmer). The radioactivity associated with each organ was quantified as a percentage of the injected dose per gram of organ (% ID/g).

Statistical Analyses

Statistical analyses were performed using GraphPad Prism version 9.5.1 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. Data are shown as mean ± SEM. For total protein, HER2 and CAV-1 protein levels between control and lovastatin groups were compared using two-way ANOVA with Šídák’s multiple comparison test. For immunofluorescence, fluorescence intensity between cell lines was compared using one-way ANOVA with Turkey’s multiple comparison test and unpaired t test to compare control and lovastatin groups in each cell line. For binding studies, total binding was compared using unpaired t-test and one-way ANOVA with Turkey’s multiple comparison test to compare membrane-bound in the three cell lines. For PET imaging, two-way ANOVA with Turkey’s comparison test and unpaired t-test were used. For blocking study, two-way ANOVA with Šídák’s multiple comparison test was used. ns = nonsignificant, * = p < 0.05 ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Results

Inverse Relationship between HER2 and CAV-1 in Cancer Cells

The efficacy of trastuzumab-based therapies depends on the density of cell-surface HER2: higher HER2 expression correlates with increased uptake of trastuzumab, thereby enhancing treatment efficacy.17,18,23−25 Previous studies have demonstrated a negative correlation between cell-surface HER2 expression and levels of CAV-1 protein within tumors.18,26−28 Others have shown that depletion of tumoral CAV-1 increases trastuzumab,18 T-DM1,17,22 T-DXd,22 and Lutetium-177-labeled trastuzumab23 uptake and efficacy in HER2-high gastric xenografts. We initially used data from the Cancer Cell Line Encyclopedia29 to determine HER2 and CAV-1 protein levels in cancer cell lines (Figure 1a). Similar to previous studies,18 total protein levels of HER2 were inversely correlated with those of CAV-1. HER2 protein levels were found to be highest in NCIN87, followed by MIAPaCa-2 and UMUC3 cell lines. NCIN87 gastric, MIAPaCa-2 pancreatic, and UMUC3 bladder cell lines were subsequently chosen to represent high, moderate, and low HER2 density, respectively.

Figure 1.

Figure 1

Open in a new tab

HER2 and CAV-1 protein levels in different cancer cells. (A) Correlation between HER2 and CAV-1 in NCIN87, MIAPaCa-2 and UMUC3 cancer cells. The data was obtained from the Cancer Cell Lines Encyclopedia database. (B) Schematic representation of the effect of lovastatin in the modulation of HER2 surface receptors. The scheme was created in BioRender. Ribeiro pereira, P. (2024) BioRender.com/j34u217. (C) Western blot images (left) and quantification (right) obtained for HER2, CAV-1, and β-actin proteins in cancer cells after incubation with or without lovastatin (25 μM, 4 h). Values are normalized to β-actin and control (mean ± S.E.M, n = 4). (D) Western blot images (left) and quantification (right) illustrating the modulation of cell-surface HER2 expression using lovastatin (25 μM, 4 h) of MIAPaCa-2 cell line (mean ± S.E.M, n = 4). Statistical analyses were performed with two-way ANOVA.

Based on previous observations using the HER2-high NCIN87 cell line,18 we hypothesized that lovastatin, a CAV-1-depleting pharmacologic approach, could potentially elevate HER2 expression at the cell membrane in cells exhibiting medium and low HER2 expression (Figure 1b). For in vitro studies, statin-induced CAV-1 depletion was performed using the active form of lovastatin as previously described.18 The total HER2 and CAV-1 protein levels were determined both with and without lovastatin treatment (Figure 1c; Supplementary Figure 1a). Western blot analyses confirmed the Cancer Cell Line Encyclopedia data, revealing an inverse relationship between total HER2 and CAV-1 expression levels (Supplementary Figure 2). The NCIN87 cell line exhibiting the highest HER2 expression demonstrated the lowest levels of CAV-1, and conversely, UMUC3 cells exhibiting the lowest HER2 expression showed the highest levels of CAV-1. We found that lovastatin resulted in a reduction in the total levels of CAV-1 ranging from 1 to 2.3-fold, and enhanced HER2 expression in total protein lysates across a range of 1.2 to 1.9-fold (Figure 1c). Similar to previous studies in NCIN87 cancer cells,18 statin incubation resulted in a 1.3-fold increase in cell-surface HER2 expression in MIAPaCa-2 cancer cells, as evidenced by cell-surface biotinylation assays (Figure 1d; Supplementary Figure 1b).

In summary, our findings validate our previous studies showing an inverse relationship between HER2 protein levels and CAV-1 protein levels in cancer cells of varying HER2 expression.

Lovastatin Enhances Trastuzumab Binding in MIAPaCa-2 HER2-Moderate and UMUC3 HER2-Low Cancer Cells

We next performed immunofluorescence assays to evaluate the effect of lovastatin on trastuzumab binding to cancer cells (Figure 2). We observed a positive correlation between trastuzumab binding and HER2 protein levels: NCIN87 >MIAPaCa-2 >UMUC3. When incubated with lovastatin, cancer cells demonstrated increased trastuzumab staining (Figure 2a), a finding supported by fluorescence quantification (Figure 2b). Notably, incubation of cancer cells with lovastatin resulted in a 1.3-fold increase in trastuzumab accumulation in MIAPaCa-2 and a 2.0-fold increase in UMUC3 compared to untreated conditions.

Figure 2.

