NIRDye 812: A molecular platform tailored for multimodal bioimaging applications of targeted fluorescence- and photoacoustic-guided surgery.

The primary treatment for malignant tumors remains to be surgical removal of the diseased tissue. The presence or absence of residual diseased tissue at the tumor margin is the strongest predictor of postoperative prognosis and recurrence. Accordingly, reliance on the ability of surgeons to visually distinguish diseased tissue from healthy tissue unambiguously in real time is crucial. Near infrared-I (NIRI) fluorescence-emitting targeting biomolecular constructs such as anticancer antibody-fluorophore conjugates, namely cetuximab-IRDye® 800CW (CTB-IRDye® 800CW), are FDA-approved for clinical trial usage in the fluorescence-guided resection of diseased tissue due to affording improved direct visualization of tumor tissue when compared to the use of either the unaided eye under standard white light illumination (WLI) surgical techniques or non-targeting fluorophores. Unfortunately, though helpful, CTB-IRDye® 800CW affords limited (i) identification of diseased tissue and (ii) tumor margin delineation, because the immunoconjugate generates suboptimal tumor-to-background ratios (TBRs) as a result of its spectral/photophysical profiles poorly aligning with the fixed optical windows of pre-/clinical setups. As such, CTB-IRDye® 800CW is more prone to affording incomplete resection compared to if TBRs were higher due to otherwise. To aid in accurately identifying deep-seated diseased tissue, photoacoustic (PA) tomography has been implemented alongside CTB-IRDye® 800CW to achieve PA signals that could result in higher TBRs. However, in clinical trial practice, using IRDye® 800CW for PA imaging also yields subpar TBRs due to it affording low PA signals. To overcome such limitations, we developed NIRDye 812, a structurally-modified topological equivalent of IRDye® 800CW, to confer it the capability to yield both higher TBRs and superior PA signal than that of the equivalent CTB-conjugate and fluorophore IRDye® 800CW itself, respectively. To do so, we substituted the oxygen atom at its meso-position with a sulfur atom. CTB-NIRDye 812 demonstrated a red-shifted absorption wavelength at 796 nm and a peak NIR-I fluorescence emission wavelength at 820 nm, which better dovetails with the fixed windows of preinstalled fixed emission filters within commercial pre-/clinical NIR-I fluorescence imaging instruments. Overall, CTB-NIRDye 812 provided a âˆ¼ 2-fold increase in TBRs compared to those of CTB-IRDye® 800CW in vivo. Also, NIRDye 812 displayed an ∼60% higher PA signal than that of IRDye® 800CW. Collectively, we achieved our goal of improving upon the spectral/photophysical and PA properties of IRDye® 800CW via introducing a subtle modification to its electronic core such that its CTB immunoconjugate could potentially allow for fast track or breakthrough designation by the FDA due to its near-identical structure displaying considerably improved efficacy.

Preclinical Evaluation of 89Zr-Panitumumab for Biology-Guided Radiation Therapy.

PURPOSE: Biology-guided radiation therapy (BgRT) uses real-time line-of-response data from on-board positron emission tomography (PET) detectors to guide beamlet delivery during therapeutic radiation. The current workflow requires 18F-fluorodeoxyglucose (FDG) administration daily before each treatment fraction. However, there are advantages to reducing the number of tracer injections by using a PET tracer with a longer decay time. In this context, we investigated 89Zr-panitumumab (89Zr-Pan), an antibody PET tracer with a half-life of 78 hours that can be imaged for up to 9 days using PET. METHODS AND MATERIALS: The BgRT workflow was evaluated preclinically in mouse colorectal cancer xenografts (HCT116) using small-animal positron emission tomography/computed tomography (PET/CT) for imaging and image-guided kilovoltage conformal irradiation for therapy. Mice (n = 5 per group) received 7 MBq of 89Zr-Pan as a single dose 2 weeks after tumor induction, with or without fractionated radiation therapy (RT; 6 × 6.6 Gy) to the tumor region. The mice were imaged longitudinally to assess the kinetics of the tracer over 9 days. PET images were then analyzed to determine the stability of the PET signal in irradiated tumors over time. RESULTS: Mice in the treatment group experienced complete tumor regression, whereas those in the control group were killed because of tumor burden. PET imaging of 89Zr-Pan showed well-delineated tumors with minimal background in both groups. On day 9 postinjection, tumor uptake of 89Zr-Pan was 7.2 ± 1.7 in the control group versus 5.2 ± 0.5 in the treatment group (mean percentage of injected dose per gram of tissue [%ID/g] ± SD; P = .07), both significantly higher than FDG uptake (1.1 ± 0.5 %ID/g) 1 hour postinjection. To assess BgRT feasibility, the clinical eligibility criteria was computed using human-equivalent uptake values that were extrapolated from preclinical PET data. Based on this semiquantitative analysis, BgRT may be feasible for 5 consecutive days after a single 740-MBq injection of 89Zr-Pan. CONCLUSIONS: This study indicates the potential of long-lived antibody-based PET tracers for guiding clinical BgRT.

