Objective. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is overexpressed in various cancers which has led to the development of therapeutic anti-Siglec-15 monoconal antibodies (mAbs). In these preclinical studies, the therapeutic mAb, NC318 (antihuman/murine Siglec-15 mAb), was labeled with zirconium-89 and evaluated in human Siglec-15 expressing cancer cells and mouse xenografts for potential use as a clinical diagnostic imaging agent. Methods. Desferrioxamine-conjugated NC318 was radiolabeled with zirconium-89 to synthesize [89Zr]Zr-DFO-NC318. Cancer cell lines expressing variable Siglec-15 levels were used for in vitro cell binding studies and tumor xenograft mouse models for biodistributions. [89Zr]Zr-DFO-NC318 biodistribution and PET imaging studies to determine tissue uptakes (tissue : muscle ratios, T : M) included pharmacokinetic evaluation in Siglec-15+tumor xenografts and immunocompetent mice, blocking with nonradioactive NC318 (20, 100, and 300 μg) and xenografts with low/negligible Siglec-15 expressing tumors. Results. [89Zr]Zr-DFO-NC318 exhibited high affinity ([Formula: see text]~4 nM) for Siglec-15 and distinguished between moderate and negligible Siglec-15 expression levels in cancer cell lines. The highest [89Zr]Zr-DFO-NC318 uptakes occurred in the spleen and lymph nodes of the Siglec-15+tumor xenografts at all time points followed by Siglec-15+tumor uptake which was lower although highly retained. In immunocompetent mice, the spleen and lymph nodes exhibited lower uptakes indicating that the athymic xenografts had increased Siglec-15+ immune cells. Specific [89Zr]Zr-DFO-NC318 binding to Siglec-15 was proven with NC318 blocking studies in which dose-dependent decreases in Siglec-15+tumor T : Ms were observed. Higher than expected, tumor T : Ms were seen in lower expressing tumors likely due to the contribution of murine Siglec-15+ immune cells in the tumor microenvironment as confirmed by immunohistochemistry. Siglec-15+tumors were identified on PET images whereas low/negligible expressing tumors showed lower uptakes. Conclusions. In vitro and in vivo [89Zr]Zr-DFO-NC318 uptakes correlated with Siglec-15 expression levels in target tissues. Despite uptake in immune cell subsets in the tumor microenvironment, these results suggest that clinical [89Zr]Zr-DFO-NC318 PET imaging may have value in selecting patients for Siglec-15-targeted therapies.

Background: Osteosarcoma (OS) is an aggressive pediatric cancer with unmet therapeutic needs. Glutaminase 1 (GLS1) inhibition, alone and in combination with metformin, disrupts the bioenergetic demands of tumor progression and metastasis, showing promise for clinical translation. Materials and Methods: Three positron emission tomography (PET) clinical imaging agents, [18F]fluoro-2-deoxy-2-D-glucose ([18F]FDG), 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT), and (2S, 4R)-4-[18F]fluoroglutamine ([18F]GLN), were evaluated in the MG63.3 human OS xenograft mouse model, as companion imaging biomarkers after treatment for 7 d with a selective GLS1 inhibitor (CB-839, telaglenastat) and metformin, alone and in combination. Imaging and biodistribution data were collected from tumors and reference tissues before and after treatment. Results: Drug treatment altered tumor uptake of all three PET agents. Relative [18F]FDG uptake decreased significantly after telaglenastat treatment, but not within control and metformin-only groups. [18F]FLT tumor uptake appears to be negatively affected by tumor size. Evidence of a flare effect was seen with [18F]FLT imaging after treatment. Telaglenastat had a broad influence on [18F]GLN uptake in tumor and normal tissues. Conclusions: Image-based tumor volume quantification is recommended for this paratibial tumor model. The performance of [18F]FLT and [18F]GLN was affected by tumor size. [18F]FDG may be useful in detecting telaglenastat's impact on glycolysis. Exploration of kinetic tracer uptake protocols is needed to define clinically relevant patterns of [18F]GLN uptake in patients receiving telaglenastat.

Complete and detailed descriptions of materials and methods are in the supplementary information, including animal studies, image registration, hyperpolarized 13C MRI, 18F-FDG PET, EPR imaging, Western blot, Immunohistochemistry, and statistical analysis. A supplementary Figure S contains H&E and CD31 staining of 100 μm thick slices from the center of the xenograft tumors to depict differences in vascularity are not the primer driver for the rim/interior difference in FDG uptake and O2 levels.

