[68 Ga]Ga-FAPI-46 PET for non-invasive detection of pulmonary fibrosis disease activity.

PURPOSE: The lack of effective molecular biomarkers to monitor idiopathic pulmonary fibrosis (IPF) activity or treatment response remains an unmet clinical need. Herein, we determined the utility of fibroblast activation protein inhibitor for positron emission tomography (FAPI PET) imaging in a mouse model of pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intratracheal administration of bleomycin (1 U/kg) while intratracheal saline was administered to control mice. Subgroups from each cohort (n = 3-5) underwent dynamic 1 h PET/CT after intravenously injecting FAPI-46 radiolabeled with gallium-68 ([68 Ga]Ga-FAPI-46) at 7 days and 14 days following disease induction. Animals were sacrificed following imaging for ex vivo gamma counting and histologic correlation. [68 Ga]Ga-FAPI-46 uptake was quantified and reported as percent injected activity per cc (%IA/cc) or percent injected activity (%IA). Lung CT density in Hounsfield units (HU) was also correlated with histologic examinations of lung fibrosis. RESULTS: CT only detected differences in the fibrotic response at 14 days post-bleomycin administration. [68 Ga]Ga-FAPI-46 lung uptake was significantly higher in the bleomycin group than in control subjects at 7 days and 14 days. Significantly (P = 0.0012) increased [68 Ga]Ga-FAPI-46 lung uptake in the bleomycin groups at 14 days (1.01 ± 0.12%IA/cc) vs. 7 days (0.33 ± 0.09%IA/cc) at 60 min post-injection of the tracer was observed. These findings were consistent with an increase in both fibrinogenesis and FAP expression as seen in histology. CONCLUSION: CT was unable to assess disease activity in a murine model of IPF. Conversely, FAPI PET detected both the presence and activity of lung fibrogenesis, making it a promising tool for assessing early disease activity and evaluating the efficacy of therapeutic interventions in lung fibrosis patients.

18F-16α-17ß-Fluoroestradiol Binding Specificity in Estrogen Receptor-Positive Breast Cancer.

Purpose To determine the binding specificity of 18F-16α-17ß-fluoroestradiol (FES) in estrogen receptor (ER) α-positive breast cancer cells and tumor xenografts. Materials and Methods Protocols were approved by the office of biologic safety and institutional animal care and use committee. By using ER-negative MDA-MB-231 breast cancer cells, clonal lines were created that expressed either wild-type (WT; 231 WT ER) or G521R mutant ERα (231 G521R ER), which is defective in estradiol binding. ERα protein levels, subcellular localization, and transcriptional function were confirmed. FES binding was measured by using an in vitro cell uptake assay. In vivo FES uptake was measured in tumor xenografts by using small-animal positron emission tomographic/computed tomographic imaging of 24 mice (17 WT ER tumors, nine mutant G521R ER tumors, eight MDA-MB-231 tumors, and four MCF-7 ER-positive tumors). Statistical significance was determined by using Mann-Whitney (Wilcoxon rank sum) test. Results ERα transcriptional function was abolished in the mutated 231 G521R ER cells despite appropriate receptor protein expression and nuclear localization. In vitro FES binding in the 231 G521R ER cells was reduced to that observed in the parental cells. Similarly, there was no significant FES uptake in the 231 G521R ER xenografts (percent injected dose [ID] per gram, 0.49 ± 0.042), which was similar to the negative control MDA-MB-231 xenografts (percent ID per gram, 0.42 ± 0.051; P = .20) and nonspecific muscle uptake (percent ID per gram, 0.41 ± 0.0095; P = .06). Conclusion This study showed that FES retention in ER-positive breast cancer is strictly dependent on an intact receptor ligand-binding pocket and that FES binds to ERα with high specificity. These results support the utility of FES imaging for assessing tumor heterogeneity by localizing immunohistochemically ER-positive metastases that lack receptor-binding functionality. © RSNA, 2017 Online supplemental material is available for this article.

Targeting angiogenesis for radioimmunotherapy with a 177Lu-labeled antibody.