Figure 2

Open in a new tab

In vitro HER2 modulation results in an increase in trastuzumab binding. Confocal images (A) and quantification (B) of immunofluorescence of HER2 in NCIN87, MIAPaCa-2, and UMUC3 cells incubated with 100 nM trastuzumab-Alexa488 for 1.5 h in the presence or absence of lovastatin (25 μM, 4 h). Lovastatin was incubated in cancer cells for 4 h, followed by incubation with the fluorescently labeled trastuzumab for 30 min at 4 °C and 90 min at 37 °C. Scale bars = 20 μm. Results are expressed as total fluorescence intensity normalized by unit area (mean ± S.E.M, n = 3), comparing fluorescence between cell lines for control groups (top graph) and between control and treated conditions (bottom graph). Statistical analyses were performed with one-way ANOVA (cell line comparison) and unpaired t-test (control versus lovastatin).

In sum, our in vitro findings showed that lovastatin enhances trastuzumab binding in vitro in HER2-moderate MIAPaCa-2 and HER2-low UMUC3 cancer cells.

89Zr- and 64Cu-labeled Trastuzumab Monitor Cell-surface HER2

The positron emitter 89Zr has demonstrated potential as a radioisotope for immuno-PET imaging because of its favorable characteristics–a half-life of 78.4 h, which matches the kinetics of monoclonal antibodies.30−32 [89Zr]Zr-DFO-trastuzumab has shown predictive value in the clinics to monitor tumor response to HER2-targeted therapies.7,8,13,33−37 In addition to 89Zr, we tested the use of 64Cu to image HER2-high, -moderate, and -low tumors, given its shorter half-life of 12.7 h compared to 78.41 h for 89Zr. Trastuzumab was first conjugated with DFO or NOTA (Figure 3a; Supplementary Figure 3 and 4).33,38 [89Zr]Zr-DFO-trastuzumab and [64Cu]Cu-NOTA-trastuzumab were obtained with radiochemical yields of 66.6 ± 13.2% and 70.6 ± 2.5% and specific activities of 0.14 ± 0.7 and 0.22 ± 1.1 MBq/μg, respectively (Figure 3b; Supplementary Figure 5).

Figure 3.

Figure 3

Open in a new tab

Radiolabeling of trastuzumab and binding studies. (A) Schematic representation of the conjugation of trastuzumab with deferoxamine (DFO) or 1,4,7-triazacyclononane-1,4,7-tricetic acid (NOTA) to be radiolabeled with zirconium-89 (89Zr) or copper-64 (64Cu), respectively. (B) Radiochemical yield and specific activity obtained from the radiolabeling of trastuzumab with 89Zr and 64Cu. (C) Membrane-bound [89Zr]Zr-DFO-trastuzumab and [64Cu]Cu-NOTA-trastuzumab after incubation with or without lovastatin (25 μM, 4 h) in NCIN87, MIAPaCa-2 and UMUC3 cancer cells. (D) Total binding of [89Zr]Zr-DFO-trastuzumab after incubation with or without lovastatin (25 μM, 4 h) in MIAPaCa-2 cancer cells.

NCIN87 cells demonstrated higher membrane-associated radioactivity (both 89Zr and 64Cu, Figure 3c), which is a result of higher HER2 expression on NCIN87 cancer cells relative to MIAPaCa-2 and UMUC3 cells. No difference in trastuzumab uptake was observed between [89Zr]Zr-DFO-trastuzumab or [64Cu]Cu-NOTA-trastuzumab in these three cancer cell lines. In the presence of lovastatin, MIAPaCa-2 cancer cells exhibited a 2.5-fold increase in 89Zr-labeled trastuzumab binding compared to untreated cells (Figure 3d).

In conclusion, our in vitro observations demonstrate the ability of 64Cu and 89Zr-labeled trastuzumab to assess HER2 expression across cancer cells with varying HER2 protein levels.

[64Cu]Cu-NOTA-trastuzumab and [89Zr]Zr-DFO-trastuzumab Monitors In Vivo HER2

We conducted preclinical PET imaging studies of [64Cu]Cu-NOTA-trastuzumab and [89Zr]Zr-DFO-trastuzumab in mice bearing HER2-high NCIN87, HER2-moderate MIAPaCa-2, and HER2-low UMUC3 tumors. PET imaging was performed at 24 and 48 h postadministration of radiolabeled trastuzumab, and PET images were quantified to determine 89Zr- or 64Cu-labeled trastuzumab uptake in xenografts (Figure 4; Supplementary Tables 2–4 and Figure 6). A blocking study was also performed with a coinjection of excess unlabeled DFO-trastuzumab. Similar to previous findings observed in NCIN87 xenografts,33 coadministration of a 125 μg DFO-trastuzumab blocking dose with the 89Zr-labeled trastuzumab demonstrated an 1.6-fold decrease in MIAPaCa-2 tumor uptake (10.8 ± 5%ID/g) at 48 h after injection (Supplementary Figure 7).

Figure 6.