High expression of prostate-specific membrane antigen (PSMA) in prostate cancers prompted the development of the PSMA-targeted PET-imaging agent [18F]DCFPyL, which was recently approved by the FDA. Fluorine-18-labeled Lys–Urea–Glu-based oxime derivatives of [18F]DCFPyL were prepared for the comparison of their in vitro and in vivo properties to potentially improve kidney clearance and tumor targeting. The oxime radiotracers were produced by condensation of an aminooxy functionalized PSMA-inhibitor Lys–Urea–Glu scaffold with fluorine-18-labeled aldehydes. The radiochemical yields were between 15–42% (decay uncorrected) in 50–60 min. In vitro saturation and competition binding assays with human prostate cancer cells transfected with PSMA, PC3(+), indicated similar high nM binding affinities to PSMA for all radiotracers. In vivo biodistribution studies with positive control PC3(+) tumor xenografts showed that the kidneys had the highest uptake followed by tumors at 60 min. The PC3(+) tumor uptake was blocked with non-radioactive DCFPyL, and PC3(−) tumor xenograft (negative control) tumor uptake was negligible indicating that PSMA targeting was preserved. The most lipophilic tracer, [18F]2a, displayed comparable tumor-targeting to [18F]DCFPyL and a desirable alteration in pharmacokinetics and metabolism, resulting in significantly lower kidney uptake with a shift towards hepatobiliary clearance and increased liver uptake.

Background The activities of MYC, the androgen receptor, and its associated pioneer factors demonstrate substantial reprogramming between early and advanced prostate cancer. Although previous studies have shown a shift in cellular metabolic requirements associated with prostate cancer progression, the epigenetic regulation of these processes is incompletely described. Here, we have integrated chromatin immunoprecipitation sequencing (ChIP-seq) and whole-transcriptome sequencing to identify novel regulators of metabolism in advanced prostate tumors characterized by elevated MYC activity. Results Using ChIP-seq against MYC, HOXB13, and AR in LNCaP cells, we observed redistribution of co-bound sites suggestive of differential KMT2A activity as a function of MYC expression. In a cohort of 177 laser-capture microdissected foci of prostate tumors, KMT2A expression was positively correlated with MYC activity, AR activity, and HOXB13 expression, but decreased with tumor grade severity. However, KMT2A expression was negatively correlated with these factors in 25 LuCaP patient-derived xenograft models of advanced prostate cancer and 99 laser-capture microdissected foci of metastatic castration-resistant prostate cancer. Stratified by KMT2A expression, ChIP-seq against AR and HOXB13 in 15 LuCaP patient-derived xenografts showed an inverse association with sites involving genes implicated in lipid metabolism, including the arachidonic acid metabolic enzyme PLA2G4F . LuCaP patient-derived xenograft models grown as organoids recapitulated the inverse association between KMT2A expression and fluorine-18 labeled arachidonic acid uptake in vitro. Conclusions Our study demonstrates that the epigenetic activity of transcription factor oncogenes exhibits a shift during prostate cancer progression with distinctive phenotypic effects on metabolism. These epigenetically driven changes in lipid metabolism may serve as novel targets for the development of novel imaging agents and therapeutics.

Positron projection imaging (PPI) of tumor-bearing mice under certain circumstances can provide accurate in vivo estimates of total tumor radioactivity, an important pharmacokinetic measurement. However, the number of images generated in these studies is typically very large and many 2D tumor regions-of-interest (ROIs) must be manually defined to obtain accurate radioactivity estimates. In this study, we compared several methods that might allow automatic quantification of tumor radioactivity content. In total, 120 images (n = 81 mice) were acquired in pairs during two separate experiments. The first experimental batch was used for development, and the second as an independent testing cohort. Four methodologies were evaluated, including deep-learning (U-net), region-growing (Level-Set), and thresholding (Otsu, mean value). For all methodologies, preprocessing of the images included uptake normalization to fixed window. Tumor radioactivity is defined as total uptake within a tumor region minus a background estimate. Performance metrics were evaluated for both segmentation results (Sorenson-Dice Coefficient) and radioactivity calculation results (Bland-Altman). Using the test batch data, DICE score for U-net segmentation was 0.82, vs. 0.5-0.6 for the other three methods. Bland-Altman plots showed a mean difference of -0.26 for U-net based calculations vs. -0.5 to -0.8 for the other methods. The U-net approach had the highest accuracy in both segmentation and subsequent radioactivity calculation.

