extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine Chemistry

text:
Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA-TATE Uptake in Neuroblastoma Xenograft Models

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro, NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for 131I-MIBG therapy, low 123I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68Ga-DOTA-TATE uptake and antitumor efficacy of 177Lu-DOTA-TATE. 68Ga-DOTA-TATE PET is superior to 123I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177Lu-DOTA-TATE therapy.

Introduction
Neuroblastoma (NB) is the most common extracranial childhood malignancy, responsible for 15% of all childhood cancer deaths [1]. Despite intensive treatment protocols including multimodal therapy with hematopoietic stem cell transplantation and immunotherapy, three-year disease-free survival is only about 60% for metastatic disease compared to 95% for localized tumors [2,3].
Somatostatin receptors (SSTRs) are expressed at relatively low levels in most organs. They are moderately expressed in the brain, gastrointestinal tract, pancreas, kidney, and spleen.
In contrast, SSTRs, especially SSTR2, have been shown to be highly expressed in various human tumors including pancreatic, small cell lung, and carcinoid tumors, as well as paraganglioma, pheochromocytoma, and neuroblastoma [4]. Georgantzi et al. demonstrated variable frequencies of somatostatin receptor (SSTR1-5) expression in 5 NB cell lines and 11 NB patient tumor biopsy samples [5], making molecular imaging and radionuclide therapy with somatostatinbased nuclear probes an attractive therapeutic option in appropriately selected patient populations [6].
DOTA-TATE is the somatostatin (cyclic peptide hormone) analog of Tyr3-octreotate (TATE) coupled with the 2 Contrast Media & Molecular Imaging macrocyclic chelator 1,4,7,10-tetraazacyclododecane,1,4,7,10tetraacetic acid (DOTA). DOTA-TATE is SSTR2 selective with a higher SSTR2 affinity (∼0.2 nM) in vitro comparing to two other commonly used somatostatin analogs [ 68 Ga-DOTA 0 -Tyr 3 ]octreotide (DOTA-TOC) and [DOTA 0 , 1NaI 3 ]octreotide (DOTA-NOC) [7]. Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs are an established and effective treatment option for adult patients with SSTR-positive neuroendocrine tumors [4]. DOTA-TOC and DOTA-TATE can also be radiolabeled with 90 Y or 177 Lu for targeted − -particle radionuclide therapy of neuroendocrine tumors. In a recent phase I trial, the safety and efficacy of 90 Y-DOTA-TOC therapy were demonstrated in 17 children and young adults with refractory SSTRpositive neuroendocrine tumors including NB [8]. The first study of 177 Lu-DOTA-TATE treatment in 35 patients with gastroenteropancreatic neuroendocrine tumors was published in 2003 where an objective response of 38% was achieved [9]. In a 2008 evaluation of 310 adult patients with neuroendocrine tumors, an overall response of 30% was reported [10]. More recently, a pilot clinical study demonstrated that 68 Ga-DOTA-TATE PET could be used to image children with relapsed or primary refractory high-risk NB, and 68 Ga-DOTA-TATE PET could be used to identify potential candidates for 177 Lu-DOTA-TATE treatment [11]. In this study, 6 out of 8 children demonstrated high uptake of 68 Ga-DOTA-TATE and proceeded to treatment. Patients received 2 or 3 administrations of 177 Lu-DOTA-TATE (0.3 GBq/kg; 8.1 mCi/kg per dose) at a median interval of 9 weeks and a median administered activity of 7.3 GBq. Five of these patients had the stable disease as assessed using the Response Evaluation Criteria in Solid Tumors (RECIST). This study, while limited in the number of patients studied, provided proof-of-principle that children with NB can be imaged and treated with somatostatin receptor-targeted agents. More interestingly, this study demonstrated that 1 patient (out of a series of 6 patients) whose disease was negative for 123 I-MIBG nevertheless demonstrated marked uptake of 68 Ga-DOTA-TATE.
The primary purpose of this study is to evaluate the uptake of 68 Ga-DOTA-TATE in NB xenograft models and correlate this uptake with the expression levels of SSTR2 and, therefore, identify biomarkers which can predict the therapeutic effects of 177 Lu-DOTA-TATE.

Materials and Reagents.
Gallium-68 and lutetium-177 radiolabeled DOTA-TATE were supplied by the Centre for Probe Development and Commercialization (CPDC, Hamilton, ON, Canada). 68 Ga-DOTA-TATE was produced with the specific activity of 41.2 ± 9.9 GBq/ mol and with the radiochemical purity of >97% at all cases. NET antibody (NET17-1) was purchased from MAb Technologies (Stone Mountain, GA), and SSTR2 antibody (ab134152) was obtained from Abcam (Cambridge, MA). Triton X-100, ethylenediaminetetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Company (St Louis, MO).

RT-PCR.
Total cellular RNA was prepared using Qiagen RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturers' instruction. Residual DNA was eliminated using the Qiagen RNase-Free DNase Set. cDNAs were synthesized from 2 g of RNA with the Superscript II6 Reverse Transcriptase (Invitrogen). PCR was performed using 1 L of cDNA in the PCR buffer supplemented with 0.2 mM of dNTP, 2.5 units of Taq polymerase (Biorad and ThermoFisher Scientific), and 0.5 M of each sense and antisense primer. The following primers were used: SSTR2 forward 5 -GGTGAAGTCCTCTGGAATCC-3 and reverse 5 -CCATTGCCAGTAGACAGAGC-3 ; NET forward 5 -CTCAAGGAGGCCACGGTATGGATCG-3 and reverse 5 -ACCTGGAAGTCATCAGCCAGTCCGG-3 ; GAPDH forward 5 -CTGTCCAGTTAATTTCTGACC-3 and reverse 5 -CTTTGTACATGGTATTCACCAC-3 . PCR products were run on a 1.5% agarose (Invitrogen) with a 100 bp marker (Thermo Fisher Scientific) and stained with ethidium bromide. Gel pictures were taken using the AlphaImager6 2200 (Alpha Innotech, Kasendorf, Germany).

Western Blot.
The protein lysates were analyzed by Western blot for SSTR2 and norepinephrine transporter (NET). Briefly, cells were lysed in lysis buffer and denatured. Samples were separated using 10% Bis-Tris precast gels (Invitrogen), followed by transfer to a PDVF membrane. After blocking, all membranes were incubated overnight at 4 ∘ C in TBST (Trisbuffered saline, 0.1% Tween 20) buffer containing the primary antibodies. Primary antibody complexes were then detected  using horseradish peroxidase-(HRP-) conjugated secondary antibodies. Protein bands were revealed with SuperSignal6 West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). NET protein expression in CHLA-15, SK-N-BE(2), and BE(2)C xenografts was quantified densitometrically using ImageJ software (NIH, USA) and normalized with respect to the corresponding expression of -actin.

Tissue Preparation for Western Blot.
Xenograft tumors were snap frozen in liquid nitrogen immediately after harvesting and stored at −80 ∘ C until ready for processing. Tumor tissue samples were homogenized in a RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5 mM EDTA, 1% Triton X-100, and 0.1% SDS) plus complete protease inhibitor cocktail (Complete Protease Inhibitor Tablets; Boehringer Mannheim, Ingelheim am Rhein, Germany). Homogenates were then centrifuged at 100,000 ×g for 45 minutes at 4 ∘ C.
The supernatants were assayed for protein content, aliquoted, and stored at −80 ∘ C. 25 g of lysate was subjected to future protein analysis by SDS-PAGE and Western blot analysis.

Mouse Xenograft Models.
All animal studies were approved by Animal Care Committee at the Hospital for Sick Children and at the University Health Network (Toronto, ON, Canada). Four-to 6-week-old, female, nonobese diabetic, severe combined immunodeficiency (NOD/SCID) mice were purchased from Jackson Laboratory (Bar Harbor, ME). CHLA-15, SK-N-BE(2) and BE(2)C cells were used to establish murine models. Briefly, tumor cells were washed three times with Hanks' Balanced Salt Solution (HBSS) before injection. Cell suspensions were mixed 1 : 1 with Growth Factor Reduced Matrigel Matrix (BD Bioscience, Mississauga, ON, Canada). Subcutaneous xenografts were developed by injecting 1 × 10 6 tumor cells subcutaneously into the dorsal upper flank of NOD/SCID mice.