Figure 6

Open in a new tab

64Cu immuno-PET imaging on mice bearing bilateral tumors of NCIN87, MIAPaCa-2 and UMUC3 cancer cells. (A) PET images (maximum intensity projections, coronal view) of NCIN87, MIAPaCa-2, and UMUC3 bilateral tumors obtained at 24 and 48 h postinjection of [64Cu]Cu-NOTA-trastuzumab (8.4–8.7 MBq, 50 μg). PBS (50 μL, control, left-sided tumor) and lovastatin (0.44 mg/kg, 50 μL, right-sided tumor) were administered intratumorally 4 h before and simultaneously with tail vein injection of [64Cu]Cu-NOTA-trastuzumab. PET images were coregistered with CT images. (B) PET image quantification of tumor uptake. Results are expressed as %ID/cm3 normalized to tumor volume (mean ± S.E.M, n = 3 per group). (C) Tumor:muscle ratio quantification in MIAPaCa-2 tumors.

At 24 h postinjection of radiolabeled trastuzumab, we observed higher trastuzumab accumulation in NCIN87 tumors (25.5%ID/cm3 for 89Zr and 19.6%ID/cm3 for 64Cu) when compared with MIAPaCa-2 (9.3%ID/cm3 for 89Zr and 10.7%ID/cm3 for 64Cu) and UMUC3 (5.4%ID/cm3 for 89Zr and 6.7%ID/cm3 for 64Cu) tumors (Figure 4b; Supplementary Tables 2–4). Comparable results were noted at 48 h postinjection of radiolabeled trastuzumab. Additionally, the results obtained showed no difference between the two radioimmunoconjugates across the three xenograft models (Figure 4c). [89Zr]Zr-DFO-trastuzumab uptake was positively correlated with that of [64Cu]Cu-NOTA-trastuzumab, showing a Spearman’s rank correlation coefficient (r) of 0.75 (P < 0.05, Spearman’s correlation, Supplementary Figure 8).

In conclusion, our study demonstrates that both [64Cu]Cu-NOTA-trastuzumab and [89Zr]Zr-DFO-trastuzumab exhibit similar ability in imaging varying levels of HER2 expression in xenograft models.

Lovastatin Enhances Trastuzumab Binding in vivo in MIAPaCa-2 HER2-Moderate Xenografts

Lovastatin has been reported as a potential pharmacologic approach to accelerate and enhance 89Zr-labeled trastuzumab accumulation in HER2-high NCIN87 tumors.18 To determine the use of lovastatin to enhance trastuzumab accumulation in HER2-moderate MIAPaCa-2 and HER2-low UMUC3 tumors, we performed PET imaging to monitor trastuzumab uptake in control PBS and statin-administered tumors. Bilateral xenografts of NCIN87 gastric cancer, MIAPaCa-2 pancreatic cancer, and UMUC3 bladder cancer were used following a previously described procedure.18 The tumor on the right flank was injected with lovastatin, and control saline was injected into the tumor located on the left flank.

As previously reported,18 NCIN87 tumors showed a 1.4-fold increase in trastuzumab accumulation after lovastatin treatment at 48 h (Figures 5 and 6; Supplementary Figure 9). Although we did not observe significant changes, the uptake of trastuzumab in MIAPaCa-2 xenografts was enhanced by 1.5-fold and 1.3-fold for 89Zr-labeled trastuzumab (Figures 5c), and 1.3-fold and 1.4-fold for 64Cu-labeled trastuzumab (Figure 6b) at 24 and 48 h, respectively, in lovastatin-administered tumors. This increased tumor uptake was confirmed by quantification of tumor/muscle ratio (Figures 5d and 6c). On the other hand, the biodistribution obtained in UMUC3 xenografts for both radiotracers was typical of an isotype control IgG, with tumor uptake <5%ID/cm3 for control and lovastatin-administered tumors (Figures 5 and 6). These results are corroborated by cell-surface Western blot analyses, showing no difference in cell-surface HER2 between control and treated UMUC3 cells (Supplementary Figure 10).

Figure 5.

Figure 5

Open in a new tab

89Zr immuno-PET imaging on mice bearing bilateral tumors of NCIN87, MIAPaCa-2 and UMUC3 cancer cells. (A) Schematic representation of the experimental setup performed for the immuno-PET study. The scheme was created in BioRender. Ribeiro pereira, P. (2023) BioRender.com/m14c002. (B) PET images (maximum intensity projections, coronal view) of NCIN87, MIAPaCa-2, and UMUC3 bilateral tumors obtained at 24 and 48 h postinjection of [89Zr]Zr-DFO-trastuzumab (7.2–7.5 MBq, 50 μg). PBS (50 μL, control, left-sided tumor) and lovastatin (0.44 mg/kg, 50 μL, right-sided tumor) were administered intratumorally 4 h before and simultaneously with tail vein injection of [89Zr]Zr-DFO-trastuzumab. PET images were coregistered with CT images. (C) Quantification of tumor uptake from PET images. (D) Tumor:muscle ratio quantification in MIAPaCa-2 tumors. Results are expressed as %ID/cm3 normalized to tumor volume (mean ± S.E.M, n = 3 per group).

Our findings show that statin administration enhances trastuzumab uptake in HER2-moderate MIAPaCa-2 tumors but does not induce significant alterations in trastuzumab uptake in HER2-low UMUC3 tumors.