PSMA overexpression has been associated with aggressive prostate cancer (PCa). However, PSMA PET imaging has revealed highly variable changes in PSMA expression in response to ADT treatment ranging from increases to moderate decreases. To better understand these PSMA responses and potential relationship to progressive PCa, the PET imaging agent, [18F]DCFPyL, was used to assess changes in PSMA expression in response to ADT using genomically characterized LuCaP patient-derived xenograft mouse models (LuCaP-PDXs) which were found to be sensitive to ADT (LuCaP73 and LuCaP136;CS) or resistant (LuCaP167;CR). [18F]DCFPyL (2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid) was used to assess PSMA in vitro (saturation assays) in LuCaP tumor membrane homogenates and in vivo (imaging/biodistribution) in LuCaP-PDXs. Control and ADT-treated LuCaPs were imaged before ADT (0 days) and 2-, 7-, 14-, and 21-days post-ADT from which tumor:muscle ratios (T:Ms) were determined and concurrently tumor volumes were measured (caliper). After the 21-day imaging, biodistributions and histologic/genomic (PSMA, AR) analysis were done. [18F]DCFPyL exhibited high affinity for PSMA and distinguished different levels of PSMA in LuCaP tumors. Post-ADT CS LuCaP73 and LuCaP136 tumor volumes significantly decreased at day 7 or 14 respectively vs controls, whereas the CR LuCaP167 tumor volumes were minimally changed. [18F]DCFPyL imaging T:Ms were increased 3–5-fold in treated LuCaP73 tumors vs controls, while treated LuCaP136 T:Ms remained unchanged which was confirmed by day 21 biodistribution results. For treated LuCaP167, T:Ms were decreased (~ 45 %) vs controls but due to low T:M values (<2) may not be indicative of PSMA level changes. LuCaP73 tumor PSMA histologic/genomic results were comparable to imaging/biodistribution results, whereas the results for other tumor types varied. Tumor responses to ADT varied from sensitive to resistant among these LuCaP PDXs, while only the high PSMA expressing LuCaP model exhibited an increase in PSMA levels in response to ADT. These models may be useful in understanding the clinical relevance of PSMA PET responses to ADT and potentially the relationship to disease progression as it may relate to the genomic signature.

Background: Patients with osteoblastic bone metastases are candidates for radium-223 (223RaCl2) therapy and may undergo sodium fluoride-18 (18F-NaF) positron emission tomography-computed tomography imaging to identify bone lesions. 18F-NaF has been shown to predict 223RaCl2 uptake, but intratumor distributions of these two agents remain unclear. In this study, the authors evaluate the spatial distribution and relative uptakes of 18F-NaF and 223RaCl2 in Hu09-H3 human osteosarcoma mouse xenograft tumors at macroscopic and microscopic levels to better quantify their correlation. Materials and Methods: 18F-NaF and 223RaCl2 were co-injected into Hu09-H3 xenograft tumor severe combined immunodeficient mice. Tumor content was determined from in vivo biodistributions and visualized by PET, single photon emission computed tomography, and CT imaging. Intratumor distributions were visualized by quantitative autoradiography of tumor tissue sections and compared to histology of the same or adjacent sections. Results: 18F and 223Ra accumulated in proportional amounts in whole Hu09-H3 tumors (r2 = 0.82) and in microcalcified regions within these tumors (r2 = 0.87). Intratumor distributions of 18F and 223Ra were spatially congruent in these microcalcified regions. Conclusions: 18F-NaF and 223RaCl2 uptake are strongly correlated in heterogeneously distributed microcalcified regions of Hu09-H3 xenograft tumors, and thus, tumor accumulation of 18F is predictive of 223Ra accumulation. Hu09-H3 xenograft tumors appear to possess certain histopathological features found in patients with metastatic bone disease and may be useful in clarifying the relationship between administered 223Ra dose and therapeutic effect.