PURPOSE: Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. METHODS: The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with 177Lu (t 1/2 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: 177Lu only, TRC105 only, 177Lu-DTPA-IgG (a nonspecific antibody), 177Lu-DTPA-TRC105 low-dose, and 177Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of 177Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose 177Lu-DTPA-TRC105. RESULTS: Biodistribution studies indicated steady uptake of 177Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p < 0.001, all groups). In most study groups (all except the nonspecific IgG group), the body weights of the mice did not decrease by more than 10%, indicating the safety of the injected agents. Serum alanine transaminase levels remained nearly constant indicating no damage to the liver (a primary clearance organ of the agent), and this was confirmed by ex vivo histological analyses. CONCLUSION: 177Lu-DTPA-TRC105, when administered at a sufficient dose, is able to curtail tumor growth and provide a significant survival benefit without off-target toxicity. Thus, this targeted agent could be used in combination with other treatment options to slow tumor growth allowing the other agents to be more effective.

Absence of the Vitamin D Receptor Inhibits Atherosclerotic Plaque Calcification in Female Hypercholesterolemic Mice.

Epidemiological and clinical data suggest adverse cardiovascular outcomes with respect to vitamin D deficiency. Here, we explored the effects of vitamin D in atherosclerotic plaque calcification in vivo by utilizing vitamin D receptor (Vdr)-deficient mice in an Apoe-/- background. Animals were fed a high-fat diet (HFD) for either 12 or 18 weeks and then examined for atherosclerotic plaque development. In order to prevent calcium deficiency, Vdr-/- and Apoe-/- ;Vdr-/- animals were fed a high-calcium rescue diet prior to initiation of the HFD feeding and supplemented with high-calcium water during HFD feeding. Although calcium supplementation improved bone mass in Vdr-/- and Apoe-/- ;Vdr-/- mice, neither strain was fully rescued. Systemic inflammatory responses observed in the absence of VDR were exaggerated in Apoe-/- mice. Whereas, hyperlipidemic profiles seen in Apoe-/- mice were ameliorated in the absence of VDR. Micro-computed tomography (µCT) analysis revealed that six out of eight Apoe-/- animals developed atherosclerotic plaque calcification following 12 weeks of HFD feeding and 100% of the mice developed plaque calcification after 18 weeks. In contrast, although atherosclerotic lesions were evident in Apoe-/- ;Vdr-/- mice at 12 and 18 weeks of HFD challenge, none of these animals developed plaque calcification at either time point. The active vitamin D hormone, 1,25(OH)2 D3 likely increased calcification in aortic smooth muscle cells perhaps by directly modulating expression of Alpl, Rankl, and Opg. Our data suggest that the absence of VDR inhibits atherosclerotic plaque calcification in hypercholesterolemic Apoe-/- mice, providing additional insight into the role of vitamin D in atherosclerotic plaque calcification. J. Cell. Biochem. 118: 1050-1064, 2017. © 2016 Wiley Periodicals, Inc.

Level-specific amputations and resulting regenerative outcomes in the mouse distal phalanx.

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

Neutrophils drive accelerated tumor progression in the collagen-dense mammary tumor microenvironment.

BACKGROUND: High mammographic density has been correlated with a 4-fold to 6-fold increased risk of developing breast cancer, and is associated with increased stromal deposition of extracellular matrix proteins, including collagen I. The molecular and cellular mechanisms responsible for high breast tissue density are not completely understood. METHODS: We previously described accelerated tumor formation and metastases in a transgenic mouse model of collagen-dense mammary tumors (type I collagen-α1 (Col1α1)(tm1Jae) and mouse mammary tumor virus - polyoma virus middle T antigen (MMTV-PyVT)) compared to wild-type mice. Using ELISA cytokine arrays and multi-color flow cytometry analysis, we studied cytokine signals and the non-malignant, immune cells in the collagen-dense tumor microenvironment that may promote accelerated tumor progression and metastasis. RESULTS: Collagen-dense tumors did not show any alteration in immune cell populations at late stages. The cytokine signals in the mammary tumor microenvironment were clearly different between wild-type and collagen-dense tumors. Cytokines associated with neutrophil signaling, such as granulocyte monocyte-colony stimulated factor (GM-CSF), were increased in collagen-dense tumors. Depleting neutrophils with anti-Ly6G (1A8) significantly reduced the number of tumors, and blocked metastasis in over 80 % of mice with collagen-dense tumors, but did not impact tumor growth or metastasis in wild-type mice. CONCLUSION: Our study suggests that tumor progression in a collagen-dense microenvironment is mechanistically different, with pro-tumor neutrophils, compared to a non-dense microenvironment.