Discussion

Our preclinical imaging studies demonstrate that both 64Cu- and 89Zr-labeled trastuzumab monitor HER2 expression levels across xenografts with varying HER2 expression (HER2-high NCIN87, HER2-moderate MIAPaCa-2, and HER2-low UMUC3). PET imaging in these xenograft models showed a positive correlation between both 64Cu and 89Zr-labeled trastuzumab uptake and HER2 protein levels (Figures 3 and 4, Supplementary Figure 11). This confirms a higher accumulation of trastuzumab in tumors with higher HER2 expression (NCIN87 > MIAPaCa-2 > UMUC3). Western blot and immunofluorescence analyses of cancer cell lines validated previous studies,17,18 showing an inverse correlation between total HER2 and CAV-1 protein expression (Figures 1 and 2). Incubation of cancer cells with lovastatin decreases CAV-1 levels and enhances trastuzumab binding to cancer cells, particularly at the cell surface of HER2-high NCIN87 and HER2-moderate MIAPaCa-2 cancer cells. PET imaging further demonstrated that optimization of this pharmacological strategy is needed to determine the potential of statins in improving antibody therapies in tumors expressing moderate levels of HER2 (Figures 5 and 6). Together, our findings demonstrate the effectiveness of both 64Cu- and 89Zr-labeled trastuzumab in monitoring HER2 protein levels across a spectrum of cancer cells with varying HER2 expression, highlighting their potential in guiding personalized therapy decisions.

In HER2-positive tumors, particularly in gastric cancer, the heterogeneity of HER2 expression levels within primary tumors and metastasis is a challenge for the effective detection of HER2 protein levels in tumors.39,40 Additionally, the accumulation of HER2-targeted therapies (such as trastuzumab) varies not only between tumors but also among individual patients. Therefore, radiolabeled trastuzumab has proven invaluable in clinical settings, showing its effectiveness in tracking its distribution throughout the body, noninvasively evaluating target engagement, and monitoring tumor response to treatment.8,10,16,35,41−43

It is generally agreed that the uptake of an antibody is most accurately determined when the majority of the radioimmunoconjugate has cleared from the bloodstream. This phase typically coincides with a peak uptake, which reflects the binding of the antibody to the tumor’s molecular target. PET imaging during this late phase requires a radiolabel with a half-life of several days or longer, making 89Zr (half-life of 78.4 h) particularly suitable for labeling trastuzumab.8−10 However, using long half-life isotopes comes with the drawback of higher radiation doses for patients.44 Although 64Cu has a short half-life (12.7 h) compared to the pharmacokinetics of antibodies, previous studies employing 64Cu-labeled trastuzumab in metastatic breast cancer patients have successfully visualized HER2-positive tumors using PET.12,15,16,42,45 These previous studies hypothesized that trastuzumab binding in metastatic breast cancer lesions is rapid enough to enable visualization of HER2-specific lesions within 1–2 days after injection.45

Due to the long plasma half-lives of antibodies such as trastuzumab, PET imaging typically requires a few days postinjection for optimal PET imaging acquisition. In contrast, smaller molecules enable imaging within hours after tracer injection. In this context, smaller biomolecules labeled with short-lived isotopes (e.g., gallium-68, half-life of 68 min) such as nanobodies (∼14 kDa)46 and affibody molecules (∼6.5 kDa)47,48 can detect HER2-positive tumors in patients. Future studies could consider directly comparing these approaches within the same settings.

While the results shown in this current study did not aim to compare 64Cu and 89Zr directly, it is important to note that achieving similar activity to 89Zr at 48 h would require significantly higher doses of 64Cu at the time of injection, leading to an overall higher dose. However, our findings suggest that 64Cu can be effectively used in preclinical studies at similar doses to 89Zr for tumor visualization. Our research in preclinical models suggests that the visualization and quantification of HER2-specific uptake are attainable for tumors with varying levels of HER2 within 1–2 days postinjection. Despite the relatively short half-life of 64Cu, here we show the ability to measure tumor uptake of trastuzumab within this time frame in these preclinical models.

In our previous work,17,18,22,23 we demonstrated the utilization of lovastatin as a pharmacological intervention to augment HER2 expression and enhance trastuzumab binding. These previous studies focused on tumors with high expression of HER2. In this current study, our preliminary investigations indicate the use of lovastatin to enhance trastuzumab accumulation in HER2-moderate tumors; however, this change did not reach statistical significance. In our previous studies, the statin was administered orally; in this pilot study, it was administered intratumorally. These findings suggest that further preclinical investigations are needed with the aim of determining the optimal administration routes, dosing, and timing of lovastatin administration in combination with HER2-targeted therapies in models of moderate and low expression.

While our study yielded promising results, it is important to acknowledge its limitations. First, employing intratumoral administration for lovastatin delivery to tumors may pose challenges.49 The nonuniform distribution of the statin within the tumor could be influenced by tumor vasculature, limiting the effective volume injected depending on tumor size. Additionally, the invasive nature of the intratumoral procedure may induce local tissue damage. Second, the dose of lovastatin used was determined based on a previous work using preclinical models of HER2-high expression,18 and additional statin optimization might be required for HER2-moderate and -low expressers. Lastly, our study used xenograft models with varying HER2 levels, which, although informative, may not fully represent clinical HER2 heterogeneity and further therapeutic studies involving additional models are planned.

Conclusions

Our study showed that both [64Cu]Cu-NOTA-trastuzumab and [89Zr]Zr-DFO-trastuzumab can monitor varying levels of HER2 expression within preclinical models. In vitro studies demonstrated that lovastatin enhances trastuzumab binding to HER2-moderate MIAPaCa-2 tumors. We are currently working to optimize statin administration in mice bearing HER2-moderate and HER2-low tumors and the potential combination of lovastatin with T-DXd or T-DM1 in HER2-moderate and HER2-low xenografts.