Discovery of a CSF-1R inhibitor and PET tracer for imaging of microglia and macrophages in the brain.

Colony stimulating factor 1 receptor (CSF-1R) is a kinase expressed on macrophages and microglia in the brain. It has been recognized as a potential drug and imaging target in treatment of neuroinflammatory diseases and glioblastoma. Despite several attempts, no validated CSF-1R PET tracer is currently available. The aim of this work was to develop a brain permeable CSF-1R PET tracer for non-invasive imaging of CSF-1R in vivo. Based on fragments of two potent and selective CSF-1R inhibitors, novel hybrid molecules were designed and synthesized. Affinity for human recombinant CSF-1R and selectivity over c-KIT and PDGFR-ß was determined using a FRET based in vitro assay. Radiosynthesis was performed by fully automated [11C]CH3I methylation of the corresponding des-methyl precursor. PET imaging was performed at baseline, efflux transporter blocking and CSF-1R blocking conditions. Moreover, tracer distribution and blood and plasma radiometabolites were determined following injection in healthy mice. The most promising CSF-1R inhibitor, compound 4, showed high selectivity and high affinity for CSF-1R (IC50: 12 ± 3 nM) and no affinity for kinase family members c-KIT and PDGFR-beta. [11C]4 was obtained in good yield (15 ± 0.2 % decay corrected yield, (2.0 ± 0.26 GBq at end of synthesis) and excellent purity. The compound demonstrated high brain penetration and good metabolic stability (>2 %ID/g at 60 min post injection and 79 ± 8 % intact [11C]4 in brain at 60 min post injection) and no strong efflux transporter substrate behavior. Blocking CSF-1R prior to imaging with [11C]4 resulted in significant decrease in brain uptake. In conclusion, [11C]4 shows good potential as a novel PET tracer for imaging of CSF-1R in the CNS and future experiments in relevant animal models are warranted.

89Zr-panitumumab Combined With 18F-FDG PET Improves Detection and Staging of Head and Neck Squamous Cell Carcinoma.

PURPOSE: Determine the safety and specificity of a tumor-targeted radiotracer (89Zr-pan) in combination with 18F-FDG PET/CT to improve diagnostic accuracy in head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: Adult patients with biopsy-proven HNSCC scheduled for standard-of-care surgery were enrolled in a clinical trial and underwent systemic administration of 89Zirconium-panitumumab and panitumumab-IRDye800 followed by preoperative 89Zr-pan PET/CT and intraoperative fluorescence imaging. The sensitivity, specificity, and AUC were evaluated. RESULTS: A total of fourteen patients were enrolled and completed the study. Four patients (28.5%) had areas of high 18F-FDG uptake outside the head and neck region with maximum standardized uptake values (SUVmax) greater than 2.0 that were not detected on 89Zr-pan PET/CT. These four patients with incidental findings underwent further workup and had no evidence of cancer on biopsy or clinical follow-up. Forty-eight lesions (primary tumor, LNs, incidental findings) with SUVmax ranging 2.0-23.6 were visualized on 18F-FDG PET/CT; 34 lesions on 89Zr-pan PET/CT with SUVmax ranging 0.9-10.5. The combined ability of 18F-FDG PET/CT and 89Zr-pan PET/CT to detect HNSCC in the whole body was improved with higher specificity of 96.3% [confidence interval (CI), 89.2%-100%] compared to 18F-FDG PET/CT alone with specificity of 74.1% (CI, 74.1%-90.6%). One possibly related grade 1 adverse event of prolonged QTc (460 ms) was reported but resolved in follow-up. CONCLUSIONS: 89Zr-pan PET/CT imaging is safe and may be valuable in discriminating incidental findings identified on 18F-FDG PET/CT from true positive lesions and in localizing metastatic LNs.