2.7.
Micro-PET/CT Mouse Imaging. Animals were prepared for imaging such that when the tumor xenografts reach a diameter of approximately 1 cm, the mice were anesthetized with 2% isoflurane in the medical air (1.0 L/min) and injected intravenously (IV) via the tail vein [13] with 11.7 MBq ± 2.5 MBq of 68 Ga-DOTA-TATE. Images were acquired on a Focus 220 micro-PET scanner (Siemens Preclinical Solutions, Knoxville, TN) at 1 hour after injection using a Minerve imaging bed (Esternay, France) to maintain body temperature at 37 ∘ C. Images were reconstructed in a 256 × 256 matrix and a zoom of 6.5 using an ordered subset expectation maximization (OSEM), followed by a maximum a posteriori probability reconstruction algorithm with no attenuation correction. Quantification was performed by volumeof-interest (VOI) analysis using Inveon Research Workplace (IRW) software (Siemens). Tumor volume was obtained by summing multiple 2-dimensional regions of interest from consecutive tomographic planes encompassing the entire tumor volume on fused PET-CT slices. Tumor uptake was expressed as the mean ± SD percentage injected dose per gram (%ID/g). Immediately after small-animal PET, the Minerve imaging bed with the mouse was transferred to an eXplore Locus Ultra Preclinical CT scanner (GE Healthcare, London, ON, Canada). The micro-CT scan of the mouse was acquired with routine acquisition parameters (80 kV, 70 mA, 16 sec per rotation). PET and CT images were coregistered using IRW software. The micro-CT scan was used for anatomic referencing and for delineating the aforementioned VOIs.
To verify the accuracy of %ID values, a 10 mL specimen of 68 Ga with known radioactivity was scanned on the micro-PET. The image-derived concentration was compared with the concentration calculated from the same radioactivity measured by the radioisotope dose calibrator (Model CRC5 -15R, Capintec Inc., Ramsay, NJ). The difference in the two concentrations was less than 10%.
2.8. 123 I-MIBG SPECT/CT Imaging. 1 × 10 6 NB tumor cells were injected subcutaneously into the shoulder area of NOD/SCID mice. When tumor xenografts grew to an approximate diameter of 1 cm or more, each mouse was injected with 17.5 MBq ± 2.3 MBq of 123 I-MIBG into a lateral tail vein. Five to six hours after injection, both CT and SPECT imaging were performed using a preclinical nanoSPECT/ CT system (Bioscan, Washington, DC). For imaging, mice were anesthetized with 1.5% isoflurane and medical air at 1.0 L/min. For anatomical reference, the cone-beam CT scan was acquired first at 45 kVp and 177 A. Image slices were reconstructed in a 176 × 176 matrix with a fast filtered back-projection algorithm using InVivoScope5 1.43 software (Bioscan, USA).
For the SPECT scan, a 20% window was set around the 159 keV principal gamma-photon of I-123. In this multiplexed multipinhole SPECT system, the 1.4 mm nine-pinhole mouse "standard" collimators were attached to each of the four detector heads consisting of NaI(Tl) crystals. Photons were acquired for about 150 s/projection and 24 projections per detector head in a 256 × 256 matrix for a total imaging time ranging from 60 to 75 minutes. SPECT data were reconstructed by ordered subset expectation maximization (OSEM) methods with four subsets of data undergoing 9 iterations each using InVivoScope5 1.43. The CT and SPECT slices were then coregistered. Image analysis and volumeof-interest (VOI) quantification were performed using Vivo-Quant5 2.5 (Mediso/inviCRO, Boston, MA). The activity concentration in the VOI for the whole tumor was divided by the activity concentration in the VOI for the hind limb muscle in order to calculate the tumor-to-muscle ratio ( / ).  (2), and BE(2)C xenografts were harvested, cut in half, embedded in Tissue-Tek5 optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura, Torrance, CA), along with a piece of forelimb muscle as control, and frozen on liquid nitrogen vapor. Frozen blocks were transferred to the STTARR correlative pathology lab on dry ice, and serial frozen sections of alternating 5 m (for immunohistochemistry) and 50 m thickness (for autoradiography) were cut, placed on glass slides, and left to dry for 20 minutes. After sections were completely dry, the 50 m sections were placed in a custombuilt 16-slide holder that held the frozen sections in close proximity to a storage phosphor screen (Cyclone Plus Storage Phosphor System, Perkin Elmer, Shelton, CT, USA), with a layer of plastic wrap separating them. In some instances, a piece of filter paper with serial dilutions of radiotracer was included in the cassette for use as a standard and to check the linearity of the film. This cassette was maintained at −20 ∘ C for a period of time equating to 10 half-lives of activity. The timing of tumor resection and contact with phosphor screen were recorded for all cases. Following completion of 10 halflives of decay, the phosphor screen was removed from the cassette and developed on the Cyclone Plus imaging system at 600 dpi resolution, with a written record of slide locations on the screen. Image quantification was performed in ImageJ software, in which regions of interest (ROIs) were drawn around each tumor and corresponding piece of muscle tissue, and mean phosphor intensity was recorded in each region. A ROI corresponding to background signal was also recorded, taken from the region of screen not containing any slides or tissue. Mean tumor-to-muscle ratios were calculated by division of mean per-pixel intensity in tumor (subtracting background) divided by mean per-pixel muscle intensity, subtracting background.

SSTR2
Immunohistochemistry. SSTR2 immunofluorescence staining was performed on 5 m thick frozen sections. Tissue sections were fixed for 10 minutes in acetone and allowed to air dry for 5 minutes. Endogenous biotin, biotin receptors, and avidin sites were blocked with the Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories, Burlingame, CA). The tissue sections were incubated with rabbit anti-SSTR2 antibody (1 : 100; ab9550, Abcam) for 1 hour at room temperature. Detection of the rabbit antibodies was performed by incubation with Texas Red goat antirabbit IgG antibody (1 : 200; TI-1000, Vector Laboratories) for 30 minutes. For the positive control of SSTR immunohistochemistry, normal pancreas islets were used. Negative controls were done by omitting the specific primary antibodies and processed in the same way. The tissue sections were washed and then mounted with Vectashield mounting medium with DAPI (H-1200, Vector Laboratories). Wholeslide scanned immunofluorescence images were acquired on a TissueScope 4000 (Huron Technologies, Waterloo ON, Canada) at 1 m/pixel resolution. Coregistration of autoradiography with immunofluorescence was performed by upsampling the autoradiography image (600 dpi, which equates to 42 m/pixel resolution) of the 50 m adjacent section to match the immunofluorescence image (imaged at 1 m/pixel resolution), followed by rigid registration using a customized MATLAB script. 177 Lu-DOTA-TATE. Drug treatment commenced when the tumor sizes reached 0.5 cm in diameter. Animals were randomized into two groups, each with 7 animals: the control group and the 177 Lu-DOTA-TATE treatment group. A single dose of 177 Lu-DOTA-TATE (20 MBq) [14] was administered as treatment. Control mice received the same volume of saline. Tumor growth was monitored by measuring tumor dimensions using a digital caliper. Tumor volume was calculated as width 2 ×length×0.5. When tumor volume reached 3 cm 3 , mice were sacrificed, and tumors were dissected and weighed. Tumor growth curves consisting of the tumor volumes at different time points were plotted. During the study, the mice were observed daily for possible adverse effects due to treatments. Morbidity signs of ill health such as ruffled/thinning fur, abnormal behaviors, or local erosion from the tumor, were observed. Animal body weight was also monitored for general toxicity.

Statistical Analysis.
Data from different experiments were presented as mean ± SD. Two-tailed, unpaired Student's -tests were performed to compare the uptake values obtained from SPECT/CT imaging and tumor growth in two different groups. / ratios of 123 I-MIBG uptake and of 123 I-MIBG autoradiography in different groups were analyzed by one-way analysis of variance (ANOVA) and Tukey's test. Comparison of NET expression between different groups was analyzed by the nonparametric Kruskal-Wallis test with Dunn's multiple comparison tests. Statistical significance was achieved with a two-sided < 0.05. All statistics were generated using GraphPad Prism software version 6. somatostatin receptors (SSTR2) and NET in NB cell lines was determined using RT-PCR (Figure 1(a)) and Western blot (Figure 1(b)). Although different NB cell lines showed similar SSTR2 mRNA expression levels (Figure 1(a)), marked variation of SSTR2 protein expression was observed (Figure 1(b)).

