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

Abstract

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

Abbreviations

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; MWB_evacizumab: Molecular Weight of Bevacizumab; e _cy7: Extinction Coefficient of Cy7 at Abs₇₄₇

Introduction

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 (Avastin^TM 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].

Results

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.

Figure 1: Flow cytometry assay. Expressions levels of VEGF in MM1S and Toledo cell lines were assessed by flow cytometry using FITC-Bevacizumab. Cell Surface (A.1 and B.1) and intracellular (A.2 and B.2) staining is shown.

    


    


    Figure 1: Flow cytometry assay. Expressions levels of VEGF in MM1S and Toledo cell lines were assessed by flow cytometry using FITC-Bevacizumab. Cell

Surface (A.1 and B.1) and intracellular (A.2 and B.2) staining is shown.

    

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In vivo biodistribution studies

In vivo biodistribution studies in healthy male BALB/c, MM1S and Toledo male BALB/c nude tumor-bearing mice are showed in Figure 2-4.

Figure 2: Healthy BALB/c and MM1S and Toledo tumor-bearing BALB/c nude mice biodistribution studies of ^99mTc-HYNIC-Bevacizumab at 6 and 24 h p.i. Values are expressed as % ID per gram (or % ID/g) (mean ± SD, n=5).

    


    


    Figure 2: Healthy BALB/c and MM1S and Toledo tumor-bearing BALB/c nude mice biodistribution studies of 99mTc-HYNIC-Bevacizumab at 6 and 24 h p.i. Values

are expressed as % ID per gram (or % ID/g) (mean ± SD, n=5).

    

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Figure 3: Healthy BALB/c and MM1S and Toledo tumor-bearing BALB/c nude mice biodistribution studies of ^99mTc-HYNIC-Bevacizumab at 6 and 24 h p.i. Values are expressed as % ID (or % ID) (mean ± SD, n=5).

    


    


    Figure 3: Healthy BALB/c and MM1S and Toledo tumor-bearing BALB/c nude mice biodistribution studies of 99mTc-HYNIC-Bevacizumab at 6 and 24 h p.i. Values

are expressed as % ID (or % ID) (mean ± SD, n=5).

    

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Figure 4: Tumor-to-blood and tumor-to-muscle ratios of ^99mTc-HYNIC-Bevacizumab in MM1S and Toledo tumor-bearing BALB/c nude mice at 6 and 24 h p.i.

    


    


    Figure 4: Tumor-to-blood and tumor-to-muscle ratios of 99mTc-HYNIC-Bevacizumab in MM1S and Toledo tumor-bearing BALB/c nude mice at 6 and 24 h p.i.

    

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Blood radioactivity levels in healthy mice were 25.44 ± 2.78 %ID/g and 11.04 ± 2.83 %ID/g at 6 and 24 h p.i., respectively. MM1S tumorbearing BALB/c nude mice blood radioactivity levels were 15.55 ± 4.48 %ID/g and 10.43 ± 0.93 %ID/g at 6 and 24 h p.i., respectively. Toledo tumor-bearing BALB/c nude mice blood radioactivity levels were 18.74 ± 1.62 ID/g and 14.34 ± 2.64 ID/g at 6 and 24 h p.i, respectively. This shows a slow clearance of radiolabeled antibody from the blood.