Evaluations of extracellular pH within in vivo tumors using acidoCEST MRI.

PURPOSE: A practical, noninvasive method is needed to measure the extracellular pH (pHe) within in vivo tumors to longitudinally monitor tumor acidosis. We have optimized a biomedical imaging method, termed acidoCEST MRI, to provide noninvasive assessments of tumor pHe in preclinical models of mammary carcinoma. METHODS: A CEST-FISP MRI method was optimized to detect the chemical exchange saturation transfer (CEST) of two amide protons of a clinically approved CT contrast agent, iopromide. The ratio of the two CEST effects was used to measure pH. Routes of administration of iopromide were evaluated to ensure sufficient delivery of the agent to the tumor. The optimized acidoCEST MRI method was then used to evaluate the change in tumor pHe following alkalinizing bicarbonate treatment. RESULTS: The acidoCEST MRI protocol measured pH between 6.2 and 7.2 pH units. Greater delivery of iopromide was shown to improve the precision of the measurement of tumor pHe, but the agent did not influence the tumor pHe. AcidoCEST MRI was used to longitudinally monitor the effect of bicarbonate treatment on the pHe of tumors and bladders. CONCLUSION: This study demonstrates that an optimized acidoCEST MRI method is a practical, noninvasive method for assessing changes in tumor acidosis.

Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

Objectives: Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Methods: Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Results: Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. Conclusion: The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.

[64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via µPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

Multimodal Cross-Device and Marker-Free Co-Registration of Preclinical Imaging Modalities.

Integrated preclinical multimodal imaging systems, such as X-ray computed tomography (CT) combined with positron emission tomography (PET) or magnetic resonance imaging (MRI) combined with PET, are widely available and typically provide robustly co-registered volumes. However, separate devices are often needed to combine a standalone MRI with an existing PET-CT or to incorporate additional data from optical tomography or high-resolution X-ray microtomography. This necessitates image co-registration, which involves complex aspects such as multimodal mouse bed design, fiducial marker inclusion, image reconstruction, and software-based image fusion. Fiducial markers often pose problems for in vivo data due to dynamic range issues, limitations on the imaging field of view, difficulties in marker placement, or marker signal loss over time (e.g., from drying or decay). These challenges must be understood and addressed by each research group requiring image co-registration, resulting in repeated efforts, as the relevant details are rarely described in existing publications. This protocol outlines a general workflow that overcomes these issues. Although a differential transformation is initially created using fiducial markers or visual structures, such markers are not required in production scans. The requirements for the volume data and the metadata generated by the reconstruction software are detailed. The discussion covers achieving and verifying requirements separately for each modality. A phantom-based approach is described to generate a differential transformation between the coordinate systems of two imaging modalities. This method showcases how to co-register production scans without fiducial markers. Each step is illustrated using available software, with recommendations for commercially available phantoms. The feasibility of this approach with different combinations of imaging modalities installed at various sites is showcased.

Theranostic cobalt-55/58m for neurotensin receptor-mediated radiotherapy in vivo: A pilot study with dosimetry.

