Thyroid Cancer Imaging In Vivo by Targeting the Anti-Apoptotic Molecule Galectin-3

Background The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. Methods The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific 99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 ?Ci of 99mTc-labeled mAb to galectin-3 (30 ?g protein/in 100 ?l saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. Findings Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. Conclusions These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed

ApoD Mediates Binding of HDL to LDL and to Growing T24 Carcinoma

<div><p>Apolipoprotein (Apo) D is an important protein produced in many parts of the body. It is necessary for the development and repair of the brain and protection from oxidative stress. The purpose of this study was to investigate the extent to which apoD interacts with lipoproteins in human plasma. By using detergent-free ELISA, we show that immobilized monoclonal antibodies against apoD very efficiently bind to low density lipoprotein (LDL) from plasma; this binding is as equally efficient as binding to an anti-apoB monoclonal antibody. Adding detergent to the plasma inhibited the binding, suggesting that the binding is dependent on the presence of intact lipoprotein particles. Reversing the system by using immobilized anti-apoB revealed that the affinity of apoD for LDL is rather low, suggesting that multiple bindings are needed for a durable connection. Biosensor experiments using purified lipoproteins also showed that purified apoD and high density lipoprotein 3 (HDL3), a lipoprotein fraction rich in apoD, were both able to bind LDL very efficiently, indicating that the HDL3-LDL interaction may be a physiological consequence of the affinity of apoD for LDL. Furthermore, we found that apoD increases the binding of HDL to actively growing T24 bladder carcinoma cells but not to quiescent, contact-inhibited, confluent T24 cells. This result is especially intriguing given that the T24 supernatant only contained detectable levels of apoD after growth inhibition, raising the possibility that alternating the expression of apoD and a putative apoD-receptor could give direction to the flow of lipids. In the current paper, we conclude that apoD mediates binding of HDL to LDL and to growing T24 carcinomas, thereby highlighting the importance of apoD in lipid metabolism.</p></div

Biosensor monitoring of apoD-mediated binding of LDL.

<p>In the Blitz biosensor analysis, the signal corresponds to the mass collected on the biosensor surface. Here we used Streptavidin-coated sensor surfaces that were loaded with biotinylated antibody (10 µg/ml for 120 seconds) followed by apoD (2 µg/ml for 300 seconds) or buffer-control (300 seconds). The surfaces were subsequently loaded with the LDL, HDL2 or HDL3 lipoprotein particles (100 µg/ml for 300 seconds). Between loading reagents, the sensors were rinsed for 30 seconds. The buffer used for all steps and dilutions was PBS, azide, 0.1% BSA. <b>A</b>) The green, black, grey and red lines represent experiments preloaded with anti-apoD D544. The blue line indicates the control preloaded with isotype control antibody 7B6 (anti-IFNγ). The green and blue lines indicate that ApoD was preloaded, while the black, grey, and red lines indicate the buffer controls. The panel shows the final step in which the lipoprotein particles bind to the antibody loaded with or without apoD. The green, black and blue lines show binding of LDL, the grey line shows binding of HDL3 and the red line shows binding of HDL2. <b>B</b>) The binding step of the biotinylated antibody was omitted. Green, black, red and grey lines indicate experiments that used D544-biotin, and the blue line indicates that E981-biotin (anti-apoE) was used. Subsequent reactions show the addition of HDL2 in red, HDL3 in grey, apoD in green and blue, or buffer in black to the preloaded antibodies. In the last step, LDL was loaded in all the conditions.</p

Detergent-free dual-specific ELISA.

<p>The absence of detergent maintains the structure of intact lipoprotein particles. The particles are immobilized using one specific antibody and the content is detected by using another.</p

Non-confluent T24 cells stain positive for apoD.

<p>Cells were seeded on objective glasses in petri dishes. After either 2 days (non-confluent) or 9 days (confluent), the cells were washed and incubated with a negative isotype-control mAb (anti-IL4), a positive control anti-thioredoxin reductase mAb (anti-Trxred), or anti-apoD D544 mAb. Bound monoclonal antibodies were stained with polyclonal anti-mouse FITC-conjugated Fab fragments. Nuclei were visualized with DAPI. Cells were photographed with a Leica DMRB fluorescent microscope (shutter speed FITC 2 sec, and DAPI 1/30sek). Staining was repeated three times.</p

Anti-apoD antibodies capture ApoB in a detergent-free dual specific ELISA.