Liver radioactivity levels in healthy mice were 8.50 ± 1.62 %ID/g and 10.25 ± 2.25 %ID/g at 6 and 24 h p.i, respectively. MM1S tumorbearing BALB/c nude mice liver radioactivity levels were 7.59 ± 0.43 %ID/g and 3.93 ± 0.56 %ID/g at 6 and 24 h p.i., respectively. Toledo tumor-bearing BALB/c nude mice liver radioactivity levels were 8.96 ± 2.48 ID/g and 4.25 ± 1.07 ID/g at 6 and 24 h p.i, respectively. Also, kidney radioactivity levels in healthy mice were 7.77 ± 1.13 %ID/g and 4.98 ± 2.61 %ID/g at 6 and 24 h p.i, respectively. MM1S tumor-bearing BALB/c nude mice kidney radioactivity levels were 7.03 ± 0.35 %ID/g and 3.79 ± 0.05 %ID/g at 6 and 24 h p.i., respectively. Toledo tumorbearing BALB/c nude mice kidney radioactivity levels were 9.31± 1.40 %ID/g and 5.23 ± 2.13 %ID/g at 6 and 24 h p.i, respectively. Liver and kidney uptake were related to radiolabeled antibody clearance. Also, gastrointestinal radioactivity levels in healthy and MM1S and Toledo tumor-bearing BALB/C nude was present related to hepatic clearance of the antibody. At 24h p.i. (19.59 ± 5.00 %ID, 32.01 ± 5.96 %ID and 11.25 ± 1.50 %ID) of ^99mTc-HYNIC-Bevacizumab had been excreted in the urine. Less than 4 %ID/g was present in muscle, bone, thyroid and stomach at all analyzed time points.

MM1S tumor-bearing BALB/c nude mice lungs and intestine radioactivity levels were 6.87 ± 2.66 %ID/g and 3.10± 0.15 %ID/g, 4.97 ± 0.05 and 1.61 ± 0.61 %ID/g at 6 and 24 h p.i, respectively. Toledo tumor-bearing BALB/c nude mice lungs and intestine radioactivity levels were 12.90 ± 4.10 and 3.02 ± 0.49 %ID/g, and 5.56 ± 1.17 and 1.47 ± 0.31 %ID/g at 6 and 24 h p.i, respectively.

MM1S and Toledo tumor-bearing BALB/c nude mice reveled relevant ^99mTc-HYNIC-Bevacizumab tumor uptake and retention. Tumor uptake in MM1S tumor-bearing BALB/c nude mice were 2.64 ± 0.22% ID/g and 2.91 ± 0.50 % ID/g at 6 and 24 h p.i, respectively. Tumor uptake in Toledo tumor-bearing BALB/c nude mice were 6.81 ± 0.87% ID/g and 8.76 ± 3.70 % ID/g at 6 and 24 h p.i., respectively.

MM1S tumor-bearing BALB/c nude mice showed ^99mTc-HYNICBevacizumab tumor-to-muscle ratios of 3.13 and 3.42 at 6 and 24 h, respectively. Toledo tumor-bearing BALB/c nude mice showed tumor-to-muscle ratios of 6.88 and 7.92 at 6 and 24 h, respectively.

Bevacizumab-Cy7 conjugation

The concentration of Cy7-Bevacizumab obtained was 2.04 mg/ mL, with a Cy7/Bevacizumab ratio of 2.6.

Molecular imaging

SPECT/CT imaging: ^99mTc-HYNIC-Bevacizumab SPECT/CT images showed an irregular liver uptake in healthy and MM1S and Toledo tumor-bearing mice at 6 and 24 h p.i. (Figures 5 and 6). Beside liver uptake there were significant heart, spleen, kidney, bladder, lungs and intestine uptake at 6 h p.i. similar to our biodistribution findings (Figure 5.A and 6.A). Discrete tumor uptake was also evident in the back of the mice where MM cells were inoculated and in the lymph nodes in the NHL model (Figure 5.B and 6.B). Tumor visualization was possible at 6 and 24 h, respectively.

Figure 5: SPECT/CT axial, sagital and coronal images at 6 and 24 h post-injection of ^99mTc-HYNIC-Bevacizumab on healthy (A) and MM1S tumor-bearing BALB/c nude mice (B). BALB/c nude mice SPECT/CT image shows non homogeneous heart, spleen, liver, kidney, bladder and gastrointestinal uptake and MM1S tumorbearing BALB/c nude mice SPECT/CT shows a remarkable uptake of the radiolabeled Bevacizumab in the site of cell inoculation (tumor uptake, yellow arrows).