Limitations of Fluorine 18 Fluoromisonidazole in Assessing Treatment-induced Tissue Hypoxia after Transcatheter Arterial Embolization of Hepatocellular Carcinoma: A Prospective Pilot Study.

Purpose To determine the variance and correlation with tumor viability of fluorine 18 (18F) fluoromisonidazole (FMISO) uptake in hepatocellular carcinoma (HCC) prior to and after embolization treatment. Materials and Methods In this single-arm, single-center, prospective pilot study between September 2016 and March 2017, participants with at least one tumor measuring 1.5 cm or larger with imaging or histologic findings diagnostic for HCC were enrolled (five men; mean age, 68 years; age range, 61-76 years). Participants underwent 18F-FMISO PET/CT before and after bland embolization of HCC. A tumor-to-liver ratio (TLR) was calculated by using standardized uptake values of tumor and liver. The difference in mean TLR before and after treatment was compared by using a Wilcoxon rank sum test, and correlation between TLR and tumor viability was assessed by using the Spearman rank correlation coefficient. Results Four participants with five tumors were included in the final analysis. The median tumor diameter was 3.2 cm (IQR, 3.0-3.9 cm). The median TLR before treatment was 0.97 (IQR, 0.88-0.98), with a variance of 0.02, and the median TLR after treatment was 0.85 (IQR, 0.79-1), with a variance of 0.01; both findings indicate a narrow range of 18F-FMISO uptake in HCC. The Spearman rank correlation coefficient was 0.87, indicating a high correlation between change in TLR and nonviable tumor. Conclusion Although there was a correlation between change in TLR and response to treatment, the low signal-to-noise ratio of 18F-FMISO in the liver limited its use in HCC. Keywords: Molecular Imaging-Clinical Translation, Embolization, Abdomen/Gastrointestinal, Liver Clinical trial registration no. NCT02695628 © RSNA, 2022.

18F-FSPG PET/CT Imaging of System xC- Transporter Activity in Patients with Primary and Metastatic Brain Tumors.

Background The PET tracer (4S)-4-(3-[18F]fluoropropyl)-l-glutamate (18F-FSPG) targets the system xC- cotransporter, which is overexpressed in various tumors. Purpose To assess the role of 18F-FSPG PET/CT in intracranial malignancies. Materials and Methods Twenty-six patients (mean age, 54 years ± 12; 17 men; 48 total lesions) with primary brain tumors (n = 17) or brain metastases (n = 9) were enrolled in this prospective, single-center study (ClinicalTrials.gov identifier: NCT02370563) between November 2014 and March 2016. A 30-minute dynamic brain 18F-FSPG PET/CT scan and a static whole-body (WB) 18F-FSPG PET/CT scan at 60-75 minutes were acquired. Moreover, all participants underwent MRI, and four participants underwent fluorine 18 (18F) fluorodeoxyglucose (FDG) PET imaging. PET parameters and their relative changes were obtained for all lesions. Kinetic modeling was used to estimate the 18F-FSPG tumor rate constants using the dynamic and dynamic plus WB PET data. Imaging parameters were correlated to lesion outcomes, as determined with follow-up MRI and/or pathologic examination. The Mann-Whitney U test or Student t test was used for group mean comparisons. Receiver operating characteristic curve analysis was used for performance comparison of different decision measures. Results 18F-FSPG PET/CT helped identify all 48 brain lesions. The mean tumor-to-background ratio (TBR) on the whole-brain PET images at the WB time point was 26.6 ± 24.9 (range: 2.6-150.3). When 18F-FDG PET was performed, 18F-FSPG permitted visualization of non-18F-FDG-avid lesions or allowed better lesion differentiation from surrounding tissues. In participants with primary brain tumors, the predictive accuracy of the relative changes in influx rate constant Ki and maximum standardized uptake value to discriminate between poor and good lesion outcomes were 89% and 81%, respectively. There were significant differences in the 18F-FSPG uptake curves of lesions with good versus poor outcomes in the primary brain tumor group (P < .05) but not in the brain metastases group. Conclusion PET/CT imaging with (4S)-4-(3-[18F]fluoropropyl)-l-glutamate (18F-FSPG) helped detect primary brain tumors and brain metastases with a high tumor-to-background ratio. Relative changes in 18F-FSPG uptake with multi-time-point PET appear to be helpful in predicting lesion outcomes. Clinical trial registration no. NCT02370563 © RSNA, 2022 Online supplemental material is available for this article.