Acknowledgments

This research was supported by internal funds provided by the Mallinckrodt Institute of Radiology, NIH (R37CA276498, P.M.R. Pereira), and in part by the American Cancer Society (IRG-21–133–64–03, P.M.R. Pereira), the Breast Cancer Alliance (P.M.R. Pereira), and a W. M. Keck Postdoctoral Fellowship (C. Simó). The Preclinical Imaging Facility was supported by NIH/NCI Siteman Cancer Center Support Grant P30CA091842, NIH instrumentation grants S10OD018515 and S10OD030403, and internal funds provided by Mallinckrodt Institute of Radiology. The authors have nonfinancial interests to disclose. We thank the Washington University School of Medicine isotope production team for the copper-64 and zirconium-89 and the small animal imaging facility for help with the small animal PET/CT

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.4c00777.

  • Experimental details for the generation of bilateral xenograft animal model; Raw data of Western blot images; correlation between the data of the Cancer Cell Line Encyclopedia and Western Blot; SDS-gel of unconjugated and conjugated trastuzumab; MALDI results; radio-TLC chromatograms; raw data of PET images quantification; PET images; blocking study; spearman correlation between radiotracers; tumor:muscle ratio quantification for NCIN87 tumors; Cell-surface HER2 Western Blot in UMUC3 (PDF)

The authors declare no competing financial interest.

Supplementary Material

mp4c00777_si_001.pdf (1.3MB, pdf)