<p>The following capture antibodies functional under detergent-free conditions were used: A) anti-apoD (D544), B) anti-apoB (LDL17), C) anti-apoE (E981), D) anti-apoH (H219), E) anti-apoB (LDL20) and F) anti-apoJ (J29). Human EDTA finger blood plasma, prepared as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115180#s2" target="_blank">methods</a>, was diluted with PBS containing 0.1% BSA. Detecting antibodies in A–D were LDL20-biotin (anti-apoB), D263-biotin (anti-apoD), H464-biotin (anti-apoH), 7B6-biotin (anti-IFNγ) and E981 (anti-apoE), and in E and F were D201-biotin (anti-apoD), D263-biotin (anti-apoD), LDL17-biotin (anti-apoB) and J84-biotin (anti-apoD). Note that E981 was also used for capture in C). The means ± SD of four replicates are shown. Results within each panel are from the same donor. Experiments A–D were repeated three times using the same donor, and experiments E and F were repeated four times using four different donors.</p

HDL3 and purified apoD facilitates the binding of LDL to D544.

<p>Anti-apoD (D544) was used as the capture antibody and anti-apoB (LDL20-biotin) was used as the detection antibody in a detergent free dual-specific ELISA. LDL (333 ng/ml) was added either alone or in combination with ApoA1 (100 ng/ml), HDL2 (1000 ng/ml), HDL3 (1000 ng/ml), apoD (native) (100 ng/ml) or recombinant apoD (100 ng/ml). As a the control, LDL was omitted, but the same amounts of apoA1, HDL2, HDL3, apoD or recombinant apoD were added. The control without LDL is shown in grey bars. Relative absorbance was calculated by dividing the absorbance value by the absorbance of LDL alone. The experiment was repeated 6 times, each time using four replicates, and mean of the 6 experiments ± SD is shown. ** p<0.01 compared with LDL alone; p-values were calculated using the Mann-Whitney test with GraphPad prism 6.</p

ApoD production in T24 cells was assayed by ELISA for culture supernatant and by FACS for intracellular staining.

<p><b>A</b>) T24 cells were grown in DMEM with 10% FBS in 25 cm² flasks. Three flasks each of confluent cells (2.5 million in 9 ml) and non-confluent cells (1.5 million in 7 ml) were sampled 4 times. ApoD content was assayed by apoD ELISA; detection limit  = 0.1 ng/ml. <b>B</b>) D544 (anti apoD) was used to stain confluent (black) and non-confluent (grey) T24 carcinoma cells. The isotype control is depicted with a dotted black line for confluent T24 cells and with a dashed grey line for non-confluent T24 cells. Experiments were repeated three times.</p

The interaction between apoD and apoB is a general mechanism.

<p>Finger blood from ten donors was assayed for A) apoD/apoB interaction in a detergent free dual-specific ELISA using D544 for capture and biotinylated LDL20 for detection B) ELISA for apoB levels and C) ELISA for apoD levels. Note that finger blood plasma, in figures B and C, was arbitrary calculated as half the finger blood volume, with the remaining volume assumed to be cells. Of the ten donors, No. 1–5 were women, and No. 6–10 were men, all between 30–62 years old. Means ± SD of four replicates are shown.</p

ApoD facilitates binding of HDL to growing T24 cells.

<p>T24 cells grown in 24-well plates were washed with protein-free RPMI two times, and 200 µl/well RPMI supplemented with 0.1%BSA was added with or without HDL (total HDL, 200 ng/well) and with or without apoD (20 ng/well) or apoA1 (20 ng/well). After incubation for 1 hour at 21°C, the plate was washed three times with protein-free RPMI, and bound apoA1 was released using detergent and assayed by apoA1 ELISA. In <b>A</b>) the cells were non-confluent and in <b>B</b>) the cells were confluent. Means ± SD of four experiments are shown. ** p<0.01, ****p<0.0001; p-values were calculated using two-way ANOVA with Holm-Sidal multiple comparison test, GraphPad Prism 6.</p