BLZ945 derivatives for PET imaging of colony stimulating factor-1 receptors in the brain.

BACKGROUND: The kinase colony stimulating factor-1 receptor (CSF-1R) has recently been identified as a novel therapeutic target for decreasing tumor associated macrophages and microglia load in cancer treatment. In glioblastoma multiforme (GBM), a high-grade cancer in the brain with extremely poor prognosis, macrophages and microglia can make up to 50% of the total tumor mass. Currently, no non-invasive methods are available for measuring CSF-1R expression in vivo. The aim of this work is to develop a PET tracer for imaging of CSF-1R receptor expression in the brain for future GBM patient selection and treatment monitoring. METHODS: BLZ945 and a derivative that potentially allows for fluorine-18 labeling were synthesized and evaluated in vitro to determine their affinity towards CSF-1R. BLZ945 was radiolabeled with carbon-11 by N-methylation of des-methyl-BLZ945 using [11C]CH3I. Following administration to healthy mice, metabolic stability of [11C]BLZ945 in blood and brain and activity distribution were determined ex vivo. PET scanning was performed at baseline, efflux transporter blocking, and CSF-1R blocking conditions. Finally, [11C]BLZ945 binding was evaluated in vitro by autoradiography on mouse brain sections. RESULTS: BLZ945 was the most potent compound in our series with an IC50 value of 6.9 ± 1.4 nM. BLZ945 was radiolabeled with carbon-11 in 20.7 ± 1.1% decay corrected radiochemical yield in a 60 min synthesis procedure with a radiochemical purity of >95% and a molar activity of 153 ± 34 GBq·µmol-1. Ex vivo biodistribution showed moderate brain uptake and slow wash-out, in addition to slow blood clearance. The stability of BLZ945 in blood plasma and brain was >99% at 60 min post injection. PET scanning demonstrated BLZ945 to be a substrate for efflux transporters. High brain uptake was observed, which was shown to be mostly non-specific. In accordance, in vitro autoradiography on brain sections revealed high non-specific binding. CONCLUSIONS: [11C]BLZ945, a CSF-1R PET tracer, was synthesized in high yield and purity. The tracer has high potency for the target, however, future studies are warranted to address non-specific binding and tracer efflux before BLZ945 or derivatives could be translated into humans for brain imaging.

Evaluation of carbon-11 labeled 5-(1-methyl-1H-pyrazol-4-yl)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)nicotinamide as PET tracer for imaging of CSF-1R expression in the brain.

Pharmacological targeting of tumor associated macrophages and microglia in the tumor microenvironment is a novel therapeutic strategy in the treatment of glioblastoma multiforme. As such, the colony stimulating factor-1 receptor (CSF-1R) has been identified as a druggable target. However, no validated companion diagnostic marker for these therapies exists to date. Towards development of a CSF-1R PET tracer, a set of six compounds based on recently reported CSF-1R inhibitor 5-(1-methyl-1H-pyrazol-4-yl)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)nicotinamide (Compound 5) was designed, synthesized and evaluated in vitro for potency and selectivity. The highest affinity for CSF-1R was found for compound 5 (IC50: 2.7 nM). Subsequent radiosynthesis of [11C]5 was achieved in 2.0 ± 0.2% yield (decay corrected to start of synthesis) by carbon-11 carbon monoxide aminocarbonylation in 40 min after end of bombardment. In vitro autoradiography with [11C]5 on rat brain sections demonstrated high specific binding, but also strong off-target binding. Ex vivo, only intact tracer was observed in blood plasma at 90 min post injection in healthy rats. PET scanning results demonstrated negligible brain uptake under baseline conditions and this brain uptake did not increase by blocking of efflux transporters using Tariquidar. To conclude, [11C]5 was successfully synthesized and evaluated in healthy rats. However, the inability of [11C]5 to cross the blood-brain-barrier excludes its use for imaging of CSF-1R expression in the brain.

A NIR fluorescent smart probe for imaging tumor hypoxia.