References

  1. Yan M.; Schwaederle M.; Arguello D.; Millis S. Z.; Gatalica Z.; Kurzrock R. HER2 expression status in diverse cancers: review of results from 37,992 patients. Cancer Metastasis Rev. 2015, 34 (1), 157–164. 10.1007/s10555-015-9552-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Slamon D. J.; Clark G. M.; Wong S. G.; Levin W. J.; Ullrich A.; Mcguire W. L. Human-Breast Cancer - Correlation of Relapse and Survival with Amplification of the Her-2 Neu Oncogene. Science 1987, 235 (4785), 177–182. 10.1126/science.3798106. [DOI] [PubMed] [Google Scholar]
  3. Baselga J.; Swain S. M. Novel anticancer targets: revisiting ERBB2 and discovering ERBB3. Nat. Rev. Cancer 2009, 9 (7), 463–475. 10.1038/nrc2656. [DOI] [PubMed] [Google Scholar]
  4. Kreutzfeldt J.; Rozeboom B.; Dey N.; De P. The trastuzumab era: current and upcoming targeted HER2+breast cancer therapies. Am. J. Cancer Res. 2020, 10 (4), 1045–1067. [PMC free article] [PubMed] [Google Scholar]
  5. Brastianos P. K.; Carter S. L.; Santagata S.; Cahill D. P.; Taylor-Weiner A.; Jones R. T.; Van Allen E. M.; Lawrence M. S.; Horowitz P. M.; Cibulskis K.; et al. Genomic Characterization of Brain Metastases Reveals Branched Evolution and Potential Therapeutic Targets. Cancer Discovery 2015, 5 (11), 1164–1177. 10.1158/2159-8290.CD-15-0369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. McNamara K. L.; Caswell-Jin J. L.; Joshi R.; Ma Z. C.; Kotler E.; Bean G. R.; Kriner M.; Zhou Z.; Hoang M.; Beechem J.; et al. Spatial proteomic characterization of HER2-positive breast tumors through neoadjuvant therapy predicts response. Nat. Cancer 2021, 2 (4), 400–413. 10.1038/s43018-021-00190-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Sanchez-Vega F.; Hechtman J. F.; Castel P.; Ku G. Y.; Tuvy Y.; Won H.; Fong C. J.; Bouvier N.; Nanjangud G. J.; Soong J.; et al. EGFR and MET Amplifications Determine Response to HER2 Inhibition in ERBB2-Amplified Esophagogastric Cancer. Cancer Discovery 2019, 9 (2), 199–209. 10.1158/2159-8290.CD-18-0598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dehdashti F.; Wu N.; Bose R.; Naughton M. J.; Ma C. X.; Marquez-Nostra B. V.; Diebolder P.; Mpoy C.; Rogers B. E.; Lapi S. E.; et al. Evaluation of [89Zr]trastuzumab-PET/CT in differentiating HER2-positive from HER2-negative breast cancer. Breast Cancer Res. Treat 2018, 169 (3), 523–530. 10.1007/s10549-018-4696-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dijkers E. C.; Munnink T. H. O.; Kosterink J. G.; Brouwers A. H.; Jager P. L.; de Jong J. R.; van Dongen G. A.; Schröder C. P.; Lub-de Hooge M. N.; de Vries E. G. Biodistribution of 89Zr-trastuzumab and PET Imaging of HER2-Positive Lesions in Patients With Metastatic Breast Cancer. Clin. Pharmacol. Ther. 2010, 87 (5), 586–592. 10.1038/clpt.2010.12. [DOI] [PubMed] [Google Scholar]
  10. Laforest R.; Lapi S. E.; Oyama R.; Bose R.; Tabchy A.; Marquez-Nostra B. V.; Burkemper J.; Wright B. D.; Frye J.; Frye S.; et al. [89Zr]Trastuzumab: Evaluation of Radiation Dosimetry, Safety, and Optimal Imaging Parameters in Women with HER2-Positive Breast Cancer. Mol. Imaging Biol. 2016, 18 (6), 952–959. 10.1007/s11307-016-0951-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. O’Donoghue J. A.; Lewis J. S.; Pandit-Taskar N.; Fleming S. E.; Schöder H.; Larson S. M.; Beylergil V.; Ruan S.; Lyashchenko S. K.; Zanzonico P. B.; et al. Pharmacokinetics, Biodistribution, and Radiation Dosimetry for 89Zr-Trastuzumab in Patients with Esophagogastric Cancer. J. Nucl. Med. 2018, 59 (1), 161–166. 10.2967/jnumed.117.194555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kurihara H.; Hamada A.; Yoshida M.; Shimma S.; Hashimoto J.; Yonemori K.; Tani H.; Miyakita Y.; Kanayama Y.; Wada Y.; et al. 64Cu-DOTA-trastuzumab PET imaging and HER2 specificity of brain metastases in HER2-positive breast cancer patients. EJNMMI 2015, 5, 8. 10.1186/s13550-015-0082-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mileva M.; de Vries E. G. E.; Guiot T.; Wimana Z.; Deleu A. L.; Schroder C. P.; Lefebvre Y.; Paesmans M.; Stroobants S.; Huizing M.; et al. Molecular imaging predicts lack of T-DM1 response in advanced HER2-positive breast cancer (final results of ZEPHIR trial). NPJ. Breast Cancer 2024, 10 (1), 4. 10.1038/s41523-023-00610-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Janjigian Y. Y.; Viola-Villegas N.; Holland J. P.; Divilov V.; Carlin S. D.; Gomes-DaGama E. M.; Chiosis G.; Carbonetti G.; de Stanchina E.; Lewis J. S. Monitoring Afatinib Treatment in HER2-Positive Gastric Cancer with 18F-FDG and 89Zr-Trastuzumab PET. J. Nucl. Med. 2013, 54 (6), 936–943. 10.2967/jnumed.112.110239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tamura K.; Kurihara H.; Yonemori K.; Tsuda H.; Suzuki J.; Kono Y.; Honda N.; Kodaira M.; Yamamoto H.; Yunokawa M.; et al. 64Cu-DOTA-trastuzumab PET imaging in patients with HER2-positive breast cancer. J. Nucl. Med. 2013, 54 (11), 1869–1875. 10.2967/jnumed.112.118612. [DOI] [PubMed] [Google Scholar]