Variable Expression Levels of SSTR2 and NET in
In some NB cell lines such as CHLA-15, CHLA-20, CHLA-90, and LAN-5, a prominent SSTR2 expression was detected, whereas, in others, a low level of SSTR2 expression was identified (Figure 1(b)). A similar variation was observed for the expression of NET, a primary transporter responsible for specific active cellular uptake of MIBG [15] (Figures 1(a) and  1(b)). Interestingly, some high SSTR2-expressing cell lines, CHLA-15, CHLA-90, and LAN-5, showed low expression levels of NET, which makes SSTR2 a potential alternative molecular target for NB imaging or treatment, especially for MIBG nonacid tumors. In addition, SSTR2 or NET expression did not appear to be correlated with MYCN amplification or p53 mutation status. In some cell lines, two bands were detected using the monoclonal SSTR2 antibody (ab134152, Abcam), which is consistent with a previous report in which two bands were also detected in IMR-32 neuroblastoma cell lysates [16].

Uptake of 68 Ga-DOTA-TATE Correlates with SSTRT2
Expression in NB Xenografts. DOTA-TATE has a high affinity for SSTR2 in vitro. In order to assess the relationship between 68 Ga-DOTA-TATE uptake and SSTR2 expression, we selected a high SSTR2-expressing NB cell line, CHLA-15, and a low SSTR2-expressing cell line, SK-N-BE(2), for in vivo PET/CT tumor imaging. Tumor uptake was expressed as Standardized Uptake Value (SUV). As shown in Figures  2(a)-2(c), we observed a significant difference in the uptake of 68 Ga-DOTA-TATE between CHLA-15 and SK-N-BE(2) xenografts. The mean tumor uptake value of 68 Ga-DOTA-TATE was significantly higher in the CHLA-15 xenografts (0.79 ± 0.10% ID/g; = 0.0003), compared to SK-N-BE(2) tumors (0.13 ± 0.02% ID/g; < 0.01).

Histological Colocalization of SSTR2 and 68 Ga-DOTA-TATE Uptake.
To further evaluate the relationship between SSTR2 expression and 68 Ga-DOTA-TATE uptake, we performed SSTR2 immunostaining and ex vivo autoradiography with CHLA-15 and SK-N-BE(2) xenograft sections. Consistent with the PET results, CHLA-15 tumors showed significantly higher accumulation of 68 Ga-DOTA-TATE as compared to SK-N-BE(2) tumors (Figures 3(a) and 3(b)). We also observed spatial heterogeneity for both SSTR2 expression (Figure 3(c)) and 68 Ga-DOTA-TATE accumulation (Figures 3(a) and 3(d)). When we merged the SSTR2 fluorescent staining and autoradiography images, we observed intratumoral colocalization of SSTR2 expression and 68 Ga-DOTA-TATE uptake (Figure 3(d)). Tumor regions with a high number of SSTR2-positive cells corresponded to focal areas of increased radioactivity. In SK-N-BE(2) tumors, both SSTR2 expression and 68 Ga-DOTA-TATE autoradiography signals were too weak to be detected (Figure 3(e)).  (Figure 4(a)). From the slope of tumor growth curve, Lu-177-DOTA-TATE treated tumors regained tumor growth rate after day 12, which indicates that single dose Lu-177-DOTA-TATE may not achieve long-lasting antitumor effects. No overall toxicity with respect to body weight loss was observed at the dose of 20 MBq/animal (Figure 4(b)). Also, in vivo 123 I-MIBG uptake is not related to in vitro NET expression levels in three selected NB cell lines. Although BE(2)C cells had a relatively higher NET expression in vitro, their in vivo 123 I-MIBG uptake is at the similar level as the background, with the / ratio of 1.13 ± 0.13. We were not able to detect positive signals from NET immunohistochemistry staining with harvested NB xenografts. In order to compare the NET expression in vivo, we ran a Western blot of NET with homogenized tumor samples ( Figure 5(d)). We did not, however, observe a significant difference of NET expression between CHLA-15, SK-N-BE(2), and BE(2)C xenografts, which indicates a possible change of NET expression profile due to in vitro cell culture ( Figure 5(e)).

Discussion
Neuroblastoma is an extremely heterogeneous disease, both biologically and clinically, comprising tumor cells with very different molecular features. Somatostatin receptors (SSTRs) are variably expressed in neuroblastoma cell lines and tumors, as demonstrated by autoradiography, Western blot, immunohistochemistry, and RT-PCR techniques [17][18][19]. In this study, we selected a panel of NBL cells lines with different biological and genetic backgrounds (Table 1). We detected SSTR2 mRNA in most of the selected cell lines, but only 4 out of 8 cell lines express high levels of SSTR2 protein, including CHLA-15, CHLA-20, CHLA-90, and LAN-5 cells. In addition, expression of SSTR2 was not related to the p53 mutation and MYCN amplification status. Since the commercially available NB cell lines are mostly derived from stage IV patient samples, we are currently conducting a large-scale Children's Oncology Group biological study (COG ANBL14B3) with both high-risk and non-high-risk NB patient specimens to investigate the relationship between SSTR2 expression and clinical features.  Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs are an established and effective treatment option for adult patients with SSTR-positive neuroendocrine tumors [4]. 111 In-[diethylenetriaminepentaacetic acid (DTPA)]octreotide (Octreoscan; Mallinckrodt), a somatostatin analog, has been used for more than 15 years in the diagnosis and staging of SSTR-positive tumors. Nevertheless, it has a restricted ability to identify lesions smaller than 1 cm and to obtain a good spatial resolution, even when using SPECT rather than planar imaging [20]. In recent years, new PET-based radiopharmaceuticals targeting somatostatin receptors have been developed to address these issues. Several studies have demonstrated that 68 Ga-labeled DOTA-TOC or DOTA-TATE PET combined with CT has distinctly higher sensitivity and improved spatial resolution for the detection of SSTR-positive neuroendocrine tumors compared to scintigraphy with conventional SPECT imaging using Octreoscan [4,21]. In this study, we successfully imaged 68 Ga-DOTA-TATE uptake in SSTR2-positive neuroblastoma xenografts with micro-PET/CT. We also observed intensive 68 Ga-DOTA-TATE uptake in MIBG-low-avidity CHLA-15 xenografts.
131 I-MIBG has been used over the past 15 years in multimodal therapy as a radiotherapeutic agent in relapsed and refractory NB patients [22]. However, only 30-40% of children with chemotherapy-refractory disease respond to 131 I-MIBG, and the responses are usually only transient [22][23][24]. In this study, we observed low 131 I-MIBG avidity in all selected NB cell lines. Although BE(2)C cells showed relatively elevated NET expression in vitro, we were not able to detect positive NET immunostaining with harvested BE(2)C xenografts. Western blot of NET with homogenized tumor samples showed similar NET expression levels between CHLA-15, SK-N-BE(2), and BE(2)C xenografts, which indicates a possible change of NET expression profile in vivo. We also demonstrated the complementary role of somatostatin receptor imaging in detecting additional sites of neuroendocrine tumors that were not visualized with 123 I-MIBG scintigraphy. Several clinical studies also confirmed discordant uptake patterns of MIBG and somatostatin receptor expression in some NB tumors [25]. Kroiss et al. [26], for example, described that somatostatin receptor imaging with 68 Ga-DOTA-TOC PET was able to detect the sites of disease in 2/4 patients with NB which were not visible by 123 I-MIBG. Numerous other studies in patients with neuroendocrine tumors have similarly demonstrated the complementary role of somatostatin receptor imaging in detecting additional sites of disease that were not visualized with 123 I-MIBG scintigraphy [27]. In this study, we observed discordant expression of NET and SSTR2 expression in NB-cell lines. CHLA-15 has a lower level of NET expression but higher SSTR2 expression compared to SK-N-BE(2) cells. In vivo PET/CT imaging demonstrated high 68 Ga-DOTA-TATE uptake in CHLA-15 xenografts compared to SK-N-BE(2) tumors. Consequently, somatostatin receptor expression and DOTA-TATE imaging could serve as an adjunct to existing diagnostic and therapeutic methods. They may provide valuable information for the pretherapeutic staging of the disease and impact patient outcomes.
One of the major goals of our study is to understand how the molecular expression of SSTR2 determined the responses of radioactively labeled DOTA-TATE. DOTA-TATE can also be radiolabeled with 177 Lu for targeted − -particle radiotherapy. 177 Lu has a similar physical half-life as 131 I. 177 Lu emits a medium-energy − -particle resulting in localized energy deposition; thus the targeted tissue receives lowdose-rate radiation exposure. Mouse studies of solid tumor xenografts found 177 Lu to be superior to other radiolanthanides in effecting tumor control with pretargeted therapy [28]. We demonstrated the antitumor effect of 177 Lu-DOTA-TATE on CHLA-15 xenografts which have high SSTR2 expression and elevated uptake of 68 Ga-DOTA-TATE. These characteristics may be valuable in future clinical trials: both 68 Ga-DOTA-TATE PET imaging and SSTR2 protein profile could serve as indicators for 177 Lu-DOTA-TATE treatment response. This avenue needs to be evaluated further in clinical scenarios.