BACKGROUND: Tumor hypoxia is a characteristic of paramount importance due to low oxygenation levels in tissue negatively correlating with resistance to traditional therapies. The ability to noninvasively identify such could provide for personalized treatment(s) and enhance survival rates. Accordingly, we recently developed an NIR fluorescent hypoxia-sensitive smart probe (NO2 -Rosol) for identifying hypoxia via selectively imaging nitroreductase (NTR) activity, which could correlate to oxygen deprivation levels in cells, thereby serving as a proxy. We demonstrated proof of concept by subjecting a glioblastoma (GBM) cell line to extreme stress by evaluating such under radiobiological hypoxic (pO2 ≤ ~0.5%) conditions, which is a far cry from representative levels for hypoxia for brain glioma (pO2  = ~1.7%) which fluctuate little from physiological hypoxic (pO2  = 1.0-3.0%) conditions. AIM: We aimed to evaluate the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia in vitro and in vivo via assessing NTR activity in diverse GBM models under relevant oxygenation levels (pO2  = 2.0%) within physiological hypoxic conditions that mimic oxygenation levels in GBM tumor tissue in the brain. METHODS: We evaluated multiple GBM cell lines to determine their relative sensitivity to oxygenation levels via measuring carbonic anhydrase IX (CAIX) levels, which is a surrogate marker for indirectly identifying hypoxia by reporting on oxygen deprivation levels and upregulated NTR activity. We evaluated for hypoxia via measuring NTR activity when employing NO2 -Rosol in in vitro and tumor hypoxia imaging studies in vivo. RESULTS: The GBM39 cell line demonstrated the highest CAIX expression under hypoxic conditions representing that of GBM in the brain. NO2 -Rosol displayed an 8-fold fluorescence enhancement when evaluated in GBM39 cells (pO2  = 2.0%), thereby establishing its robustness and suitability for imaging hypoxia under relevant physiological conditions. We demonstrated the feasibility of NO2 -Rosol to afford tumor hypoxia imaging in vivo via it demonstrating a tumor-to-background of 5 upon (i) diffusion throughout, (ii) bioreductive activation by NTR activity in, and (iii) retention within, GBM39 tumor tissue. CONCLUSION: We established the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia under relevant oxygenation levels in vitro and in vivo via assessing NTR activity in GBM39 models.

[18F]-C-SNAT4: an improved caspase-3-sensitive nanoaggregation PET tracer for imaging of tumor responses to chemo- and immunotherapies.

Positron emission tomography (PET) imaging of apoptosis can noninvasively detect cell death in vivo and assist in monitoring tumor response to treatment in patients. While extensive efforts have been devoted to addressing this important need, no apoptosis PET imaging agents have yet been approved for clinical use. This study reports an improved 18F-labeled caspase-sensitive nanoaggregation tracer ([18F]-C-SNAT4) for PET imaging of tumor response to chemo- and immunotherapies in preclinical mouse models. METHODS: We rationally designed and synthesized a new PET tracer [18F]-C-SNAT4 to detect cell death both in vitro and in vivo. In vitro radiotracer uptake studies were performed on drug-sensitive and -resistant NSCLC cell lines (NCI-H460 and NCI-H1299, respectively) treated with cisplatin at different doses. In vivo therapy response monitoring by [18F]-C-SNAT4 PET imaging was evaluated with two treatment modalities-chemotherapy and immunotherapy in two tumor xenografts in mice. Radiotracer uptake in the tumors was validated ex vivo using γ-counting and cleaved caspase-3 immunofluorescence. RESULTS: This [18F]-C-SNAT4 PET tracer was facilely synthesized and displayed improved serum stability profiles. [18F]-C-SNAT4 cellular update was elevated in NCI-H460 cells in a time- and dose-dependent manner, which correlated well with cell death. A significant increase in [18F]-C-SNAT4 uptake was measured in NCI-H460 tumor xenografts in mice. In contrast, a rapid clearance of [18F]-C-SNAT4 was observed in drug-resistant NCI-H1299 in vitro and in tumor xenografts. Moreover, in BALB/C mice bearing murine colon cancer CT26 tumor xenografts receiving checkpoint inhibitors, [18F]-C-SNAT4 showed its ability for monitoring immunotherapy-induced apoptosis and reporting treatment-responding mice from non-responding. CONCLUSION: The uptake of [18F]-C-SNAT4 in tumors received chemotherapy and immunotherapy is positively correlated with the tumor apoptotic level and the treatment efficacy. [18F]-C-SNAT4 PET imaging can monitor tumor response to two different treatment modalities and predict the therapeutic efficacy in preclinical mouse models.

In Vivo Imaging of Methionine Aminopeptidase II for Prostate Cancer Risk Stratification.

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18-labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. SIGNIFICANCE: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.