  16. Mortimer J. E.; Joanne E. M.; Bading J. R.; James R. B.; Park J. M.; Jinha M. P.; Frankel P. H.; Paul H. F.; Carroll M. I.; Mary I. C.; Tran T. T.; Tri T. T.; Poku E. K.; Erasmus K. P.; Rockne R. C.; Russell C. R.; Raubitschek A. A.; Andrew A. R.; Shively J. E.; John E. S. Tumor Uptake of 64Cu-DOTA-Trastuzumab in Patients with Metastatic Breast Cancer. J. Nucl. Med. 2018, 59 (1), 38. 10.2967/jnumed.117.193888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pereira P. M. R.; Mandleywala K.; Monette S.; Lumish M.; Tully K. M.; Panikar S. S.; Cornejo M.; Mauguen A.; Ragupathi A.; Keltee N. C.; et al. Caveolin-1 temporal modulation enhances antibody drug efficacy in heterogeneous gastric cancer. Nat. Commun. 2022, 13 (1), 2526 10.1038/s41467-022-30142-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pereira P. M. R.; Sharma S. K.; Carter L. M.; Edwards K. J.; Pourat J.; Ragupathi A.; Janjigian Y. Y.; Durack J. C.; Lewis J. S. Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy. Nat. Commun. 2018, 9 (1), 5137 10.1038/s41467-018-07608-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Panikar S. S.; Keltee N.; Berry N. K.; Shmuel S.; Fisher Z. T.; Brown E.; Zidel A.; Mabry A.; Pereira P. M. R. Metformin-Induced Receptor Turnover Alters Antibody Accumulation in HER-Expressing Tumors. J. Nucl. Med. 2023, 64 (8), 1195–1202. 10.2967/jnumed.122.265248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pereira P. M. R.; Mandleywala K.; Ragupathi A.; Lewis J. S. Acute Statin Treatment Improves Antibody Accumulation in EGFR- and PSMA-Expressing Tumors. Clin. Cancer Res. 2020, 26 (23), 6215–6229. 10.1158/1078-0432.CCR-20-1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sekhar S. C.; Kasai T.; Satoh A.; Shigehiro T.; Mizutani A.; Murakami H.; El-Aarag B. Y. A.; DS S.; Salomon D. S.; Massaguer A.; de Llorens R. Identification of Caveolin-1 as a Potential Causative Factor in the Generation of Trastuzumab Resistance in Breast Cancer Cells. J. Cancer 2013, 4 (5), 391–401. 10.7150/jca.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Brown E. L.; Shmuel S.; Mandleywala K.; Panikar S. S.; Berry N. K.; Rao Y.; Zidel A.; Lewis J. S.; Pereira P. M. R. Immuno-PET Detects Antibody-Drug Potency on Coadministration with Statins. J. Nucl. Med. 2023, 64 (10), 1638–1646. 10.2967/jnumed.122.265172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rao Y.; Samuels Z.; Carter L. M.; Monette S.; Panikar S. S.; Pereira P. M. R.; Lewis J. S. Statins enhance the efficacy of HER2-targeting radioligand therapy in drug-resistant gastric cancers. Proc. Natl. Acad. Sci. U.S.A. 2023, 120 (14), e2220413120 10.1073/pnas.2220413120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Akyildiz A.; Guven D. C.; Yildirim H. C.; Ismayilov R.; Yilmaz F.; Tatar O. D.; Chalabiyev E.; Kus F.; Yalcin S.; Aksoy S. Do statins enhance the antitumor effect of trastuzumab emtansine (T-DM1)?: Real-life cohort. Medicine 2023, 102 (18), e33677. 10.1097/Md.0000000000033677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pereira P. M. R.; Ragupathi A.; Shmuel S.; Mandleywala K.; Viola N. T.; Lewis J. S. HER2-Targeted PET Imaging and Therapy of Hyaluronan-Masked HER2-Overexpressing Breast Cancer. Mol. Pharmaceutics 2020, 17 (1), 327–337. 10.1021/acs.molpharmaceut.9b01091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sung M.; Tan X.; Lu B.; Golas J.; Hosselet C.; Wang F.; Tylaska L.; King L.; Zhou D.; Dushin R.; et al. Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1). Mol. Cancer Ther. 2018, 17 (1), 243–253. 10.1158/1535-7163.MCT-17-0403. [DOI] [PubMed] [Google Scholar]
  27. Sekhar S. C.; Kasai T.; Satoh A.; Shigehiro T.; Mizutani A.; Murakami H.; El-Aarag B. Y. A.; DS S.; Salomon D. S.; Massaguer A.; de Llorens R. Identification of Caveolin-1 as a Potential Causative Factor in the Generation of Trastuzumab Resistance in Breast Cancer Cells. J. Cancer 2013, 4 (5), 391–401. 10.7150/jca.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Chung Y. C.; Kuo J. F.; Wei W. C.; Chang K. J.; Chao W. T. Caveolin-1 Dependent Endocytosis Enhances the Chemosensitivity of HER-2 Positive Breast Cancer Cells to Trastuzumab Emtansine (T-DM1). PLoS One 2015, 10 (7), e0133072 10.1371/journal.pone.0133072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Barretina J.; Caponigro G.; Stransky N.; Venkatesan K.; Margolin A. A.; Kim S.; Wilson C. J.; Lehar J.; Kryukov G. V.; Sonkin D.; et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012, 483 (7391), 603–607. 10.1038/nature11003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Fischer G.; Seibold U.; Schirrmacher R.; Wangler B.; Wangler C. 89Zr, a radiometal nuclide with high potential for molecular imaging with PET: chemistry, applications and remaining challenges. Molecules 2013, 18 (6), 6469–6490. 10.3390/molecules18066469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van de Watering F. C. J.; Rijpkema M.; Perk L.; Brinkmann U.; Oyen W. J.; Boerman O. C. Zirconium-89 labeled antibodies: a new tool for molecular imaging in cancer patients. Biomed Res. Int. 2014, 2014, 203601 10.1155/2014/203601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sharma S. K.; Glaser J. M.; Edwards K. J.; Khozeimeh Sarbisheh E.; Salih A. K.; Lewis J. S.; Price E. W. A Systematic Evaluation of Antibody Modification and 89Zr-Radiolabeling for Optimized Immuno-PET. Bioconjug. Chem. 2021, 32 (7), 1177–1191. 10.1021/acs.bioconjchem.0c00087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Janjigian Y. Y.; Viola-Villegas N.; Holland J. P.; Divilov V.; Carlin S. D.; Gomes-DaGama E. M.; Chiosis G.; Carbonetti G.; de Stanchina E.; Lewis J. S. Monitoring afatinib treatment in HER2-positive gastric cancer with 18F-FDG and 89Zr-trastuzumab PET. J. Nucl. Med. 2013, 54 (6), 936–943. 