    


    


    Figure 5: SPECT/CT axial, sagital and coronal images at 6 and 24 h post-injection of 99mTc-HYNIC-Bevacizumab on healthy (A) and MM1S tumor-bearing BALB/c

nude mice (B). BALB/c nude mice SPECT/CT image shows non homogeneous heart, spleen, liver, kidney, bladder and gastrointestinal uptake and MM1S tumorbearing

BALB/c nude mice SPECT/CT shows a remarkable uptake of the radiolabeled Bevacizumab in the site of cell inoculation (tumor uptake, yellow arrows).

    

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Figure 6: SPECT/CT axial and coronal images at 6 and 24 h post-injection of ^99mTc-HYNIC-Bevacizumab on healthy (A) and Toledo tumor-bearing BALB/c nude mice (B). BALB/c nude mice SPECT/CT image shows non homogeneous heart, spleen, liver, kidney, bladder and gastrointestinal uptake and Toledo tumor-bearing BALB/c nude mice SPECT/CT shows a remarkable uptake of the radiolabeled Bevacizumab in the lymph nodes (tumor uptake, yellow arrows).

    


    


    Figure 6: SPECT/CT axial and coronal images at 6 and 24 h post-injection of 99mTc-HYNIC-Bevacizumab on healthy (A) and Toledo tumor-bearing BALB/c nude

mice (B). BALB/c nude mice SPECT/CT image shows non homogeneous heart, spleen, liver, kidney, bladder and gastrointestinal uptake and Toledo tumor-bearing

BALB/c nude mice SPECT/CT shows a remarkable uptake of the radiolabeled Bevacizumab in the lymph nodes (tumor uptake, yellow arrows).

    

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Fluorescent imaging: Real-time imaging studies using Cy7- Bevacizumab corroborated the previously quantified biodistribution results (Figure 7). Two MM1S and Toledo tumor-bearing mice were injected intravenously with 100μg of Cy7-Bevacizumab and were followed and quantified at 2, 24, 48, 72 and 96 h. Healthy BALB/c nude mice were used as control. Images showed clear liver and tumor (site of MM1S inoculation and lymph nodes) uptake of Cy7- Bevacizumab fluorescence (Figure 7). Also, it was possible to observe increased retention and fluorescence of Cy7-Bevacizumab in the tumor in both models up to 96h p.i. Ex-vivo analysis at 96h p.i. in Toledo tumor-bearing mice reveled a significant retention of Cy7- Bevacizumab in the lymph nodes corroborates the biodistribution results and SPECT/CT images.

Figure 7: Fluorescence imaging. Healthy BALB/c nude mice (control) and MM1S and Toledo tumor-bearing BALB/c nude mice fluorescence imaging showed clear liver (white arrows) and tumor uptake (site of MM1S line cells inoculation and lymph nodes, yellow arrows) of Cy7-Bevacizumab fluorescence. Also, it was possible to observe increased retention and fluorescence of Cy7-Bevacizumab in tumor in both models up to 96h p.i. Ex-vivo fluorescence imaging of lymph node at 96h p.i. in Toledo tumor-bearing mice reveled a significant retention of Cy7-Bevacizumab corroborates the biodistribution results and SPECT/CT images.

    


    


    Figure 7: Fluorescence imaging. Healthy BALB/c nude mice (control) and MM1S and Toledo tumor-bearing BALB/c nude mice fluorescence imaging showed clear

liver (white arrows) and tumor uptake (site of MM1S line cells inoculation and lymph nodes, yellow arrows) of Cy7-Bevacizumab fluorescence. Also, it was possible

to observe increased retention and fluorescence of Cy7-Bevacizumab in tumor in both models up to 96h p.i. Ex-vivo fluorescence imaging of lymph node at 96h p.i.

in Toledo tumor-bearing mice reveled a significant retention of Cy7-Bevacizumab corroborates the biodistribution results and SPECT/CT images.

    

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Discussion

Tumor VEGF in vivo real time imaging expression has the potential to open the path to novel antiangiogenic diagnostic and therapeutic options. In this way, we evaluate in vivo VEGF expression in liquid hematological tumors such as MM and LNH through ^99mTc via HYNIC as bifunctional chelating agent and Cy7-labeled Bevacizumab. We designed and evaluated ^99mTc-HYNIC and Cy7 Bevacizumab as a specific LNH and MM imaging agent, taking into account ^99mTc widespread availability and our experience labeling antibodies with ^99mTc-HYNIC [68-70,90-94].