Conclusions
This study has allowed us to demonstrate the association between SSTR2 expression and 68 Ga-DOTA-TATE uptake, which potentially leads to the antitumor activity of 177 Lu-DOTA-TATE in NB preclinical models. Histological colocalization of SSTR2 and 68 Ga-DOTA-TATE was also observed in our study. SSTR2 expression therefore could be used as a potential biomarker for predicting drug response to 177 Lu-DOTA-TATE radiotherapy. Moreover, in our model, we demonstrated that 68 Ga-DOTA-TATE PET is superior to 123 I-MIBG SPECT imaging in detecting NB xenograft tumors. The absence of significant difference of NET expression between various NB xenografts models and discordant in vitro and in vivo NET expression represent a limitation of our study which will require further investigation.
The ongoing COG study looking at the prevalence of SSTR2/NET expression in high-risk NB patients may allow us to identify a subset of patients who could benefit from this new SSRT2 targeted radiotherapeutic modality, in particular for the small number of patients demonstrating MIBGnonavid tumors.

Conflicts of Interest
No potential conflicts of interest were disclosed by the authors.


extfieldsofstudy: Biology Medicine

text:
Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, we describe an EMC virus isolate, identified as EMCV C15, which was successfully obtained from the serum of dogs from animal hospitals. Virus production in cell culture was confirmed by EMCV-specific real-time RT-PCR, indirect immunofluorescence assays and electron microscopy. In addition, the open reading frame sequence (ORF) of the EMCV C15 virus was determined. From sequence comparison and phylogenetic analysis among 24 reference EMCV strains, it appears that the EMCV C15 strain is closely genetically related to strain BEL2887A/91 (>99.0% nucleotide identity). In artificially challenged dogs, the heart and brain were important targets of EMCV C15. This study provides genetic and pathogenic characterization of the EMCV C15 strain isolated in Beijing and calls for sustained surveillance of EMCV infection in China to support better prevention and control of the disease.

Real time RT-PCR for EMCV detection.
Real-time RT-PCR assay utilized the SYBR Premix Ex TaqTM kit (Takara, Dalian, China) in a total volume of 25 μL. The assay was performed following the manufacturer's instructions. The EMCV-specific primer set described by Wang et al. 16 was used as follows: forward primer (position 1116-1140) 5′-GACGCTTGAAGACGTTGTCTTCTTA-3′; reverse primer (position 1302-1326) 5′-CCCTACCTCACGGAATGGGGCAAAG-3′. The primers were designed based on a 3D sequence and have been confirmed to be highly specific for EMCV. Real-time RT-PCR was conducted on an ABI H7900 Fast instrument (Life Technologies, Carlsbad, CA) and the results were analyzed with the included system software.
Clinical Samples. A total of 69 serum specimens were collected from dogs at pet hospitals in Beijing in 2015. The dogs presented high fever symptoms (69/69), diarrhea (45/69) and dyspnea (32/69). Colloidal gold strip detection was used to detect canine parvovirus (CPV) and canine distemper virus (CDV). The detection results showed that 44 and 36 of 69 dogs were infected with CPV and CDV, respectively, while 22 of 69 dogs were co-infected with CPV and CDV. The results of real-time RT-PCR showed that 4 of 69 dogs were co-infected with CPV and EMCV.
Virus isolation, propagation, and titration. The EMCV-positive serum samples were incubated with 1.0 IU/mL penicillin/streptomycin for 10 min at 37 °C. Then, serum samples were filtered through a 0.22-μm filter and used as an inoculum for EMC virus isolation. Isolation of EMCV C15 was attempted using BHK21 cells as previously described with some modifications 17 . Confluent BHK21 cells in 6-well plates were washed twice with postinoculation medium and inoculated with 300 μL of sample and 100 μL of postinoculation medium. After 45 min, another 1.0 mL of DMEM supplemented with 10% FBS and 1.0 IU/mL of penicillin and streptomycin was added to each well in the 6-well plate. Inoculated cell (passages 0 [P 0]) were incubated at 37 °C with 5% CO 2 . When a 70% cytopathic effect (CPE) developed, the plates were subjected once to freezing and thawing. The mixtures were centrifuged at 3,000× g for 15 min at 4 °C. The supernatants were harvested for further propagation or saved at −80 °C. If no CPE was observed three days post-inoculation, the plates were frozen and thawed once, after which the supernatants were inoculated on new BHK21 cells for a second passage. Inoculated cells at each passage were also tested using a real-time RT-PCR assay. If the CPE tests and real-time RT-PCR results were negative after four passages, the virus isolation result was considered negative. Virus titration was performed in 96-well plates with 10-fold serial dilutions performed in triplicate per dilution. Virus titers were determined according to the Reed and Muench method and expressed as the 50% tissue culture infective dose (TCID 50 )/100 μL. The virus isolated and characterized in this study was designated EMCV C15.
Electron Microscopy (EM). Samples were prepared for negative staining examination by electron microscopy (EM) following previously described procedures with some modifications 18 . BHK21 cells infected with the EMCV-C15 P3 virus isolate were frozen and thawed 48 h post-infection, after which they were centrifuged at 3,000× g for 15 min. A total of 5.0 mL of supernatant was prepared in this section. The virus was pelleted from the supernatant by ultracentrifugation at 159,000× g for 1.5 h at 4 °C and resuspended in PBS. The resulting pellet was resuspended in 500 μL of 0.01 M PBS (pH 7.2 to 7.4), which was nebulized onto coated EM grids. The grids were stained with 1% phosphotungstic acid (pH 7.0) and observed with a transmission electron microscope. instructions. An equal amount of each sample was loaded onto a 12% SDS-PAGE gel, followed by protein transfer to a PVDF membrane (Millipore). Each membrane was blocked for 1 h with blocking buffer (5% skimmed milk and 0.1% Tween-20 in PBS) and incubated with anti-VP1 primary antibody (1:1,000 dilution) 19 or with mouse anti-GAPDH antibodies (Cell Signaling Technology, Inc.) to detect endogenous GAPDH, which served as a protein-loading control. Each membrane was washed three times with washing buffer (0.1% Tween-20 in PBS) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) for 1 h, after which the membranes were washed and exposed to the detection agent 3,3′-N-diaminobenzidine tertrahydrochloride (DAB, CWBIO, China). Protein sizes were determined by comparison with prestained protein ladders (Thermo Scientific, U.S.A.).

Animal experiments and detection of viral load in dogs.
Eight 25-to-30-day-old dogs were obtained from a commercial breeding herd that was free of CPV, CDV, CPIV, CHV, ICHV and EMCV. The dogs were observed for two weeks to ensure that none were symptomatic and were then randomly assigned to two groups: group A (n = 5) and group B (n = 3). Each dog in group A was treated with 1.0 mL (10 5 TCID 50 ) of EMCV-C15, whereas each dog in group B was treated with 1.0 mL of DMEM. At 35 DPI, the animals in group A and group B were euthanized and the viral load in several organs (heart, liver, spleen, lung, kidney and brain) was determined by real-time RT-PCR performed as described above. Each sample was tested three times.
Sanger sequencing and phylogenetic analysis. EMCV-specific primers (Table 1) were designed based on the ORF of the BJC3 strain (DQ464062). DNA fragments corresponding to the complete ORFs of EMCV C15 strain were amplified and the amplicons were sequenced commercially (HuaDa, Beijing). Sequence assembly was carried out using the SeqMan program included with the DNASTAR software package (Madison, WI). The complete nucleotide sequence of the EMCV-C15 ORF was deposited in GenBank under accession number KU664327. Phylogenetic analysis was performed using the nucleotide sequence of the EMCV-C15 virus from this study, as well as the sequences of 24 reference strains for which full genome sequences were available in GenBank. A phylogenetic tree was constructed based on the ORF sequences of EMCV-C15 using the

Results
Virus isolation and characterization. An EMCV isolate designated EMCV-C15 was successfully obtained from a mixture of four serum samples from pet hospitals in Beijing (collected in March 2015) which had shown positive real-time RT-PCR results. The EMCV-C15 particles in infected BHK21 cells were examined by EM 48 h post-infection. As shown in Fig. 1, negatively stained samples contained a small virus with a diameter of 27-30 nm 18 . Virus growth was confirmed by IFA using an anti-EMCV VP1 specific monoclonal antibody 19 . The EMCV VP1 protein stained by the monoclonal antibody was distributed in the cytoplasm but not in the nucleus ( Fig. 2A). Negative control cells are shown in Fig. 2B. Western blot analysis demonstrated that capsid protein VP1 expression was similar in BHK-21 cells infected with EMCV-C15 or EMCV HB10, which was used as a positive control (Fig. 3). Taken together, these results show that EMCV-C15 and EMCV HB10 are indistinguishable with regard to viral replication and spreading in BHK-21 cells and that the monoclonal antibody recognizes an epitope common to both strains.