10.2967/jnumed.112.110239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. O’Donoghue J. A.; Lewis J. S.; Pandit-Taskar N.; Fleming S. E.; Schoder H.; Larson S. M.; Beylergil V.; Ruan S.; Lyashchenko S. K.; Zanzonico P. B.; et al. Pharmacokinetics, Biodistribution, and Radiation Dosimetry for 89Zr-Trastuzumab in Patients with Esophagogastric Cancer. J. Nucl. Med. 2018, 59 (1), 161–166. 10.2967/jnumed.117.194555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ulaner G. A.; Hyman D. M.; Ross D. S.; Corben A.; Chandarlapaty S.; Goldfarb S.; McArthur H.; Erinjeri J. P.; Solomon S. B.; Kolb H.; et al. Detection of HER2-Positive Metastases in Patients with HER2-Negative Primary Breast Cancer Using 89Zr-Trastuzumab PET/CT. J. Nucl. Med. 2016, 57 (10), 1523–1528. 10.2967/jnumed.115.172031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ulaner G. A.; Hyman D. M.; Lyashchenko S. K.; Lewis J. S.; Carrasquillo J. A. 89Zr-Trastuzumab PET/CT for Detection of Human Epidermal Growth Factor Receptor 2-Positive Metastases in Patients With Human Epidermal Growth Factor Receptor 2-Negative Primary Breast Cancer. Clin Nucl. Med. 2017, 42 (12), 912–917. 10.1097/RLU.0000000000001820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lumish M. A.; Maron S. B.; Paroder V.; Chou J. F.; Capanu M.; Philemond S.; O’Donoghue J. A.; Schöder H.; Lewis J. S.; Lyashchenko S. K.; et al. Noninvasive Assessment of Human Epidermal Growth Factor Receptor 2 (HER2) in Esophagogastric Cancer Using 89Zr-Trastuzumab PET: A Pilot Study. J. Nucl. Med. 2023, 64 (5), 724–730. 10.2967/jnumed.122.264470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Woo S. K.; Jang S. J.; Seo M. J.; Park J. H.; Kim B. S.; Kim E. J.; Lee Y. J.; Lee T. S.; An G. I.; Song I. H.; et al. Development of 64Cu-NOTA-Trastuzumab for HER2 Targeting: A Radiopharmaceutical with Improved Pharmacokinetics for Human Studies. J. Nucl. Med. 2019, 60 (1), 26–33. 10.2967/jnumed.118.210294. [DOI] [PubMed] [Google Scholar]
  39. Oh D. Y.; Bang Y. J. HER2-targeted therapies - a role beyond breast cancer. Nat. Rev. Clin Oncol 2020, 17 (1), 33–48. 10.1038/s41571-019-0268-3. [DOI] [PubMed] [Google Scholar]
  40. Rüschoff J.; Hanna W.; Bilous M.; Hofmann M.; Osamura R. Y.; Penault-Llorca F.; van de Vijver M.; Viale G. HER2 testing in gastric cancer: a practical approach. Modern Pathol. 2012, 25 (5), 637–650. 10.1038/modpathol.2011.198. [DOI] [PubMed] [Google Scholar]
  41. Gebhart G.; Lamberts L. E.; Wimana Z.; Garcia C.; Emonts P.; Ameye L.; Stroobants S.; Huizing M.; Aftimos P.; Tol J.; et al. Molecular imaging as a tool to investigate heterogeneity of advanced HER2-positive breast cancer and to predict patient outcome under trastuzumab emtansine (T-DM1): the ZEPHIR trial. Ann. Oncol. 2016, 27 (4), 619–624. 10.1093/annonc/mdv577. [DOI] [PubMed] [Google Scholar]
  42. Mortimer J. E.; Bading J. R.; Park J. M.; Frankel P. H.; Carroll M. I.; Tran T. T.; Poku E. K.; Rockne R. C.; Raubitschek A. A.; Shively J. E.; Colcher D. M. Tumor Uptake of 64Cu-DOTA-Trastuzumab in Patients with Metastatic Breast Cancer. J. Nucl. Med. 2018, 59 (1), 38–43. 10.2967/jnumed.117.193888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sanchez-Vega F.; Hechtman J. F.; Castel P.; Ku G. Y.; Tuvy Y.; Won H.; Fong C. J.; Bouvier N.; Nanjangud G. J.; Soong J.; et al. EGFR and MET Amplifications Determine Response to HER2 Inhibition in ERBB2 -Amplified Esophagogastric Cancer. Cancer Discovery 2019, 9 (2), 199–209. 10.1158/2159-8290.CD-18-0598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yoon J. K.; Park B. N.; Ryu E. K.; An Y. S.; Lee S. J. Current Perspectives on 89Zr-PET Imaging. Int. J. Mol. Sci. 2020, 21 (12), 4309. 10.3390/ijms21124309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mortimer J. E.; Bading J. R.; Frankel P. H.; Carroll M. I.; Yuan Y.; Park J. M.; Tumyan L.; Gidwaney N.; Poku E. K.; Shively J. E.; Colcher D. M. Use of 64Cu-DOTA-Trastuzumab PET to Predict Response and Outcome of Patients Receiving Trastuzumab Emtansine for Metastatic Breast Cancer: A Pilot Study. J. Nucl. Med. 2022, 63 (8), 1145–1148. 10.2967/jnumed.121.262940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Keyaerts M.; Xavier C.; Heemskerk J.; Devoogdt N.; Everaert H.; Ackaert C.; Vanhoeij M.; Duhoux F. P.; Gevaert T.; Simon P.; et al. Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma. J. Nucl. Med. 2016, 57 (1), 27–33. 10.2967/jnumed.115.162024. [DOI] [PubMed] [Google Scholar]
  47. Altena R.; Buren S. A.; Blomgren A.; Karlsson E.; Tzortzakakis A.; Brun N.; Moein M. M.; Jussing E.; Frejd F. Y.; Bergh J.; et al. Human Epidermal Growth Factor Receptor 2 (HER2) PET Imaging of HER2-Low Breast Cancer with [68Ga]Ga-ABY-025: Results from a Pilot Study. J. Nucl. Med. 2024, 65 (5), 700–707. 10.2967/jnumed.123.266847. [DOI] [PubMed] [Google Scholar]
  48. Zhou N.; Guo X.; Yang M.; Zhu H.; Yang Z. 68Ga-ZHER2 PET/CT Reveals HER2-Positive Metastatic Gastric Cancer With Better Image Quality Than 18F-FDG. Clin Nucl. Med. 2020, 45 (2), e101–e102. 10.1097/RLU.0000000000002859. [DOI] [PubMed] [Google Scholar]
  49. Yun W. S.; Kim J.; Lim D. K.; Kim D. H.; Jeon S. I.; Kim K.. Recent Studies and Progress in the Intratumoral Administration of Nano-Sized Drug Delivery Systems. Nanomaterials 2023, 13 ( (15), 2225. 10.3390/nano13152225. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mp4c00777_si_001.pdf (1.3MB, pdf)

Articles from Molecular Pharmaceutics are provided here courtesy of American Chemical Society

RESOURCES