Introduction: [227Th]Th-3,2-HOPO-MSLN-mAb, a mesothelin (MSLN)-targeted thorium-227 therapeutic conjugate, is currently in phase I clinical trial; however, direct PET imaging using this conjugate is technically challenging. Thus, using the same MSLN antibody, we synthesized 3,2-HOPO and deferoxamine (DFO)-based zirconium-89 antibody conjugates, [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb, respectively, and compared them in vitro and in vivo. Methods: [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb were evaluated in vitro to determine binding affinity and immunoreactivity in HT29-MSLN and PDX (NCI-Meso16, NCI-Meso21) cells. For both the zirconium-89 conjugates, in vivo studies (biodistribution/imaging) were performed at days 1, 3, and 6, from which tissue uptake was determined. Results: Both the conjugates demonstrated a low nanomolar binding affinity for MSLN and >95% immunoreactivity. In all the three tumor types, biodistribution of [89Zr]Zr-DFO-MSLN-mAb resulted in higher tumor uptake(15.88-28-33%ID/g) at all time points compared with [89Zr]Zr-3,2-HOPO-MSLN-mAb(7–13.07%ID/g). [89Zr]Zr-3,2-HOPO-MSLN-mAb femur uptake was always higher than [89Zr]Zr-DFO-MSLN-mAb, and imaging results concurred with the biodistribution studies. Conclusions: Even though the conjugates exhibited a high binding affinity for MSLN, [89Zr]Zr-DFO-MSLN-mAb showed a higher tumor and lower femur uptake than [89Zr]Zr-3,2-HOPO-MSLN-mAb. Nevertheless, [89Zr]Zr-3,2-HOPO-MSLN-mAb could be used to study organ distribution and lesion uptake with the caveat of detecting MSLN-positive bone lesions. Clinical trial (NCT03507452).

1236 Objectives: Programmed cell death-ligand 1 (PD-L1), a cell surface immune checkpoint ligand, is overexpressed in several cancers and suppresses the host’s immune system, thereby allowing cancer cells to evade detection and proliferate. Anti-PD-L1 monoclonal antibodies (mAb) have emerged as effective therapeutics in reversing this immunosuppression in certain subsets of patients; however, in some responsive patients, tumor cell PD-L1 expression is not detected by immunohistochemistry. Therefore, selecting patients for anti-PD-L1 therapies based on histological characterization of tumor cells alone is insufficient, which may result from not accounting for PD-L1 expression in the tumor microenvironment (TME). Non-invasive imaging agents can detect and provide a real-time readout of PD-L1 expression in the TME and may be a better predictor of patient response to anti-PD-L1 therapies. Previous work has shown that an anti-PD-L1 mAb (Avelumab) can be radiolabeled with zirconium-89 (89Zr) to produce a conjugate, [89Zr]Zr-DFO-Avelumab (ZPD), which has high affinity (sub nM) for both human and murine PD-L1 [1]. ZPD previously determined that there were moderate levels of PD-L1 expression in human MDA-MB-231 tumor cells (MB) and undetectable PD-L1 expression in human MKN-45 tumor cells (MK) [1]. Herein, ZPD was further evaluated in these human cancers using xenograft mouse models to provide further insights into PD-L1 expression in the TME. Methods: ZPD was synthesized as previously described [1]. MKN-45 [MK; human gastric carcinoma; PD-L1(-)] and MDA-MB-231 [MB; human breast carcinoma; PD-L1(+)] engrafted athymic nude mice were injected with ZPD + 20 µg of Avelumab. Positron emission tomography (PET) imaging was acquired after 1 day and 3 days with in vivo biodistributions performed after 3 days. Blood and tissue uptakes were quantitated as percent injected dose per gram (%ID/g) and expressed as tissue-to-muscle (T:M) ratios. Excised tumors were then sectioned and analyzed: 1) by ex vivo autoradiography (for ZPD localization); 2) for histology and expression of murine/human PD-L1, CD45 and CD11b with immunohistochemistry (IHC). Results: Immuno-PET imaging (Figure 1A) and in vivo biodistributions of MK and MB xenograft mice demonstrated the highest ZPD uptake was present in the spleen (~25 %ID/g) and lymph nodes (~30 %ID/g) with no differences in non-target tissues. Interestingly, ZPD tumor uptake was similar between the PD-L1(-) MK tumors (7.2 %ID/g) and the PD-L1(+) MB tumors (9.8 %ID/g), and T:M ratios for MB tumors were 2-fold higher compared to MK tumors (Table 1). Ex vivo autoradiography showed that ZPD localization was heterogeneously distributed in MK tumor sections and homogeneously distributed in MB tumor sections (Figure 1B). Molecular pathology demonstrated that PD-L1 expression in MK tumors occurred in infiltrating immune cells and not tumor cells, whereas PD-L1 expression in MB tumors represented a combination of both tumor cells and infiltrating immune cell expression (Table 1). PD-L1(+) cell populations in the TME colocalize with cells expressing CD45 and CD11b, consistent with myeloid-derived suppressor cells or other infiltrating immune cells. Conclusions: These studies indicate that PD-L1 expression on both tumor cells and cells within the TME can contribute significantly to ZPD uptake, offering a potential source of variability in patient responses to anti-PD-L1 therapy that can be quantified clinically. ZPD Immuno-PET imaging may serve as a reliable diagnostic tool for selecting and monitoring patient response to anti-PD-L1 therapies. Although PD-L1(+) cell types found in xenograft mouse models are likely different from those in humans, the TME may need to be considered when predicting patients likely to benefit from anti-PD-L1 therapies.