EMCV-C15 infection in artificially challenged mice. Mice injected with EMCV-C15 showed clinical
symptoms including anorexia, paralysis and sudden death, and displayed high mortality. At 15 DPI, the mice that were still alive were bled and subjected to pathological examination. The presence of EMCV-C15 was confirmed by real-time RT-PCR. The LD 50 of EMCV-C15 in mice was 10 2.98 TCID 50 (Table 2).

Quantity and distribution of EMCV-C15 in artificially challenged dogs. The five dogs in group A,
which had been inoculated with EMCV-C15, presented with pulmonary edema (Fig. 4A), actuated pericardial effusion, myocarditis, hind limb paralysis and encephalitis (pictures not shown). However, the dogs in group B displayed no clinical signs (Fig. 4B). The presence of EMCV-C15 in the organs of each group was detected by real-time PCR. In group A, EMCV was detected in the heart, liver, spleen, lung, kidney and brain, which had average viral loads of approximately 4.37 × 10 5 , 3.52 × 10 4 , 3.64 × 10 4 , 9.72 × 10 3 , 9.27 × 10 3 , and 2.68 × 10 5 EMCV genomes, respectively (Fig. 5).  otide sequences were performed to examine the degree of sequence similarity between EMCV C15 isolate and 24 EMCV reference strains retrieved from GenBank nucleotide database (Table 3). The complete 6,879-nt EMCV C15 ORF GenBank accession number (KU664327) encoding 2,292 amino acids with no insertions or deletions was examined. Multiple sequence alignment based on the ORF sequence of EMCV-C15 and other EMCV reference strains was performed using the Clustal X program 17 . The results showed the newly isolated virus (EMCV C15) shared the highest sequence similarity (99.0-99.75%) with GX0601, BJC3, NJ08, GS01, HB10 and BEL-2887A/91. However, it shared lower similarity (80-83.52%) with PV2, EMCV-B, EMCV-D, and the D variant. Phylogenetic analyses of the ORF sequence of the EMCV C15 in this study demonstrated that all EMCV strains could be divided into two mian groups (groups 1 and 2). Group 1 was subdivided into group 1a and b (Fig. 6). Except EMCV-R and EMCV C15 strains, the rest of strains were isloated form pigs in group 1a. The EMCV-C15 strain was clearly most closely related to the EMCV strain BEL2887A/91 which was detected in 1991 in Belgium. Group 2 comprises five EMCV isolates from sus scrofa.  Table 2. Outcome of infection with EMCV C15 in mice. a Data were analyzed using SPSS 17.0 software. Oneway ANOVA followed by Duncan's multiple range test was used to compare the parameters among the different groups.

Discussion
EMCV has often been described as a potential zoonotic agent with a wide range of hosts. Reports have clearly shown that humans are susceptible to EMCV infection; indeed, such infection is likely to be fairly common, but most human cases are probably asymptomatic and/or unrecognized 21 . Although human exposure to EMCV is quite common and associated with low morbidity, the virus is a potential threat to public health security. Therefore, it is essential that researchers focus on EMCV infection in humans and dogs. Infection of dogs with EMCV was reported by Schwab et al., who described a high proportion of dogs and cats with non-suppurative meningoencephalitis, for which immunohistochemical examination with antibodies against 18 different infectious agents failed to reveal a cause. However, the significance of positive immunoreactions obtained with antibodies against proteins produced by West Nile virus (WNV) and EMCV requires further investigation 22 .
In this study, 4 of 69 dog serum specimens tested positive for EMCV using real-time RT-PCR. EMCV-C15 was isolated from a mixture of four serum samples that were co-infected with CPV and EMCV. The EMCV-C15 isolate was serially propagated via cell culture methods and characterized. By examining CPE development, IFA staining, EM, western blot analysis, infectious virus titers and complete ORF sequences, we clearly demonstrated that the C15 isolate is phenotypically and genetically stable in cell culture.
Experiments in mice showed that the EMCV isolate was highly virulent and caused hind limb paralysis. In addition, we revealed that the LD 50 of EMCV-C15 in mice was 10 2.98 TCID 50 . To enhance our understanding of EMCV loads in various organs in dogs, real-time RT-PCR was used to quantify the presence of the EMCV genome in various tissues from artificially challenged dogs. The heart and brain are the most important targets for the isolated EMCV C15 in challenged dogs. Inflammation of the heart and brain were obvious in dogs infected with EMCV-C15 and might have been important in the development of acute myocarditis and pulmonary edema.
Phylogenetic analysis showed that the C15 strain is closely genetically related to strain BEL2887A/91, which circulated in Belgium in 2002. The hosts of BEL2887A/91 are pigs or piglets; however, the mechanism by which EMCV was introduced to dogs remains unknown. The history of the spread of EMCV among species and geographical areas merits further investigation.
In this study, the EMCV isolate EMCV-C15 was obtained and characterized. To our knowledge, this is the first report describing the isolation and characterization of EMCV from dogs. All experimental data indicate that the dog is a host for EMCV. Therefore, people living in areas with dogs, especially owners of pet dogs, should be aware of this potential health threat.


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Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

Background Multicellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. Here we exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia. Methods Activation of DNA damage repair markers, proliferation, cell death, glycogen accumulation and developing hypoxia were investigated using immunofluorescence, immuno-histochemistry, EdU incorporation, Western blots, COMET assays, and pharmacological agents in A673 Ewing sarcoma spheroids and monolayer cultures. Results DNA damage marker γ-H2AX is observed in the hypoxic, peri-necrotic region of growing spheroids. While most proliferating cells are seen on the spheroid surface, there are also a few Ki-67 positive cells in the hypoxic zone. The hypoxia-induced phosphorylation of H2AX to form γ-H2AX in spheroids is attenuated by the ATM inhibitor KU55933, but not the ATR inhibitor VE-821. Conclusion Tumor spheroids mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable, proliferating, and undergoing DNA damage repair processes under these different micro-environmental conditions. ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 spheroids. Spheroids could offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment.

Background
The growth of solid tumors is accompanied by the development of central hypoxic or anoxic regions, an acidic extracellular pH and modified cellular glucose and energy metabolism. Tumor cells either adapt to the unfavorable microenvironment or die, leading to areas of necrosis. Continued growth of the tumor then becomes dependent on the delivery of oxygen and nutrients through vascularization. A primary response of tumor cells to hypoxia is to upregulate HIF1-α and its downstream targets, principally the pro-angiogenic VEGF. However, because of its leaky and inefficient nature, tumor angiogenesis does not completely counter tumor hypoxia which can range from near anoxia (0.02% oxygen tension) at distances over 150 μm from vessels, to moderate (8% oxygen tension) hypoxia [1]. Clinically relevant consequences of tumor hypoxia include increased chemo-and radiation-resistance, increased metastatic potential, genomic instability and poorer prognosis.
Another defining feature of vascularized tumors is the dynamic variation in localized oxygen tension as the unstable tumor vasculature transiently provides reoxygenation. A consequence of these cycles of ischemia and reperfusion is the localized formation of reactive oxygen species (ROS) [2]. The effect of hypoxia and ROS on cell survival, replication and DNA damage has been extensively studied in both tumor and non-tumor cells (reviewed in [3][4][5][6][7]). Severe hypoxia (<0.2% O2) can cause S-phase arrest, ATR activation and phosphorylation of H2AX-Ser139 (γ-H2AX), p53-Ser15, Chk1-Ser345, and Chk2-Thr68. Moderate hypoxia can lead to replication stress and activation of the DNA Damage Repair (DDR) pathway proteins even in the absence of measurable DNA damage [8][9][10]. In contrast, hypoxiareoxygenation induces DNA damage through ROS and leads to ATM-dependent H2AX and p53 phosphorylation [2]. The cellular response to DNA damage is to limit proliferation while initiating repair. The coordinated regulation of the ATM/ATR pathways is critical to this process.
Extensive studies associating DNA damage, the upregulation of DNA damage repair pathways, and hypoxia have been conducted on a variety of cell types in 2D-cell culture systems. However, a better representation of the tumor microenvironment is provided by 3D culture models such as multi-cellular tumor spheroids (MCTS). In such spheroids oxygenation status is determined by the rates of oxygen diffusion and consumption alone, without the complicating influence of the tumor vasculature. MCTS are also a preferred platform for testing in-vitro drug sensitivity which can be either potentiated or suppressed by MCTS-specific features such as the 3D microenvironment (hypoxia, acidosis) and cell aggregation [11]. These features of MCTS lead us to the hypothesis that MCTS could be used to study the relationship between clinically relevant features of a hypoxic microenvironment such as proliferation and activation of DNA damage repair pathways. To date, there is no good model available for the study of hypoxia induced changes within spatially defined cell populations. The lack of such a model limits our ability to study relevant mechanisms of cell survival and to accurately predict efficacy of targeted therapeutics in the unique hypoxic environment present only in 3D and in vivo settings. To recapitulate features of the hypoxia-induced cellular response in avascular tumors (or at sites distant from blood vessels), here we use MCTS to study the spatial correlation between the development of hypoxia, cell proliferation, cell death, and the activation of DDR components. We show that developing hypoxia in the core of growing MCTS is accompanied by a distinctive distribution of proliferating cells, glycogen accumulation, HIF1-α, activated ATM kinase, and induction of γ-H2AX. Using pharmacological inhibitors of ATM and ATR we distinguish between their contributions to H2AX phosphorylation and cell proliferation in the differing microenvironments represented within an MCTS. Our results demonstrate that MCTS can be a physiologically relevant and convenient system for studying the activation of DNA damage-associated pathways in heterogeneous tumor cell populations present within solid tumors, and their susceptibility to pharmacological intervention. Cell culture A673 and LLC were maintained in standard culture conditions (37°C with 5% CO 2 ) in DMEM with 1% vv −1 penicillin (100 IU ml −1 ) and streptomycin 100 mg ml −1 supplemented with 10% vv −1 heat inactivated FBS.