Neurotensin receptor 1 (NTSR1) can stimulate tumor proliferation through neurotensin (NTS) activation and are overexpressed by a variety of cancers. The high binding affinity of NTS/NTSR1 makes radiolabeled NTS derivatives interesting for cancer diagnosis and staging. Internalization of NTS/NTSR1 also suggests therapeutic application with high LET alpha particles and low energy electrons. We investigated the therapeutic efficacy of [58mCo]Co-NOTA-NT-20.3 in vivo using murine models xenografted with NTSR1-positive HT29 human colorectal adenocarcinoma cells, and utilized [55Co]Co-NOTA-NT-20.3 for dosimetry. METHODS: Targeting properties and cytotoxicity of [55/58mCo]Co-NOTA-NT-20.3 were assessed with HT29 cells. Female nude mice were xenografted with HT29 tumors and administered [55Co or 58mCo]Co-NOTA-NT-20.3 to evaluate pharmacokinetics or for therapy, respectively. Dosimetry calculations followed the Medical Internal Radiation Dose (MIRD) formalism and human absorbed dose rate per unit activity were obtained from OpenDose. The pilot therapy study consisted of two groups (each N = 3) receiving 110 ± 15 MBq and 26 ± 6 MBq [58mCo]Co-NOTA-NT-20.3 one week after tumor inoculation, and control (N = 3). Tumor sizes and masses were measured twice a week after therapy. Complete blood count and kidney histology were also performed to assess toxicity. RESULTS: HPLC measured radiochemical purity of [55,58mCo]Co-NOTA-NT-20.3 > 99 %. Labeled compounds retained NTS targeting properties. [58mCo]Co-NOTA-NT-20.3 exhibited cytotoxicity for HT29 cells and was >15× more potent than [58mCo]CoCl2. Xenografted tumors responded modestly to administered doses, but mice showed no signs of radiotoxicity. Absorbed dose to tumor and kidney with 110 MBq [58mCo]Co-NOTA-NT-20.3 were 0.6 Gy and 0.8 Gy, respectively, and other organs received less than half of the absorbed dose to tumor. Off-target radiation dose from cobalt-58g was small but reduces the therapeutic window. CONCLUSION: The enhanced in vitro cytotoxicity and high tumor-to-background led us to investigate the therapeutic efficacy of [58mCo]Co-NOTA-NT-20.3 in vivo. Although we were unable to induce tumor response commensurate with [177Lu]Lu-NT127 (NLys-Lys-Pro-Tyr-Tle-Leu) studies involving similar time-integrated activity, the absence of observed toxicity may constitute an opportunity for targeting vectors with improved uptake and/or retention to avoid the aftereffects of other high-LET radioactive emissions. Future studies with higher uptake, activity and/or multiple dosing regimens are warranted. The theranostic approach employed in this work was crucial for dosimetry analysis.

PET Imaging of Estrogen Receptors Using 18F-Based Radioligands.

In vivo molecular imaging of estrogen receptor alpha (ER) can be performed via positron emission tomography (PET) using ER-specific radioligands, such as 16α-[18F]fluoro-17ß-estradiol (18F-FES). 18F-FES is a radiopharmaceutical recently approved by the United States Food and Drug Administration for use with PET imaging to detect ER+ lesions in patients with recurrent or metastatic breast cancer as an adjunct to biopsy. 18F-FES PET imaging has been used in clinical studies and preclinical research to assess whole-body ER protein expression and ligand binding function across multiple metastatic sites, to demonstrate inter-tumoral and temporal heterogeneity of ER expression, to quantify the pharmacodynamic effects of ER antagonist treatment, and to predict endocrine therapy response. 18F-FES PET has also been studied for imaging ER in endometrial and ovarian cancer. This chapter details the experimental protocol for 18F-FES PET imaging of ER in preclinical tumor xenograft models. Consistent adherence to key methodologic details will facilitate obtaining meaningful and reproducible 18F-FES PET preclinical imaging results, which could yield additional insight for clinical trials regarding imaging biomarkers and oncologic therapy.

18F-SynVesT-1 PET/MR Imaging of the Effect of Gut Microbiota on Synaptic Density and Neurite Microstructure: A Preclinical Pilot Study.

The gut microbiome profoundly influences brain structure and function. The gut microbiome is hypothesized to play a key role in the etiopathogenesis of neuropsychiatric and neurodegenerative illness; however, the contribution of an intact gut microbiome to quantitative neuroimaging parameters of brain microstructure and function remains unknown. Herein, we report the broad and significant influence of a functional gut microbiome on commonly employed neuroimaging measures of diffusion tensor imaging (DTI), neurite orientation dispersion and density (NODDI) imaging, and SV2A 18F-SynVesT-1 synaptic density PET imaging when compared to germ-free animals. In this pilot study, we demonstrate that mice, in the presence of a functional gut microbiome, possess higher neurite density and orientation dispersion and decreased synaptic density when compared to age- and sex-matched germ-free mice. Our results reveal the region-specific structural influences and synaptic changes in the brain arising from the presence of intestinal microbiota. Further, our study highlights important considerations for the development of quantitative neuroimaging biomarkers for precision imaging in neurologic and psychiatric illness.

Longitudinal Molecular Imaging of Progesterone Receptor Reveals Early Differential Response to Endocrine Therapy in Breast Cancer with an Activating ESR1 Mutation.