INTRODUCTION:A study of Pb contamination caused by the outgassing of Rn from Ra in dry, liquid, and murine tissues samples has been made to help design proper handling procedures for Ra in preclinical biodistribution work. MATERIALS AND METHODS:Pb activity levels were measured from Ra in dry, liquid, and tissue samples using aspiration and autoradiography techniques. RESULTS:Using aspiration techniques on dry samples of Ra, an average Rn outgassing rate of 51% ± 21% was measured with one measurement reaching as high as 81%. 31% ± 4% Pb contamination was measured within a 4.3 cm radius of a dry Ra source placed inside a 10-cm-diameter petri dish where the lip of the petri dish contained the Rn dissemination. Without the containment of the petri dish, Rn can reach as far as 7.8 cm from the source with trace levels spreading further. Using aspiration techniques on liquid samples of Ra, outgassing rates of Rn were 0.9% ± 0.3%. The outgassing levels in harvested organs from a biodistribution were as high as 10.1% ± 0.4% for an intraperitoneally injected mouse and 0.204% ± 0.006% for an intravenously injected mouse. The outgassing of the intravenously injected mouse carcass was less than 0.1%. CONCLUSION:In dry form, the high levels of Rn outgassing from a Ra source necessitate the use of ventilated biohoods when handling or preparing dry Ra from source vials. The very low levels of Rn outgassing from Ra liquid sources reduces exposure to Rn by a factor of 50. Rn exposure from murine organ tissue reaches levels of 10% when handling organs from an intraperitoneal injection and less than 0.2% for an intravenous injection.