MCTS culture
Spheroids were formed through use of the liquid overlay method wherein single cell suspensions (100 μl of A673 or LLC cell suspensions in DMEM supplemented with 2% FBS or 10% FBS were added at a density of 5 × 10 4 or 5 × 10 3 cells ml −1 respectively) were added to 96-well plates previously coated with agar. The agar coating prevents cell adhesion. Plates containing single cell suspensions were incubated stationary in standard cell culture conditions (37°C, 5% CO 2 ) for 48 h. During this time, tumor cells aggregated forming a single, connected mass of cells that could be moved as a singular unit. After this 48-h formation period, 100 μl of fresh media was added to each well. This makes for a total of 200 μl of total media in each well. After this point, 100 μl of media was removed and replaced with fresh media every 48 h such that total volume within the wells was maintained at 200 μl. In experiments with test compounds or hypoxia treatment, spheroids were individually washed with DMEM and transferred to freshly coated agar plates with 200 μl of fresh medium containing test inhibitors when applicable.

Hypoxia treatment
Hypoxia treatment was performed at 37°C for 12 h with 1% O 2 , 5% CO 2 in a hypoxia chamber followed by collection in a hypoxic glove bag pre-equilibrated at 1% O 2 .

ATM and ATR inhibition
10 μM KU55933 or 2.5 μM VE-821 in DMEM supplemented with 2% FBS and containing 0.025% vv −1 DMSO Fig. 1 Growth characteristics of A673 and LLC spheroids. a Growth curves for spheroids generated with A673 (red) and LLC (black) cells. The mean diameter and standard deviation from >8 spheroids are plotted over time, from initial formation to final size before spheroid collapse. b H&E staining of spheroid cryo-sections reveals the onset of necrosis in spheroids larger than 550 μm. The three sections shown come from spheroids of <500 μm, 550-650 μm and >750 μm in diameter. Shrinkage during processing as well as location of section within spheroids account for the smaller diameter in the final images shown were used. Fresh media containing inhibitors was added to MCTS cultures every 48 h. The vehicle control contained 0.025% vv −1 DMSO in DMEM supplemented with 2% FBS.

Image acquisition and analysis
Fluorescently stained sections were imaged on a Zeiss confocal microscope at 0.25× magnification across a 1000 μm grid. To determine the number of positive nuclei per spheroid, nuclei were counted as Hoechstpositive cells by using watershed separation and quantification with particle analysis in ImageJ software [12] followed by cell counting to determine the percentage of marker positive nuclei.

Statistics
Results represent the average of at least 2 separate experiments with a total of at least 8 spheroids per condition ± standard deviation (SD). Statistical analyses were performed using Graphpad PRISM version 5.0 for Mac OSX, http://www.graphpad.com. A t-test was used when two samples/conditions were compared and ANOVA for more than two groups. Significance represents P < 0.05.

Results
Spheroid growth, the onset of hypoxia and necrosis Spheroids grown with Lewis lung carcinoma (LLC) and Ewing sarcoma (A673) cell lines were characterized. In each case cells were seeded in appropriate medium (DMEM +2% FBS for A673, and DMEM +10% FBS for LLC). Both A673 and LLC formed loose aggregates within 24 h, and discrete 3D spheroids that could be manipulated within 48 h. Spheroid growth was monitored by light microscopy and plotted in Fig. 1a. A673 cells formed the most tightly packed and well-formed spheroids that plateaued at an average diameter of >950 μm 12 days after seeding. LLC spheroids grew to about 800 μm over 11-12 days, with a less tightly packed surface cell layer that disintegrated as spheroids grew larger.
To monitor the temporal and spatial onset of necrosis and hypoxia, spheroids were harvested and sectioned when they were <500 μm, 500-650 μm and >750 μm in diameter. These time-points were chosen to represent the first formation of a discrete spheroid, an exponential growth phase, and spheroids that had almost reached a size plateau. An initial analysis of cell and spheroid morphology was conducted using haemotoxylin and eosin (H&E) staining (Fig. 1b). A673 and LLC spheroids consisted of uniformly and densely packed cells with onset of necrosis apparent in the center of spheroids that were 550-650 μm in diameter. By the time spheroids were approaching their maximum diameter, large areas of central necrosis were present (accounting for 50-60% of spheroid volume).
Pimonidazole staining was used as a surrogate marker of hypoxia (Fig. 2a). Pimonidazole, a 2-nitroimidazole, is reductively activated in hypoxia and can form stable adducts with proteins, peptides and amino acids. Pimonidazole binding to cellular glutathione occurs at oxygen concentrations below 1.3% [13] and is therefore used as a marker of severe hypoxia. Pimonidazole staining was seen in the core of growing spheroids (>500 μm diameter), and in the necrotic and peri-necrotic regions of the largest spheroids (Fig. 2a). The relationship(s) between spheroid size and the onset of hypoxia and necrosis are similar in both A673 and LLC spheroids. The more detailed analyses described below were restricted to A673, since these cells form more robust and uniform spheroids.
Metabolic adaptations, including glycogen accumulation, are a well-recognized feature of tumors. To examine the spatial relationships between hypoxia in tumor MCTS and glycogen accumulation we evaluated the patterns of periodic acid-Schiff staining in spheroids (Fig. 2b). This is a routine histochemical method for detecting glycogen in tumor samples. In small (~400-500 μm) A673 spheroids PAS staining was observed sporadically throughout the spheroid, with sometimes a greater concentration near the center. In intermediate sized spheroids (550-650 μm) PAS staining became much more restricted to the perinecrotic region typically denoted as a hypoxic, quiescent zone. In the largest spheroids intense PAS staining was seen at the periphery. Amylase treatment confirmed that PAS staining in these studies corresponded to glycogen accumulation.