Activating mutations in the estrogen receptor (ER) α-gene (ESR1) result in constitutive transcriptional activity in the absence of estrogen and are associated with endocrine resistance in metastatic ER-positive (+) breast cancer. It is not known how activating ESR1 mutations may alter the predictive values of molecular imaging agents for endocrine therapy response. This study investigated the effect of an activating ESR1 mutation on pretreatment 18F-fluoroestradiol (18F-FES) uptake and early assessment of endocrine therapy response using 18F-FDG and 18F-fluorofuranylnorprogesterone (18F-FFNP) PET/CT imaging of tumor glucose metabolism and progesterone receptor (PR) expression, respectively. Methods: ER+, PR+ T47D breast cancer cells expressing wild-type (WT) ER or an activating ESR1 mutation, Y537S-ER, were used to generate tumor xenografts in ovariectomized female immunodeficient mice supplemented with 17ß-estradiol. Tumor growth curves were determined in the presence or absence of estrogen and for ethanol vehicle control or fulvestrant treatment, a selective ER degrader. Pretreatment 18F-FES uptake was compared between Y537S-ER and WT-ER tumors. Longitudinal PET/CT imaging with 18F-FFNP and 18F-FDG was performed before and 7-9 d after the start of endocrine therapy with fulvestrant. Radiopharmaceutical uptake in Y537S-ER and WT-ER tumors was compared between baseline and follow-up scans. Statistical significance was determined using paired t testing for longitudinal imaging and 2-way ANOVA for the 18F-FFNP tissue biodistribution assay. Results: Y537S-ER xenografts showed estrogen-independent growth, whereas WT-ER tumors grew only with estrogen. Fulvestrant treatment for 28 d significantly reduced tumor volumes for WT-ER but only stabilized volumes for Y537S-ER. Baseline 18F-FES uptake did not significantly differ between WT-ER and Y537S-ER tumors. Fulvestrant treatment induced a similar early metabolic response for both WT-ER and Y537S-ER tumors. 18F-FFNP uptake in WT-ER tumors was significantly reduced after 7 d of fulvestrant treatment; however, this reduction did not occur in Y537S-ER tumors, which showed no significant change between baseline and follow-up PET/CT. Conclusion: Molecular imaging of PR expression dynamics could be a noninvasive approach for early identification of reduced effectiveness of endocrine therapy resulting from activating ESR1 mutations.

Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain.

Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2+, immunologically "cold") melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50-150 mm3) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 µg daily, treatment days 6-10), and anti-CTLA-4 (100 µg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8+ and CD4+) and monocytes/macrophages (F4/80+) but no changes in FOXP3+ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (86Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic ß-particle-emitting 90Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.

18F-Fluoroestradiol PET Imaging of Activating Estrogen Receptor-α Mutations in Breast Cancer.

The purpose of this study was to determine the effect of estrogen receptor-α gene (ESR1) mutations at the tyrosine (Y) 537 amino acid residue within the ligand binding domain on 18F-fluoroestradiol (18F-FES) binding and in vivo tumor uptake compared with wild-type (WT)-estrogen receptor α (ER). Methods: ER-negative MDA-MB-231 breast cancer cells were used to generate stable cell lines that express WT-ER, Y537S, or Y537C mutant ER. Receptor expression and localization were confirmed by Western blot and immunofluorescence, respectively. ER transcriptional function was measured using an estrogen response element-luciferase reporter gene assay and quantitative polymerase chain reaction analysis of ER-regulated endogenous target genes. Saturation binding and competition assays were performed to determine equilibrium dissociation constant (Kd) and half maximal inhibitory concentration (IC50) values. 18F-FES uptake was measured in tumor xenografts grown in female athymic nude mice by small-animal PET/CT imaging and tissue biodistribution using 5.55 MBq (150 µCi) of 18F-FES. A 10-fold-lower injected dose of 0.555 MBq (15 µCi) of 18F-FES was also used for tissue biodistribution. Statistical significance was determined using ANOVA. Results: Y537S and Y537C mutations resulted in increased ER transcriptional activity in the absence of estrogen compared with WT-ER (11.48 ± 2.42 fold; P = 0.0002, and 5.89 ± 0.94 fold; P = 0.04, respectively). Constitutive ER activation of two target genes (PGR and TFF1) in the absence of estrogen was also observed in Y537S- and Y537C-ER cells compared with WT-ER. Kd values for 18F-FES were 0.98 ± 0.54 nM for Y537S-ER (P = 0.27) and 0.24 ± 0.03 nM for Y537C-ER (P = 0.95) compared with 0.07 ± 0.03 nM for WT-ER. IC50 values were 0.22 ± 0.09 nM for Y537S-ER (P = 0.97), 0.18 ± 0.09 nM for Y537C-ER (P = 0.99), and 0.19 ± 0.11 nM for WT-ER. Tumor xenografts expressing Y537S-ER (mean percentage injected dose per gram, 1.45 ± 0.06; P = 0.77) and Y537C-ER (2.09 ± 0.20; P = 0.21) had similar 18F-FES uptake compared with WT-ER (1.68 ± 0.12). Comparable 18F-FES uptake between Y537S-, Y537C-, and WT-ER xenografts was also observed using a 10-fold-lower injected dose with the tissue biodistribution assay. Conclusion: Since tumoral uptake of 18F-FES is not significantly impacted by Y537S-ER or Y537C-ER mutations, the potential diagnostic utility of 18F-FES PET imaging is expected to be equally valid for patients with or without these activating ESR1 mutations.

Cellular Metabolic Heterogeneity In Vivo Is Recapitulated in Tumor Organoids.

Heterogeneous populations within a tumor have varying metabolic profiles, which can muddle the interpretation of bulk tumor imaging studies of treatment response. Although methods to study tumor metabolism at the cellular level are emerging, these methods provide a single time point "snapshot" of tumor metabolism and require a significant time and animal burden while failing to capture the longitudinal metabolic response of a single tumor to treatment. Here, we investigated a novel method for longitudinal, single-cell tracking of metabolism across heterogeneous tumor cell populations using optical metabolic imaging (OMI), which measures autofluorescence of metabolic coenzymes as a report of metabolic activity. We also investigated whether in vivo cellular metabolic heterogeneity can be accurately captured using tumor-derived three-dimensional organoids in a genetically engineered mouse model of breast cancer. OMI measurements of response to paclitaxel and the phosphatidylinositol-3-kinase inhibitor XL147 in tumors and organoids taken at single cell resolution revealed parallel shifts in metaboltruic heterogeneity. Interestingly, these previously unappreciated heterogeneous metabolic responses in tumors and organoids could not be attributed to tumor cell fate or varying leukocyte content within the microenvironment, suggesting that heightened metabolic heterogeneity upon treatment is largely due to heterogeneous metabolic shifts within tumor cells. Together, these studies show that OMI revealed remarkable heterogeneity in response to treatment, which could provide a novel approach to predict the presence of potentially unresponsive tumor cell subpopulations lurking within a largely responsive bulk tumor population, which might otherwise be overlooked by traditional measurements.

Sensitivity and Isoform Specificity of 18F-Fluorofuranylnorprogesterone for Measuring Progesterone Receptor Protein Response to Estradiol Challenge in Breast Cancer.

The purpose of this study was to evaluate the ability of 21-18F-fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione (18F-FFNP) to measure alterations in progesterone receptor (PR) protein level and isoform expression in response to estradiol challenge. Methods: T47D human breast cancer cells and female mice-bearing T47D tumor xenografts were treated with 17ß-estradiol (E2) to increase PR expression. 18F-FFNP uptake was measured using cell uptake and tissue biodistribution assays. MDA-MB-231 breast cancer clonal cell lines were generated that express the A or B isoforms of human PR. PR protein levels, transcriptional function, and subcellular localization were determined. In vitro 18F-FFNP binding was measured via saturation and competitive binding curves. In vivo 18F-FFNP uptake was measured using tumor xenografts and positron emission tomography. Statistical significance was determined using analysis of variance and t-tests. Results: After 48 and 72 h of E2, 18F-FFNP uptake in T47D cells was maximally increased compared to both vehicle and 24 h E2 treatment (p<0.0001 vs ethanol; P = 0.02 and P = 0.0002 vs 24 h for 48 and 72 h, respectively). T47D tumor xenografts in mice treated with 72 h E2 had maximal 18F-FFNP uptake compared to ethanol-treated mice (11.3±1.4 vs 5.2±0.81 %ID/g; P = 0.002). Corresponding tumor-to-muscle uptake ratios were 4.1±0.6, 3.9±0.5, and 2.3±0.4 for 48 h E2, 72 h E2, and ethanol-treated mice, respectively. There was no significant preferential 18F-FFNP binding or uptake by PR-A versus PR-B in the PR isoform-specific cell lines and tumor xenografts. Conclusion:18F-FFNP is capable of measuring estrogen-induced shifts in total PR expression in human breast cancer cells and tumor xenografts with equivalent isoform binding.