1201 Objectives: Prostate cancer commonly metastasizes to bone and becomes osteoblastic. Radium-223 dichloride (223RaCl2) alpha therapy has been used to treat patients with metastatic prostate cancer by targeting hydroxyapatite in calcified bone lesions. Therapeutic responses of patients receiving 223RaCl2 have varied and, in some patients previously treated with standard of care therapies, an increase in bone fractures have occurred suggesting greater bone toxicity from these therapies when administered in combination. This variability in therapeutic response may be related to 223RaCl2 dosing, which is adjusted by patient body weight as opposed to total tumor burden and responsiveness of individual lesions. Thus, the bone-seeking PET imaging agent sodium fluoride-18, [18F]NaF, may be useful to select patients for 223RaCl2 therapy and determine appropriate 223RaCl2 doses per patient. Although [18F]NaF PET/CT has shown clinical utility in identifying and monitoring metastatic bone disease, the relationship of [18F]NaF uptake in bone lesions for predicting the uptake of bone-targeted therapies such as 223RaCl2 has not been established. Herein, the in vivo biodistribution of [18F]NaF and 223RaCl2 were evaluated in human osteosarcoma tumor xenograft mouse models (Hu09-H3), and then ex vivo autoradiography and confirmatory histology were performed with the tumors to determine whether the spatial distribution of [18F]NaF and 223RaCl2 coincided with calcified osteoblastic tissue. Methods: In vivo biodistributions were performed using Hu09-H3 xenografts at 1 hour post single or co-injection of [18F]NaF and 223RaCl2 from which percent injected doses per gram of tissue (% ID/g) were determined. [18F]NaF PET/CT and 223RaCl2 SPECT/CT imaging were performed at similar times using the same tumor model. Following biodistribution, tumors were further evaluated by ex vivo autoradiography to determine the regional distribution of [18F]NaF and 223RaCl2 within the same tumor sections (223RaCl2 determined after [18F]NaF decay), which were then validated by histological staining for calcifications (Von Kossa, Alizarin Red). Results: In co-injected Hu09-H3 xenografts, the highest [18F]NaF uptake occurred in target tissues, bone (28.7% ID/g) and tumor (9.7% ID/g) (Table 1). In contrast, 223RaCl2 had 1.5-fold lower target tissue uptake in bone (18.5% ID/g) and tumor (6.3% ID/g) with the highest uptakes occurring in non-target tissues, spleen (40.0% ID/g) and kidney (33.6% ID/g) (Table 1). [18F]NaF uptakes were lower in the blood and other non-target tissues compared to 223RaCl2 indicating faster clearance. The co-injected [18F]NaF and 223RaCl2 biodistribution results were comparable to biodistributions in Hu09-H3 xenograft cohorts injected with the single agents ([18F]NaF only or 223RaCl2 only), indicating the presence of both agents did not alter targeting. The high non-targeted 223RaCl2 uptake in the spleen, kidneys, and intestines are consistent with previous reports [1, 2]. [18F]NaF PET/CT and 223RaCl2 SPECT/CT imaging showed uptake in discrete regions within the tumor and throughout the skeleton with additional 223RaCl2 uptake in the kidneys and spleen, which compared favorably with the biodistribution results. Ex vivo autoradiograms of tumors from co-injected Hu09-H3 xenografts demonstrated [18F]NaF and 223RaCl2 were spatially congruent and selective for hydroxyapatite-containing regions within the tumor; histological staining verified the regional distribution of [18F]NaF and 223RaCl2 in the tumor corresponded with calcified osteoblastic tissue (Figure 1). Conclusions: Uptake of [18F]NaF and 223RaCl2 spatially co-localize in bone-forming tumor tissue and are similar in tumors containing hydroxyapatite. [18F]NaF PET/CT imaging may be useful in predicting uptake of 223RaCl2 in metastatic bone disease and potentially assess 223RaCl2 dosimetry to individual malignant bone lesions.

Background: Prostate-specific membrane antigen (PSMA) has emerged as a promising target for developing radionuclide therapy (RNT) in prostate cancer; however, accumulation of PSMA-RNT in salivary glands can result in irreversible xerostomia. Methods to prevent PSMA-RNT-related xerostomia could be clinically useful; however, little is known about PSMA expression in salivary glands of preclinical animal models. Using [18F]DCFPyL autoradiography/biodistribution, PSMA expression levels were determined in salivary glands of various preclinical monkey and rodent species and compared with humans. Methods: Binding affinities (Kd) and PSMA levels (Bmax) were determined by in vitro [18F]DCFPyL autoradiography studies. In vivo rodent tissue uptakes (%ID/g) were determined from [18F]DCFPyL biodistributions. Results: [18F]DCFPyL exhibited low nanomolar Kd for submandibular gland (SMG) PSMA across all the species. PSMA levels in human SMG (Bmax = 60.91 nM) were approximately two-fold lower compared with baboon SMG but were two- to three-fold higher than SMG PSMA levels of cynomolgus and rhesus. Rodents had the lowest SMG PSMA levels, with the mouse being 10-fold higher than the rat. In vivo rodent biodistribution studies confirmed these results. Conclusions: SMG of monkeys exhibited comparable PSMA expression to human SMG whereas rodents were lower. However, the results suggest that mice are relatively a better small animal preclinical model than rats for PSMA salivary gland studies.

Abstract Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18F-FDG-PET and lactate production, while pO2 was inversely related to lactate production and 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18F-FDG uptake negatively correlated with pO2 in the center of the tumor and positively correlated with pO2 on the periphery. In contrast to pO2 and 18F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. Significance: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis.