Cell proliferation becomes restricted to the surface as MCTS grow
Previous studies have noted that the thickness of the viable cell layer in MCTS remains approximately constant as the spheroid grows, while the necrotic core increases proportionally with the size of the spheroid [14]. The viable layer is comprised of an outer proliferative rim that has the best access to nutrients and oxygen, and an inner layer of quiescent non-proliferative but viable cells. A frequently used clinical parameter in tumors is the Ki-67 labeling index (fraction of Ki-67 positive cells). Ki-67 protein is present in all phases of the cell cycle except G0, and is thus a marker for the growth fraction of a cell population. Ki-67 staining was present throughout small (~400 μm) spheroids, but was more restricted to the cells near the spheroid surface once the diameter exceeded 500 μm (Fig. 3a). Ki-67positive cells were present approximately 150 μm from the spheroid surface in medium sized spheroids, and 70-80 μm in the largest spheroids. This transition appears to coincide with the onset of hypoxia and necrosis in the central core. Interestingly EdU staining, which marks cells in the S-phase, was concentrated in a narrower band along the spheroid surface, in a much more restricted zone than was seen with Ki-67 (Fig. 3b). The failed incorporation of EdU, despite Ki-67 marker expression, indicates a failure to progress through Sphase, perhaps due to checkpoint activation. The apoptosis marker cleaved caspase 3 was used to monitor the onset of cell death in growing spheroids. Paralleling the   Fig. 3a. c Cell death in A673 spheroids of <500 μm, 550-650 μm and >750 μm in diameter indicated by staining for cleaved caspase-3 (green, with blue Hoechst 33,342 counterstain) observations with H&E staining (Fig. 1b), an extensive region of apoptosis was evident in the central core of the largest spheroids (Fig. 3c).
ATM is primarily responsible for γ-H2AX formation in the hypoxic, peri-necrotic zone of growing spheroids Nuclear γ-H2AX staining was used to monitor activation of DNA damage repair pathways. γ-H2AX positive nuclei were present throughout the smallest (<500 μm) spheroids (Fig. 4a). It is likely that low levels of replicative stress that occur stochastically during the cell cycle, and is often elevated in tumor cells, could account for this. In larger spheroids two distinct regions of γ-H2AX staining were seen: one prominent band of γ-H2AX positive cells was present in the peri-necrotic hypoxic area, and another smaller population of γ-H2AX positive cells was variably present on the surface of the spheroids (Fig. 4a). Co-staining showed coincidence between most Ki-67-positive and γ-H2AX-positive cells in the perinecrotic area, indicating that DDR was occurring in proliferating cells (Fig. 4b).
Based on previous studies in 2D culture systems [2,4,5,[15][16][17][18] it is reasonable to hypothesize that the γ-H2AX staining in the interior of spheroids is a result of the hypoxia-induced activation of the ATR/ATM kinases. Growing spheroids (500-650 μm diameter) were stained with antibodies towards the hypoxia marker HIF1-α, pATM (phospho-serine 1981), pATR (phospho-serine 428), and pChk1 (phospho-serine 345). The hypoxic, HIF1-α-positive region (Fig. 5a) coincided with extensive  phosphorylation of ATM (Fig. 5b) in the peri-necrotic zone. We were unable to detect either pATR (pSer428) or pChk1 (pSer345) in spheroids. This observation is consistent with compelling evidence that phosphorylation at ATR Ser-428 is not associated with activation of its kinase activity [19,20], although Ser-428 phosphorylation has been associated with DNA damage. To further evaluate the roles of ATM and ATR in γ-H2AX formation we used pharmacological inhibitors; KU55933 specific to ATM [21] and VE-821 specific to ATR [22]. Pre-formed spheroids (<450 μm) were chosen for the start of inhibitor treatment as it represented a time-point just before the development of hypoxia or significant necrosis. Spheroids were maintained in either vehicle or ATM/ATR inhibitor. Both KU55933 and VE-821 treatment inhibited the growth of spheroids. After 5 days of treatment with either KU55933 or VE-821 spheroids stopped growing any further (reaching a plateau at an average diameter of 660 μm), while vehicle-treated spheroids typically increased in diameter by about 25% over 5 more days. Spheroids were stained for γ-H2AX after 96 h of inhibitor treatment. Spheroids treated with the ATM inhibitor had fewer γ-H2AX positive cells, but the spatial distribution did not change (Fig. 5c, e). VE-821 (ATR inhibition) had negligible effect on the percentage of γ-H2AX positive cells (Fig. 5c, e). Interestingly, while KU55933 had no significant effect on the percentage of Ki-67-positive cells in the spheroids, VE-821 decreased it by over 50% suggesting a strong effect on the proliferative index (Fig. 5d, f). Together these observations support a role for ATM in H2AX phosphorylation in the hypoxic zone of spheroids, while ATR plays a role in proliferation.

Hypoxia-induced γ-H2AX formation is differentially affected by ATM and ATR inhibition in spheroids versus two-dimensional cultures
To specifically query whether the changes in distribution of DDR markers with increasing spheroid size were a direct result of hypoxia at the core of the spheroid, we examined the effect of incubating small (<500 μm) spheroids (before the onset of hypoxia or necrosis) in 1% O 2 for 12 h. They were then sectioned and probed for γ-H2AX and Ki-67. Interestingly, the percentage or distribution of cells staining positive for Ki-67 did not change significantly when maintained in 1% O 2 (Fig. 6a). Spheroids subject to hypoxia had significantly more γ-H2AX staining in the central core while there was no change in γ-H2AX staining on the surface (Fig. 6b). Oxygen diffusion into a spheroid is limited by inter-cellular tight junctions and senescent/necrotic cells. Direct measurements of oxygen levels within spheroids have shown that as spheroid size increases there is a gradient of oxygen tension from the surface to the center [23]. Hence it is likely that, while 1% O 2 is not sufficient to trigger phosphorylation of H2AX, the core of the spheroid has reached a threshold level of O 2 required for γ-H2AX formation. A similar trend was seen for the activation of ATM kinase; there was a significant increase in pATM staining in the core of spheroids subject to hypoxia (Fig. 6c).
To further probe the role of ATM kinase activity in hypoxia-induced γ-H2AX formation spheroids were treated with the ATM inhibitor KU55933 during the 12 h in which they were maintained in 1% O 2 (Fig. 7a). Control spheroids maintained in vehicle for the 12 h of hypoxia displayed an increase in γ-H2AX-positive cells, while there was no increase in γ-H2AX when the spheroids were subject to 1% O 2 in the presence of KU55933 (Fig. 7b). These observations, along with the results presented in Fig. 5c are consistent with hypoxia-induced activation of ATM contributing to the H2AX Ser-139 phosphorylation in the spheroid center. The ATR inhibitor VE-821 also inhibited hypoxia-induced γ-H2AX formation in spheroids maintained in 1% O 2 (Fig. 7b). 400 μm A673 spheroids in 1% O 2 for 12 h was monitored. Spheroid sections were stained for the proliferation marker Ki-67 (green). The % Ki-67-positive cells is plotted (mean and standard deviation from >12 spheroids and two independent experiments). Hypoxia had no effect on cell proliferation. b The DDR marker γ-H2AX (red) is upregulated in the core of spheroids maintained in 1% O 2 for 12 h. Bar graph shows the mean and standard deviation from >45 spheroids and >5 independent experiments. c Activation of ATM kinase is indicated by staining for pATM (red) in spheroids maintained in 1% O 2 for 12 h. Bar graph shows the mean and standard deviation from >15 spheroids and 4 independent experiments. One way ANOVA with Dunnett's post-test. ***P < .001, ns P > .05 This contrasts with the results seen with VE-821 in Fig. 5c, raising the possibility that ATR plays a role in γ-H2AX formation only under conditions not attained when spheroids are maintained at 20% O 2 .
Previous studies have shown that moderate hypoxia (defined as >0.1% O2), such as that used here, can lead to activation of ATR and ATM kinases, without inducing COMET assay-detectable DNA damage in monolayer cultures [2,16]. In these instances, γ-H2AX is not localized in foci consistent with the absence of nucleating DNA damage. Sections from spheroids do not provide the resolution necessary to clearly identify γ-H2AX foci, if they are present. We examined how A673 cells in 2D culture respond to hypoxia (1% O 2 for 12 h). Western blots showed hypoxia-induced γ-H2AX formation (Fig. 8b), and examination of cells by immunofluorescence revealed that γ-H2AX foci are formed (Fig. 8a). Further, alkaline COMET assays showed that A673 cells maintained in 1% O 2 for 12 h had significantly increased tail moments consistent with the induction of DNA damage (Fig. 8c). In 2D culture, treatment of A673 cells with KU55933 attenuated the hypoxia-induced increase in the percentage of γ-H2AX-positive cells (Fig. 8d, e). In contrast, presence of the ATR inhibitor, VE-821, led to much higher levels of γ-H2AX induction under hypoxia, characterized by cells with both distinct foci and pannuclear γ-H2AX staining (Fig. 8d, e).