Two Patient Studies of a Companion Diagnostic Immuno-Positron Emission Tomography (PET) Tracer for Measuring Human CA6 Expression in Cancer for Antibody Drug Conjugate (ADC) Therapy.

An antigen binding fragment (BFab) derived from a tumor-associated mucin 1-sialoglycotope antigen (CA6) targeting antibody (huDS6) was engineered. We synthesized a companion diagnostic positron emission tomography (PET) tracer by radiolabeling BFab with [64Cu] to measure CA6 expression on cancer tissues prior to anti-human CA6 (huDS6-DM4 antibody-drug conjugate) therapy for ovarian and breast cancer patients. After chemotherapy, the ovarian patient received PET scan with 18F-2-fluoro-2-deoxyglucose ([18F]FDG: 10 mCi), followed by [64Cu]-DOTA-BFab ([64Cu]BFab; 5.5 mCi) 1 week later for PET scanning of CA6 expression and subsequent surgery. The breast cancer patient was treated with chemotherapy before primary tumor resection and subsequent [18F]FDG-PET scan. 4 weeks later the patient received of [64Cu]BFab (11.7 mCi) for CA6 PET scan. Whole body [18F]FDG-PET of the breast cancer patient indicated FDG-avid tumor metastases to the liver, bilateral hila and thoracic spine, but no uptake was observed for the ovarian patient. Each patient was also imaged by PET/CT with [64Cu]BFab at 1 and 24 hours after tracer administration. The [64Cu]BFab tracer was well tolerated by both patients without adverse effects, and no significant tracer uptake was observed in both patients. Immunohistochemistry (IHC) data indicated CA6 expressions were weak to intermediate and matched with the [64Cu]BFab-PET signals.

Initial evaluation of (4S)-4-(3-[18F]fluoropropyl)-L-glutamate (FSPG) PET/CT imaging in patients with head and neck cancer, colorectal cancer, or non-Hodgkin lymphoma.

PURPOSE: (4S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid ([18F]FSPG) measures system xC- transporter activity and shows promise for oncologic imaging. We present data on tumor uptake of this radiopharmaceutical in human subjects with head and neck cancer (HNC), colorectal cancer (CRC), and non-Hodgkin lymphoma (NHL). METHODS: A total of 15 subjects with HNC (n = 5), CRC (n = 5), or NHL (n = 5) were recruited (mean age 66.2 years, range 44-87 years). 301.4 ± 28.1 MBq (8.1 ± 0.8 mCi) of [18F]FSPG was given intravenously to each subject, and 3 PET/CT scans were obtained 0-2 h post-injection. All subjects also had a positive [18F]FDG PET/CT scan within 1 month prior to the [18F]FSPG PET scan. Semi-quantitative and visual comparisons of the [18F]FSPG and [18F]FDG scans were performed. RESULTS: [18F]FSPG showed strong uptake in all but one HNC subject. The lack of surrounding brain uptake facilitated tumor delineation in the HNC patients. [18F]FSPG also showed tumor uptake in all CRC subjects, but variable uptake in the NHL subjects. While the absolute [18F]FDG SUV values were comparable or higher than [18F]FSPG, the tumor-to-background SUV ratios were greater with [18F]FSPG than [18F]FDG. CONCLUSIONS: [18F]FSPG PET/CT showed promising results across 15 subjects with 3 different cancer types. Concordant visualization was mostly observed between [18F]FSPG and [18F]FDG PET/CT images, with some inter- and intra-individual uptake variability potentially reflecting differences in tumor biology. The tumor-to-background ratios were greater with [18F]FSPG than [18F]FDG in the cancer types evaluated. Future studies based on larger numbers of subjects and those with a wider array of primary and recurrent or metastatic tumors are planned to further evaluate the utility of this novel tracer.

Reactive oxygen species and enzyme dual-responsive biocompatible drug delivery system for targeted tumor therapy.

Spurred by newly developed drug delivery systems (DDSs), side effects of cancer chemotherapy could be reduced by using multifunctional nanoplatforms. However, the facile synthesis of effective DDSs remains a challenge. Here, a six-arginine-tailed anti-epidermal growth factor receptor (EGFR) affibody was employed to easily synthesize the highly reactive oxygen species (hROS)- and trypsin-responsive 11-mercaptoundecanoic acid-modified gold nanoclusters (MUA-Au NCs) for tumor-targeted drug delivery. The polyarginine moiety of affibody sealed methotrexate (MTX)-loaded MUA-Au NCs through charge effect, as well as leaving the rest targeting fragment of the affibody to specifically bind tumor overexpressed EGFR. As the shell of MUA-Au NCs-MTX-Affibody (MAMA), polyarginine chains of affibody could be digested by trypsin, helping to release MTX from MAMA. The released MTX accelerated destroying MUA-Au NCs through inducing the generation of hROS. Specifically targeting EGFR-overexpressed tumors, quickly delivering a sufficient amount of drug to the tumor, subsequently increasing the local MTX and hROS levels, and safely eliminating the biocompatible structure from kidney, endowed MAMA greater treatment effectiveness and lower side effect than chemotherapy, especially in pancreatic cancer due to its high trypsin level. This simply fabricated DDS may find applications in high effective cancer therapy, especially for tumors with high trypsin activity.

An Activatable NIR Fluorescent Rosol for Selectively Imaging Nitroreductase Activity.

Hypoxia (pO2 ≤ ~1.5%) is an important characteristic of tumor microenvironments that directly correlates with resistance against first-line therapies and tumor proliferation/infiltration. The ability to accurately identify hypoxic tumor cells/tissue could afford tailored therapeutic regimens for personalized treatment, development of more-effective therapies, and discerning the mechanisms underlying disease progression. Fluorogenic constructs identifying aforesaid cells/tissue operate by targeting the bioreductive activity of primarily nitroreductases (NTRs), but collectively present photophysical and/or physicochemical shortcomings that could limit effectiveness. To overcome these limitations, we present the rational design, development, and evaluation of the first activatable ultracompact xanthene core-based molecular probe (NO 2 -Rosol) for selectively imaging NTR activity that affords an "OFF-ON" near-infrared (NIR) fluorescence response (> 700 nm) alongside a remarkable Stokes shift (> 150 nm) via NTR activity-facilitated modulation to its energetics whose resultant interplay discontinues an intramolecular d-PET fluorescence-quenching mechanism transpiring between directly-linked electronically-uncoupled π-systems comprising its components. DFT calculations guided selection of a suitable fluorogenic scaffold and nitroaromatic moiety candidate that when adjoined could (i) afford such photophysical response upon bioreduction by upregulated NTR activity in hypoxic tumor cells/tissue and (ii) employ a retention mechanism strategy that capitalizes on an inherent physical property of the NIR fluorogenic scaffold for achieving signal amplification. NO 2 -Rosol demonstrated 705 nm NIR fluorescence emission and 157 nm Stokes shift, selectivity for NTR over relevant bioanalytes, and a 28-/12-fold fluorescence enhancement in solution and between cells cultured under different oxic conditions, respectively. In establishing feasibility for NO 2 -Rosol to provide favorable contrast levels in solutio/vitro, we anticipate NO 2 -Rosol doing so in preclinical studies.

Off-Peak Near-Infrared-II (NIR-II) Bioimaging of an Immunoconjugate Having Peak Fluorescence Emission in the NIR-I Spectral Region for Improving Tumor Margin Delineation.

The primary treatment for malignant tumors remains to be resection. The strongest predictor of recurrence and postoperative prognosis is whether diseased tissue/cells remain(s) at the surgical margin. Cancer surgery entails surgeons having the capability to visually distinguish between subtle shades of color in attempts of differentiating between diseased tissue and healthy tissue under standard white-light illumination, as such tissue states appear identical at the meso-/macroscopic level. Accordingly, enhancing the capability of surgeons to do so such that they can accurately delineate the tumor margin is of paramount importance. Fluorescence-guided surgery facilitates in enhancing such capability by color-coding the surgical field with overlaid contrasting pseudo-colors from real-time intraoperative fluorescence emission via utilizing fluorescent constructs in tandem. Constructs undergoing clinical trials or that are FDA-approved provide peak fluorescence emission in the visible (405 - 700 nm) or near-infrared-I (NIR-I) spectral region (700-900 nm), whereby differentiation between tissue states progressively improves in sync with using constructs that emit longer wavelengths of light. Here, we repurpose the usage of such fluorescent constructs by establishing feasibility of a tumor-targeting immunoconjugate (cetuximab-IRDye800) having peak fluorescence emission at the NIR-I spectral region to provide improved tumor margin delineation by affording higher tumor-to-background ratios (TBRs) when measuring its off-peak fluorescence emission at the near-infrared-II (NIR-II) spectral region (1000-1700 nm) in in vivo applications. We prepared murine tumor models, administered such immunoconjugate, and imaged such models pre-/post-administration via utilizing imaging systems that separately afforded acquisition of fluorescence emission in the NIR-I or NIR-II spectral region. On doing so, we determined in vivo TBRs, ex vivo TBRs with/-out skin, and ex vivo biodistribution, all via measuring the fluorescence emission of the immunoconjugate at tumor site(s) at both spectral regions. Collectively, we established feasibility of using the immunoconjugate to afford improved tumor margin delineation by providing 2-fold higher TBRs via utilizing the NIR-II spectral region to capture off-peak fluorescence emission from a fluorescent construct having NIR-I peak fluorescence emission.

Targeting intracranial patient-derived glioblastoma (GBM) with a NIR-I fluorescent immunoconjugate for facilitating its image-guided resection.

Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor type and is associated with a high mortality rate borne out of such affording a survival rate of only 15 months. GBM aggressiveness is associated with the overexpression of epidermal growth factor receptor (EGFR) and its mutants. Targeting GBM with therapeutics is challenging because the blood-brain barrier (BBB) permits primarily select small-molecule entities across its semipermeable blockade. However, recent preclinical data suggest that large biomolecules, such as the anti-EGFR antibody therapeutic, cetuximab, could be capable of bypassing the BBB despite the relative enormity of its size. As such, we set forth to establish the feasibility of utilizing an EGFR-targeting near-infrared-I (NIR-I) fluorescent construct in the form of an immunoconjugate (cetuxmimab-IRDye800) to achieve visual differentiation between diseased brain tissue arising from a low-passage patient-derived GBM cell line (GBM39) and healthy brain tissue via utilizing orthotopic intracranial murine GBM39 tumor models for in vivo and ex vivo evaluation such that by doing so would establish proof of concept for ultimately facilitating its in vivo fluorescence-guided resection and ex vivo surgical back-table pathological confirmation in the clinic. As anticipated, we were not capable of distinguishing between malignant tumor tissue and healthy tissue in resected intact and slices of whole brain ex vivo under white-light illumination (WLI) due to both the diseased tissue and healthy tissue appearing virtually identical to the unaided eye. However, we readily observed over an average 6-fold enhancement in the fluorescence emission in the resected intact whole brain ex vivo when performing NIR-I fluorescence imaging (FLI) on the cohort of GBM39 tumor models that were administered the immunoconjugate compared to controls. In all, we laid the initial groundwork for establishing that NIR-I fluorescent immunoconjugates (theranostics) such as cetuximab-IRDye800 can bypass the BBB to visually afford GBM39 tumor tissue differentiation for its image-guided surgical removal.

Theranostic nanoparticles enhance the response of glioblastomas to radiation.

Despite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan-Meier curves and a log rank (Mantel-Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs.

Evaluation of integrin αvß6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.

FLT-PET-CT for the Detection of Disease Recurrence After Stereotactic Ablative Radiotherapy or Hyperfractionation for Thoracic Malignancy: A Prospective Pilot Study.

Differentiating local recurrence from post-treatment changes on PET scans following stereotactic ablative radiotherapy (SABR) or hyperfractionation for lung tumors is challenging. We performed a prospective pilot study of 3-deoxy-3-[18F]-fluorothymidine (FLT)-PET-CT in patients with equivocal post-radiation FDG-PET-CT to assess disease recurrence. Methods: We prospectively enrolled 10 patients, 9 treated with SABR and 1 with hyperfractionated external beam radiotherapy for thoracic malignancy with subsequent equivocal follow-up FDG-PET-CT, to undergo FLT-PET-CT prior to biopsy or serial imaging. FLT-PET scans were interpreted by a radiologist with experience in reading FLT-PET-CT and blinded to the results of any subsequent biopsy or imaging. Results: Of the 10 patients enrolled, 8 were evaluable after FLT-PET-CT. Based on the FLT-PET-CT, a blinded radiologist accurately predicted disease recurrence vs. inflammatory changes in 7 patients (87.5%). The combination of higher lesion SUVmax and higher ratio of lesion SUVmax to SUVmax of mediastinal blood pool was indicative of recurrence. Qualitative assessment of increased degree of focality of the lesion also appears to be indicative of disease recurrence. Conclusion: Adjunctive FLT-PET-CT imaging can complement FDG-PET-CT scan in distinguishing post-treatment radiation changes from disease recurrence in thoracic malignancies. These findings support the investigation of FLT-PET-CT in a larger prospective study.