1223 Background: Mesothelin (MSLN) targeted thorium-227 (MSLN-TTC; [227Th]Th-3,2-HOPO-MSLN-mAb) has demonstrated in vivo efficacy in MSLN positive tumors1,2. This MSLN antibody (MSLN-mAb, BAY 861903) based TTC is currently being evaluated in Phase I clinical trial (NCT03507452). Due to the low gamma emission with low abundance of measurable photons in the decay chain of thorium-227, tumor imaging using thorium-227 based conjugate is technically challenging3,4. Besides, forming a complex with thorium-227, the 3-hydroxypyridin-2-one (3,2-HOPO) chelator can also form complex with zirconium-89.,6. This approach thus would allow for radiolabeling the 3,2-HOPO-MSLN-mAb either with therapeutic or imaging radionuclide. Thus, the 3,2-HOPOH-MSLN-mAb was labeled with zirconium-89 to produce [89Zr]Zr-3,2-HOPO-MSLN-mAb. In parallel, the MSLN-mAb was conjugated to deferoxamine (DFO; [89Zr]Zr-DFO-MSLN-mAb), one of the widely used chelator of zirconium-89. Both the zirconium-89 conjugates were compared in vitro and in vivo using MSLN positive tumor xenograft mouse models. Methods: 3,2-HOPO-MSLN-mAb (BAY 2287409) and DFO-MSLN-mAb conjugates were labeled with zirconium-89 to yield [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb respectively. Radiochemical purity (RCP) was determined by size exclusion HPLC. The zirconium-89 conjugates were evaluated in vitro (binding affinity) and in vivo for biodistribution and PET imaging of HT29-MSLN and patient-derived (PDXs, NCI-Meso21 and NCI-Meso16) tumor xenografts. After injecting (i.v) tumor bearing mice (Athymic, nu/nu, female) with the radioactive conjugates, biodistribution and imaging was performed on days 1, 3, and 6 for HT29-MSLN xenografts and on day 3 for PDXs. Tissue associated radioactivity was determined by gamma counter and used to calculate % injected dose/g (%ID/g), tissue:blood (T:B), and tissue:muscle (T:M) ratios. Results: The RCP of [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb was 52-76% (n=20) and 90-92% (n=8) respectively. [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb exhibited a low nanomolar binding affinity (Kd=0.16-2.3 nM) for MSLN. Pharmacokinetics over the time-course was similar for both the zirconium-89 conjugates except for blood, tumor, and femur. [89Zr]Zr-DFO-MSLN-mAb showed higher HT29-MSLN tumor uptake (28-33 %ID/g) at all time-points compared to [89Zr]Zr-3,2-HOPO-MSLN-mAb (7-11 %ID/g). Similarly, on day 3, PDX tumor accumulation of [89Zr]Zr-DFO-MSLN-mAb (15.88 -19.49%ID/g) was higher than [89Zr]Zr-3,2-HOPO-MSLN-mAb (7.95-13.07%ID/g). T:B and T:M ratios were also lower for [89Zr]Zr-3,2-HOPO-MSLN-mAb than the zirconium-89 DFO conjugate. However, femur uptake of [89Zr]Zr-3,2-HOPO-MSLN-mAb (6.74%ID/g) was ~2-fold higher compared to [89Zr]Zr-DFO-MSLN-mAb (3.57%ID/g) at day 1 and then increased to ~3-4-fold over 6 days. At all times, PET imaging results paralleled the biodistribution pattern of both the zirconium-89 conjugates. Conclusions: In vitro, both conjugates exhibited a high binding affinity for MSLN. In vivo, [89Zr]Zr-DFO-MSLN-mAb showed higher tumor uptake and lower femur uptake than [89Zr]Zr-3,2-HOPO-MSLN-mAb. As [89Zr]Zr-3,2-HOPO-MSLN-mAb uses the same chelator as [227Th]Th-3,2-HOPO-MSLN-mAb), the same 3,2-HOPO-MSLN-mAb conjugate could be better at studying organ distribution and lesion uptake of the MSLN-TTC, with the caveat that detection of MSLN positive tumors in the lower extremity might be more difficult if high femur uptake is also seen in humans.

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[18F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[18F]SFPy) have been prepared using ‘fluorination on the Sep-Pak’ method. Conjugation of 6-[18F]SFPy or 6-[18F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[18F]FPy-T140. The overall radiochemical yields were 6–17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[18F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [18F]SFPy has been prepared using ‘fluorination on the Sep-Pak’ method that allows rapid automated synthesis of 6-[18F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.