Discussion
Solid tumors are characterized by the presence of both oxic and hypoxic regions. The hypoxic tumor microenvironment stimulates neo-angiogenesis, and tumor growth becomes linked to the availability of vasculature-supplied oxygen and nutrients. For this reason anti-angiogenics have been a major focus of cancer drug development efforts. Recent evidence also links hypoxia, DNA damage repair and pathological angiogenesis in both tumors and proliferative retinopathies [10,24,25]. While there have been some spectacular clinical successes with anti-angiogenics in cancer treatment, the overall and progression-free survival rates have been disappointing [26]. Among the factors contributing to this are adaptations of cancer cells in the hypoxic tumor microenvironment that allow them to proliferate and to become more invasive and resistant to chemo-and radio-therapy. Multiple studies have tried to correlate tumor tissue pO 2 , distance from blood vessels, and proliferation indices in attempts to derive a correlation between oxygen tension and proliferative or quiescent state of tumor cells. No clear consensus has emerged from these studies [27]. Multicellular tumor spheroids offer a convenient system in which to model such hypoxic tumor niches in vitro.
Here we sought to examine markers of DNA damage in spheroids and their spatial correlation with hypoxia, glycogen accumulation, proliferation, necrosis and apoptosis.
Typically, two distinct zones are formed as spheroids grow: a viable ring of cells on the surface and a central necrotic core. The H&E staining pattern and the distribution of Ki-67 staining described here is consistent with this model and with previous studies using MCTS to examine the regionalization of cell proliferation [28,29]. We also see incorporation of EdU in a much more restricted ring of cells on the spheroid surface than that seen with Ki-67 staining (Fig. 3), as previously reported in spheroids formed with HCT116 (colon adenocarcinoma) cells [29] and Capan-2 (pancreatic cancer) cells [28]. Experimental measurements and mathematical modeling have shown that spheroids larger than about 200 μm have steep oxygen gradients; the center of the spheroid is anoxic and the interface of the hypoxic and proliferating zones is mildly hypoxic (oxygen partial pressure is predicted to be about 10 mmHg or 1.3% O 2 ) [30]. The presence of tumor cells with proliferative capacity under such intermediate hypoxia has been associated with tumor aggressiveness and lower disease-free survival rates [31]. The 3D culture system used here identifies such a proliferative cell population in the peri-necrotic, hypoxic region of growing spheroids (Fig. 4). Our data show that these cells also stain positive for γ-H2AX (Ser-139 phosphorylated H2AX) indicating the assembly of DDR complexes. γ-H2AX has been used extensively as a marker of DNA double strand breaks due to its early formation at sites of DNA lesions following irradiation or other means of double strand break induction. H2AX Ser-139 phosphorylation allows it to recruit DNA damage repair proteins, facilitating repair and survival of cells (reviewed in [32]). The coincidence of DDR markers with proliferation in spheroids is indicative of a productive DDR process that permits survival of some hypoxic cells. The peri-necrotic, proliferating cells (Fig. 4) are also within a ring of HIF1-α -positive cells (Fig. 5a), confirming the hypoxic micro-environment and identifying the region within a spheroid where transcriptional programs regulated by HIF1-α are likely to be upregulated. HIF1-α is known to promote glycogen accumulation in both tumor and non-tumor cells [33][34][35]. Furthermore, treatment of xenografts with the anti-angiogenic agent c Alkaline COMET assay shows induction of COMET tails (a direct measure of DNA damage) when A673 cells were maintained in 1% O 2 for 12 h. Tail moments were quantitated using OpenCOMET [43] and are plotted as the mean and standard deviation. d Bar graph showing the percentage of γ-H2AX-positive A673 cells when monolayers were maintained for 12 h in either 20% O 2 or 1% O 2 in vehicle, 1% O 2 in KU55933 (ATM inhibitor), or 1% O2 in VE-821 (ATR inhibitor). Mean and standard deviation from two independent experiments are shown. e Immunofluorescence for γ-H2AX (red) in A673 monolayers maintained for 12 h in either 20% O 2 or 1% O 2 in vehicle, 1% O2 in KU55933 (ATM inhibitor), or 1% O2 in VE-821 (ATR inhibitor). ATR inhibition under hypoxia results in cells with either intense pan-nuclear γ-H2AX or distinct foci, while only distinct γ-H2AX foci are seen under hypoxia in the presence of either vehicle or ATM inhibitor. One way ANOVA with Tukey's multi-comparison post-test. ***P < .001, **P < .01, *P < .05, ns P > .05 Bevacizumab led not only to increased hypoxia, but also to an increase in glycogen and upregulation of genes involved in glycogen metabolism. Glycogen is linked to increased proliferation and to survival under conditions of hypoxia and glucose deprivation [34,35]. In agreement with these reports, we noted glycogen accumulation in the peri-necrotic region of growing spheroids (Fig. 2b) coincident with the ring of HIF1-α staining seen in Fig. 5a. Curiously in the largest spheroids examined, PAS-staining (hence glycogen accumulation) was present in the entire viable zone, implying a broader cellular adaptation to use glycogen as an energy store. These observations regarding the regionalization of glycogen accumulation within a tumor spheroid are of particular relevance given the interest in targeting glycogen metabolism for cancer treatment [36]. Also pertinent is the fact that the cells used in this study derive from a human Ewing sarcoma, a malignancy for which cytoplasmic glycogen accumulation (as detected by PAS-staining) is a diagnostic parameter.
The (phosphatidylinositol 3-kinase)-like (PI3KK; includes ATM, ATR and DNA-PK) family of kinases are responsible for H2AX-Ser-139 phosphorylation, a critical early step in the DDR pathway. While there is crosstalk between these kinases, they are each associated with distinct stress conditions. ATM typically phosphorylates H2AX in response to double-strand breaks while ATR is activated in replication stress and the presence of single-stranded DNA. DNA-PK is most commonly associated with hypertonic conditions and apoptotic fragmentation. Both ATM and ATR are known to be activated by severe (O 2 < 0.1%) hypoxia [2,5,15,16,18]. In the case of ATR, this is likely to be a result of replication stress leading to regions of single-strand DNA. The exact mechanism by which ATM is activated under hypoxia is not known, although some evidence suggests that it is a response to the combined presence of replication stress and heterochromatin modifications stimulated by hypoxia [9]. The distribution of activated ATM shown here in growing spheroids with a hypoxic and necrotic center is informative. pATM is concentrated in the perinecrotic, hypoxic area (Fig. 5b). ATM is known to phosphorylate and stabilize HIF1-α under hypoxic conditions [8] and this pATM-positive region is also populated by HIF1-α-positive cells. We were unable to detect activated ATR (pATR) in growing spheroids, despite it's known role in unstressed replication [18,37]. Consistent with these observations pharmacological inhibition of ATM reduced γ-H2AX levels, whereas ATR inhibition altered cell proliferation (Ki-67 staining) rather than the levels or distribution of H2AXpositive cells (Fig. 5). In 2D culture, ATR inhibition under hypoxic conditions led to increased γ-H2AX as well as a significant number of cells showing pan-nuclear γ-H2AX staining. UV-irradiation has previously been shown to induce similar pan-nuclear γ-H2AX staining that represented a pre-apoptotic state [38]. As with results reported here, ATR inhibition increased UV-induced pan-nuclear γ-H2AX. The ATR inhibitor VE-821 is also known to decrease the percentage of cells in the S-phase of the cell cycle under normal conditions, and to abrogate the DNA damage-induced checkpoint (G2/M) after irradiation and hypoxia [22,39], thus reducing cell survival. This effect is particularly pronounced in p53-deficient cells; the Ewing Sarcoma cell line A673 used herein, contains both the oncogenic transcription factor EWS/FlI1 and mutations in the TP53 gene making them p53-null [40]. This combination of increased replication and impaired G1 checkpoint signaling may explain why hypoxic stress resulted in observable DNA damage. Further, the EWS/FLI1 transcription factor induces replication stress and the formation of stalled replication forks. Hence Ewing sarcoma cells are highly sensitive to ATR inhibitors [41].
Hypoxic activation of ATR also suppresses DNA replication through a cdc25a-CDK2-APC/C Cdh1 -cdc6 mechanism [42]. Both ATR and ATM inhibition retarded spheroid growth in our studies; a similar effect on spheroid growth has previously been reported using glioblastoma cells [37]. Taken together these results indicate that ATM activity contributes to hypoxiainduced γ-H2AX formation in the MCTS core and that ATR activity is important for cell proliferation. The observation that ATR and ATM inhibitors have different effects on DDR in monolayer cells compared to MCTS highlights the importance of recreating a 3D tumor environment when validating therapeutic targets.

Conclusions
MCTS can be used to mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable and proliferating under these different oxygen tensions. Using this in vitro model system we show that ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 MCTS, and that targeting either ATM or ATR kinase inhibits spheroid growth, albeit through different mechanisms. The results obtained using MCTS are distinct and complimentary to those seen with 2D culture. MCTS offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment.