Molecular Imaging of VEGF Expression in Multiple Myeloma and Non-Hodgkin Lymphoma

Research Article

J Mol Biol & Mol Imaging. 2022; 7(1): 1033.

Molecular Imaging of VEGF Expression in Multiple Myeloma and Non-Hodgkin Lymphoma

Camacho X1*, Perroni C1, Carneiro CG2, Junqueira MS2, Machado CL2, Faria D2, García MF1, Fernández M1, Buchpiguel CA2, Cerecetto H1, Chammas R3, Riva E4, Cabral P1 and Gambini JP5

1Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

2Nuclear Medicine Medical Investigation Laboratory LIM43, Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo-HCFMUSP, Sao Paulo, Brazil

3Laboratório de Oncologia Experimental, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Arnaldo N° 455- Cerqueira César - CEP: 01246903, São Paulo, Brazil

4Clínica Hematológica, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

5Centro de Medicina Nuclear e Imagenología Molecular, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

*Corresponding author: Camacho X, Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Mataojo 2055, P.O. 11400, Montevideo, Uruguay

Received: January 25, 2022; Accepted: February 21, 2022; Published: February 28, 2022


Angiogenesis is a crucial process in the growth, development, and metastasis of many tumor types, including Non-Hodgkin’s lymphoma (NHL) and Multiple Myeloma (MM). Vascular endothelial growth factor (VEGF) overexpression is known to be associated with poor prognosis in both pathologies, representing a rational target for anti-angiogenic therapy in NHL and MM. The monoclonal antibody Bevacizumab binds to VEGF with high affinity and blocks its action. We aim to evaluate Bevacizumab as a potential radioactive and fluorescence agent for imaging VEGF expression in MM and NHL.

Flow cytometry analysis revealed VEGF expression in MM and NHL cell lines is mainly intracellularly. Biodistribution and Single-photon emission computed tomography/computed tomography (SPECT/CT) studies of 99mTc-HYNICBevacizumab showed a slow blood clearance and supradiaphragmatic, head, axial and appendicular skeleton can be evaluated without much interference. Tumor-to-muscle ratio increased with time and is similar to the ones reported with other 99mTc-radiolabeled antibodies. Cy7-Bevacizumab fluorescent imaging allowed MM and NHL tumor visualization with greater spatial resolution than SPECT/CT.

We successfully synthesized 99mTc and Cy7-labeled anti-VEGF mAb (Bevacizumab) to be used to target VEGF expression in vivo in MM and LNH. Our encouraging results, although working with 99mTc, highlight the importance of radioinmuno-oncology as a potential tool to fight these diseases. Optical imaging of these tracers would enhance tumor sampling and guide surgical removal.

Keywords: Bevacizumab; Molecular Imaging; VEGF; Multiple Myeloma; Non-Hodgkin Lymphoma; 99mTechnetium- or Cy7-lableled Bevacizumab


NHL: Non-Hodgkin’s Lymphoma; MM: Multiple Myeloma; VEGF: Vascular Endothelial Growth Factor; SPECT/CT: Single- Photon Emission Computed Tomography/Computed Tomography; VEGFRs: Vascular Endothelial Growth Factor Receptors; RTKs: Recetor Tyrosine Kinases; NIR: Near Infrared; NHS-HYNIC-Tfa: Trifluoroacetyl Hydrazino-Protected Form of the Succinimidyl Ester of HYNIC; BCA: Bifunctional Chelating Agent; ATCC: American Type Culture Collection; PBS: Phosphate Buffered Saline; PFA: Paraformaldehyde; BSA: Bovine Serum Albumin; FITC: Fluorescein Isothiocyanate; RT: Room Temperature; SEC: Size-Size Exclusion Chromatography; MALDI TOF/TOF: Matrix- Assisted Laser Desorption/Ionization/Time-of-Flight; ITLC: Instant Thin Layer Chromatography; HPLC: High Performance Liquid Chromatography; % ID: Percentage of the Injected Dose; % ID/g: Percentage of the Injected Dose per Gram of Tissue; Cy7-NHS ester: Cy7-Monofunctional N-Hydroxysuccinimide ester; DMSO: Dimetyilsufoxide; MWBevacizumab: Molecular Weight of Bevacizumab; e cy7: Extinction Coefficient of Cy7 at Abs747


NHL and MM are lymphoproliferative diseases. VEGF overexpression occurs in many human tumors types, including lymphoproliferative disorders such as NHL and MM, which have been associated with poor prognosis [1-9].

VEGF family includes a large number of factors: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor. Each one of them has their own receptor specificities and biological properties. VEGF family most known factor is VEGF-A which has different variants (VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, VEGF189, and VEGF206), having each one diferente function and receptor specificity [10]. One of VEGF most important properties is to promote vascular endothelial cells growth and can prevent their apoptosis. It can also induce endothelial fenestration, modulating vascular permeability [11]. It has been established that many cytokines and grow factors could be responsible of VEGF mRNA expression upregulation or induce VEGF release [12]. Also VEGF has been shown to influence immune and cancer cells, although the exact mechanisms behind them are yet to be discovered [13].

Once VEGF role in angiogenesis was discovered, many inhibitors were developed in order to treat cancer [14-31]. In this way we can find anti-VEGF or anti-VEGFR monoclonal antibodies, small molecular inhibitors of recetor tyrosine kinases (RTKs) of VEGFRs [16,21,24,27,32-36]. One of the most popular anti VEGF antibodies is Bevacizumab (rhuMAb-VEGF, Avastin®, Genentech, USA) [37,38].

Some of these antiangiogenic molecules have been radiolabeled in order to produce a diagnostic and/or therapeutic agent [39-87], having the potential to detect emerging tumors, monitor the response to treatment and predict treatment outcomes as well as refer patients that could benefit from anti angiogenic therapy. These molecules can also be labeled with a fluorescent dye in order to provide highresolution, real-time imaging of VEGF tumor expression [72,73,83], allowing to guide surgeries. NIR fluorophores have been increasingly used in this setting due to their reasonable penetration with almost no tissue autofluorescence [88,89]. Taking these facts into consideration the aim of this work was to develop new potential radioactive and fluorescent agents for imaging VEGF expression in NHL and MM. To this end, we labeled Bevacizumab with Cy7 and with 99mTc via NHS-HYNIC-Tfa as BCA.

Materials and Methods

Cell culture

Human MM and B-cell NHL cell lines (MM1S and Toledo) were obtained from ATCC and from Banco de Células do Rio de Janeiro, respectively. All cell lines were grown in RPMI-1640, pH 7.4, supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100μg/mL streptomycin. All cells were maintained at 37°C in a 5% CO2 incubator.

Flow cytometry analyses

Bevacizumab (AvastinTM anti-VEGF monoclonal antibody) produced by Genetech, Inc., was provided by Roche Laboratories, Uruguay.

Surface staining: After culture disaggregation the cells were washed 3 times in PBS (5min, 600g) and fixed in 2% cold and freshly prepared PFA in PBS. Samples were then incubated at 4°C for 15min. Following cross-linking fixation, cells were blocked for 1h at 4°C with PBS-3% BSA, and then incubated with 2μg of Bevacizumab-FITC (2mg/mL) in PBS-1% BSA. After 2h of incubation in the dark at 37°C, the cells were washed 3 times in PBS (5min, 600g). Data were acquired in a FACSCALIBUR® flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Becton Dickinson & Company, Franklin Lakes, NJ, USA) [42].

Intracellular staining: After culture disaggregation in PBS, fixed in 2% PFA and washed, cells were permeabilized with 200μl of 0.2% (v/v) Tween-20 in PSA for 30min at 4°C. Then, cells were washed 3 times in PBS-1% BSA (5min, 600g) to remove 0.2% (v/v) Tween-20 from the medium, blocked for 1h at 4°C with PBS-BSA 3% and incubated with 2μg of Bevacizumab-FITC for1 h at 37°C in the dark. Then the cells were washed 3 times in PBS (5min, 600g). Data were acquired and analyzed as previously described in 2.2.1 section.

Controls for surface and intracellular staining included cells alone, isotype-FITC control (2μg for each batch) to determine autofluorescence levels, or unspecific reactions [42].

Linker Formation between HYNIC and Bevacizumab, radiolabeling with 99m-Technetium and quality controls

NHS-HYNIC-Tfa was synthesized and conjugated to Bevacizumab as previously described by our group [39-43,54]. Briefly, 0.067μmol of Bevacizumab was mixed at RT for 30min with 0.33μmol of NHS-HYNIC-Tfa. The conjugate was purified by SEC and used MALDI TOF/TOF lineal to determine the level of conjugation.

Radiolabeling Bevacizumab with 99m-Technetium and quality controls were performed as previously described by our group [39- 43,54]. For this purpose, 44.6mol of Tricine, 44.3mol of SnCl2.2H2O and 6.7nmol of antibody conjugate were mixed, and immediately a Na 99mTcO4 solution was added. The mixture was incubated at RT for 30min and the radiochemical purity was evaluated by ITLC and HPLC [39,41]. The integrity of radiolabeled Bevacizumab was analyzed by HPLC by incubation at 37°C in 0.9% NaCl, serum and in different concentrations of L-Cysteine.

Animals and tumor induction

Healthy male BALB/c and BALB/c nude mice, 8-10-weeks-old (20-24 g), were obtained from the Animal House Facility of the Universidad de la República, Uruguay and from Animal House Facility of the Faculdade de Medicina da Universidade de São Paulo. All animals were maintained in ventilated cages in ventilated racks with sterilized food and water ad libitum, in a 12/12 h light/dark cycle.

Toledo and MM1S cells at a 0.5 x 107 concentration (with at least 95% of viable cells) were subcutaneously injected in male BALB/c nude mice. The animals were followed daily for at least 1 month, evaluating tumor growth.

All procedures were in accordance with ethical principles adopted by Uruguayan Animal Experimentation Ethics Committee (procedure approval number 240011-002308-14) and Brazilian College of Animal Experimentation and approved by the Ethical Committee for Animal Research of the Faculdade de Medicina da Universidade de São Paulo (procedure approval number 279/12) [42,43,54].

In vivo biodistribution studies

99mTc-HYNIC-Bevacizumab biodistribution studies were performed on healthy male BALB/c mice and Toledo and MM1S tumor-bearing BALB/c nude mice (n=5 per group per time) as previously described by our group [42,43,54]. Briefly, animals were injected via intravenous tail with approximately 1.8MBq/100ug of radiolabeled Bevacizumab and euthanized by anesthetic drugs (xilazin-100mg/Kg and ketamin-300mg/Kg) after 2, 6 and 24 h. Selected tissues (heart, liver, lungs, thyroid, kidneys, stomach, spleen, gastrointestinal tract and bladder) were excised, rinsed of residual blood, weighed and their radioactivity measured in an dose Calibrator Capintec CRC7, Solid Scintillation counter with 3”x3” NaI(Tl) crystal detector associated to a ORTEC multichannel analyzer. Urine, blood, tumor (site of inoculation of the MM1S cell line in the MM model and lymph nodes in LNH model) were also collected together and measure. Organ activity was expressed as % ID and as % ID/g.

Bevacizumab-Cy7 conjugation

Bevacizumab-Cy7 conjugation was performed as previously described by our group [42,43]. Briefly, a solution of 500μL of Bevacizumab (0.5mg/mL) and 500μL of 0.15M NaCl was mixed and centrifuged at 14,000g for 10min at 4°C using a Centricom-30 ultrafiltration device. The buffer was then changed to 0.1M NaHCO3 (pH 8.3). One milliliter of Bevacizumab solution (0.5mg/mL) was mixed with a solution of Cy7-NHS ester diluted in DMSO. The reaction was carried out for 2h in complete darkness. To separate the free dye, the mixture was centrifuged at 14,000 xg for 10min at 4°C with a Centricom-30 ultrafiltration device and the sample was subsequently replaced with PBS.

The protein concentration in mg/mL was calculated according to the following formula: mg/mL Protein = (Abs280 - 0.04 x Abs747)/1.4

The ratio of Cy7: Bevacizumab in the final conjugate was calculated according to the following formula: ratio of Cy7/ Bevacizumab = (Abs747 x MWBevacizumab)/mg/mL Protein x e cy7 (e cy7 = 210000cm-1M-1)

Molecular imaging

SPECT/CT imaging: SPECT/CT images were performed on a μPET/SPECT/CT instrument (Triumph, Trifoil Imaging Inc.) as previously described by our group [42,43,54]. After 7 days of inoculation of MM1S and Toledo cell lines into female BALB/c nude mice, a mixture of 2-2.5% isoflurane and oxygen were used for anesthesia, followed by an intravenous tail injection of 99mTc-HYNICBevacizumab (100μg, 74-111 MBq/mice). After 6 and 24 h SPECT/ CT images were acquired with a five-pin hole collimator (0.8mm spatial resolution, 55 x 55 mm trans-axial field of view, 64 projections, FOV=46mm) and reconstructed with an OSEM filter (5 interactions with 8 subsets) correction in a 20% of a 99mTc-window followed by a DICOM generation by the Amira 4.1 software and the co-registration were analyzed by Amide software [55].

Fluorescent imaging: In vivo fluorescent imaging of MM1S and Toledo tumor-bearing mice with Cy7-Bevacizumab (100μg) was performed to assess tumor uptake up 96h p.i [42,43]. A healthy, Cy7- Bevacizumb uninjected Balb/c nude mouse was used as a control. Images were acquired with 745nm excitation and 800nm emission filters in an iVis Spectrum charge-couples device camera. Fluorescence images was quantified by total radiant efficiency quantification ((photons/s)/(μW/cm²)) using Living Image 4.3.1 software. During fluorescence examination, all animals were anesthetized with a 1-2% of isoflurane-oxygen mixture to enable imaging studies to be performed.

Statistical analysis

Data was analyzed using one-way ANOVA followed by Bonferroni post-hoc tests using GraphPad Prism 4.0 software. Differences were considered significant when p<0.5 [43,54].


Flow cytometry analyses

The VEGF expression levels of human MM1S and Toledo cells lines were analyzed by flow cytometry using FITC-Bevacizumab. Figure 1 shows the surface marker (A.1 and B.1) and intercellular profile (A.2 and B.2) of MM1S and Toledo cell lines. Therefore, since VEGF was mostly detected by intracellular staining, this confirms that its expression is at this level.