Preclinical Pharmacokinetics and Dosimetry Studies of 124I/131I-CLR1404 for Treatment of Pediatric Solid Tumors in Murine Xenograft Models.

Cancer is the second leading cause of death for children between the ages of 5 and 14 y. For children diagnosed with metastatic or recurrent solid tumors, for which the utility of external-beam radiotherapy is limited, the prognosis is particularly poor. The availability of tumor-targeting radiopharmaceuticals for molecular radiotherapy (MRT) has demonstrated improved outcomes in these patient populations, but options are nonexistent or limited for most pediatric solid tumors. 18-(p-iodophenyl)octadecylphosphocholine (CLR1404) is a novel antitumor alkyl phospholipid ether analog that broadly targets cancer cells. In this study, we evaluated the in vivo pharmacokinetics of 124I-CLR1404 (CLR 124) and estimated theranostic dosimetry for 131I-CLR1404 (CLR 131) MRT in murine xenograft models of the pediatric solid tumors neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Methods: Tumor-bearing mice were imaged with small-animal PET/CT to evaluate the whole-body distribution of CLR 124 and, correcting for differences in radioactive decay, predict that of CLR 131. Image volumes representing CLR 131 provided input for Geant4 Monte Carlo simulations to calculate subject-specific tumor dosimetry for CLR 131 MRT. Pharmacokinetics for CLR 131 were extrapolated to adult and pediatric humans to estimate normal-tissue dosimetry. In neuroblastoma, a direct comparison of CLR 124 with 124I-metaiodobenzylguanidine (124I-MIBG) in an MIBG-avid model was performed. Results: In vivo pharmacokinetics of CLR 124 showed selective uptake and prolonged retention across all pediatric solid tumor models investigated. Subject-specific tumor dosimetry for CLR 131 MRT presents a correlative relationship with tumor-growth delay after CLR 131 MRT. Peak uptake of CLR 124 was, on average, 22% higher than that of 124I-MIBG in an MIBG-avid neuroblastoma model. Conclusion: CLR1404 is a suitable theranostic scaffold for dosimetry and therapy with potentially broad applicability in pediatric oncology. Given the ongoing clinical trials for CLR 131 in adults, these data support the development of pediatric clinical trials and provide detailed dosimetry that may lead to improved MRT treatment planning.

Significant Suppression of CT Radiation-Induced DNA Damage in Normal Human Cells by the PrC-210 Radioprotector.

While computed tomography (CT) is now commonly used and considered to be clinically valuable, significant DNA double-strand breaks (γ-H2AX foci) in white blood cells from adult and pediatric CT patients have been frequently reported. In this study to determine whether γ-H2AX foci and X-ray-induced naked DNA damage are suppressed by administration of the PrC-210 radioprotector, human blood samples were irradiated in a CT scanner at 50-150 mGy with or without PrC-210, and γ-H2AX foci were scored. X-ray-induced naked DNA damage was also studied, and the DNA protective efficacy of PrC-210 was compared against 12 other common "antioxidants." PrC-210 reduced CT radiation-induced γ-H2AX foci in white blood cells to near background ( P < 0.0001) at radiation doses of 50-150 mGy. PrC-210 was most effective among the 13 "antioxidants" in reducing naked DNA X-ray damage, and its addition at 30 s before an •OH pulse reduced to background the •OH insult that otherwise induced >95% DNA damage. A systemic PrC-210 dose known to confer 100% survival in irradiated mice had no discernible effect on micro-CT image signal-to-noise ratio and CT image integrity. PrC-210 suppressed DNA damage to background or near background in each of these assay systems, thus supporting its development as a radioprotector for humans in multiple radiation exposure settings.