MM1S multiple myeloma and Toledo non-Hodgkin’s lymphoma cell lines have been reported to express VEGF [98-101], so these cell lines were used for in vitro characterization of FITC-labeled Bevacizumab to confirm its VEGF binding affinity and specificity. Flow cytometric analysis using FITC-Bevacizumab showed differences in the level of expression between membrane-bound and intracellular VEGF, clearly demonstrating that in the cell lines tested, this factor is mainly expressed intracellularly. These results allowed us to perform in vivo studies and for this purpose we developed MM and NHL tumor models based on the induction of MM1S and Toledo cell lines in female BALB/c nude mice.

Biodistribution studies with ^99mTc-HYNIC-Bevacizumab in healthy and tumor-bearing BALB/c mice show, that blood clearance is slow and that a longer-lived radioisotope would be an interesting option to work with. Although until now, we are not aware that there were any prior studies that combined MM, NHL and ^99mTc-HYNICBevacizumab. We must remember that these are liquid tumors, not solid ones, and SPECT-CT imaging of a tumor-bearing Balb/C Nude mice does not exactly reflect what happens in these diseases and their distributions, but they are a standardized model to work with. From our results we can see that supradiafragmatic, head and axial and appendicular skeleton can be evaluated without much interference. Abdomen, due to radiotracer elimination has its limitations to image interpretation that could be enhanced with hybrid SPECT/CT imaging. These results were also seen in the biodistributions studies in both tumor models. Tumor-to-muscle ratio increased with time and are similar to the ones reported with other ^99mTc-radiolabeled antibodies [69,91,95,96].

We also performed in vivo VEGF expression of these liquid tumors using Cy7-Bevacizumab. Labeling and images allowed us to visualize these tumors with greater spatial resolution than SPECT.

We have already reported ^99mTc-labeled Bevacizumab in solid tumors [68-70] and also Cy7-Bevacizumab in LNH [97], and we would be very interested in performing a hybrid (double-labeled) agent, and thus combine the strengths of both modalities to apply the concepts of Guided intraOperative Scintigraphic Tumor Targeting and Guided Hybrid Intra Operative Specific Targeting for the assessment of VEGF expression levels associated with NHL and MM [102-110].

We believe that these fluorescent and radiolabeled antibodies have great diagnostic and therapeutic potential that has yet to be explored. These are the first steps towards it implementation, our results are promising, the work is not finished but show that it is feasible although possible limitations due to suboptimal ^99mTc properties.

Conclusion

MM and LNH is a deadly, frequent disease, and molecular imaging is used to monitor their progression.

The development of specific targeting probes such as Bevacizumab that targets VEGF opens the path to new ways to understand this disease, and provide novel diagnostic and therapeutic options. We successfully developed ^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.

Declaration

Ethics approval and consent to participate: All the procedures were approved by the Ethical Committee for Animal Research of the Faculdade de Medicina da Universidade de São Paulo (procedure approval number 279/12), Brazil, and the Uruguayan Animal Experimentation Ethics Committee (procedure approval number 240011-002308-14), Uruguay.

Funding: This work was supported by Agencia Nacional de Innovación e Investigación (Uruguay) [POS_NAC_2015_1_109490] and Comisión Sectorial de Investigación Científica-Universidad de la República (Uruguay) [240600-000148-18].

Acknowledgments: The authors are grateful to Roche^® for providing Bevacizumab, Pro.In.Bio (Uruguay), PEDECIBA Química (Uruguay), Agencia Nacional de Innovación e Investigación (Uruguay) and Comisión Sectorial de Investigación Científica - Universidad de la República (Uruguay).

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Citation: Camacho X, Perroni C, Carneiro CG, Junqueira MS, Machado CL, Faria D, et al. Molecular Imaging of VEGF Expression in Multiple Myeloma and Non-Hodgkin Lymphoma. J Mol Biol & Mol Imaging. 2022; 7(1): 1033.

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