Microenvironment-triggered multimodal precision diagnostics.

Therapeutic outcomes in oncology may be aided by precision diagnostics that offer early detection, localization and the opportunity to monitor response to therapy. Here, we report a multimodal nanosensor engineered to target tumours through acidosis, respond to proteases in the microenvironment to release urinary reporters and (optionally) carry positron emission tomography probes to enable localization of primary and metastatic cancers in mouse models of colorectal cancer. We present a paradigm wherein this multimodal sensor can be employed longitudinally to assess burden of disease non-invasively, including tumour progression and response to chemotherapy. Specifically, we showed that acidosis-mediated tumour insertion enhanced on-target release of matrix metalloproteinase-responsive reporters in urine. Subsequent on-demand loading of the radiotracer 64Cu allowed pH-dependent tumour visualization, enabling enriched microenvironmental characterization when compared with the conventional metabolic tracer 18F-fluorodeoxyglucose. Through tailored target specificities, this modular platform has the capacity to be engineered as a pan-cancer test that may guide treatment decisions for numerous tumour types.

Glypican-3-Targeted Alpha Particle Therapy for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is expressed in 75% of hepatocellular carcinoma (HCC), but not normal liver, making it a promising HCC therapeutic target. GC33 is a full-length humanized monoclonal IgG1 specific to GPC3 that can localize to HCC in vivo. GC33 alone failed to demonstrate therapeutic efficacy when evaluated in patients with HCC; however, we posit that cytotoxic functionalization of the antibody with therapeutic radionuclides, may be warranted. Alpha particles, which are emitted by radioisotopes such as Actinium-225 (Ac-225) exhibit high linear energy transfer and short pathlength that, when targeted to tumors, can effectively kill cancer and limit bystander cytotoxicity. Macropa, an 18-member heterocyclic crown ether, can stably chelate Ac-225 at room temperature. Here, we synthesized and evaluated the efficacy of [225Ac]Ac-Macropa-GC33 in mice engrafted with the GPC3-expressing human liver cancer cell line HepG2. Following a pilot dose-finding study, mice (n = 10 per group) were treated with (1) PBS, (2) mass-equivalent unmodified GC33, (3) 18.5 kBq [225Ac]Ac-Macropa-IgG1 (isotype control), (4) 9.25 kBq [225Ac]Ac-Macropa-GC33, and (5) 18.5 kBq [225Ac]Ac-Macropa-GC33. While significant toxicity was observed in all groups receiving radioconjugates, the 9.25 kBq [225Ac]Ac-Macropa-GC33 group demonstrated a modest survival advantage compared to PBS (p = 0.0012) and 18.5 kBq [225Ac]Ac-IgG1 (p = 0.0412). Hematological analysis demonstrated a marked, rapid reduction in white blood cells in all radioconjugate-treated groups compared to the PBS and unmodified GC33 control groups. Our studies highlight a significant disadvantage of using directly-labeled biomolecules with long blood circulation times for TAT. Strategies to mitigate such treatment toxicity include dose fractionation, pretargeting, and using smaller targeting ligands.

Noninvasive imaging of tumor hypoxia after nanoparticle-mediated tumor vascular disruption.

We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, including tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disruption. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nanoparticles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model.

In Vitro Performance of Published Glypican 3-Targeting Peptides TJ12P1 and L5 Indicates Lack of Specificity and Potency.

Background: Glypican 3 (GPC3), a plasma membrane heparan sulfate proteoglycan, is overexpressed on human hepatocellular carcinoma and may represent a promising biomarker. Several studies have reported peptides that selectively bind to GPC3 and could serve as scaffolds for imaging or therapeutic agents. Materials and Methods: We synthesized variants of two previously published peptides, DHLASLWWGTEL (TJ12P1) and RLNVGGTYFLTTRQ (L5), and evaluated their in vitro binding performance in paired isogenic cell lines, A431(GPC3-) and A431-GPC3+ (G1), as well as the liver cancer cell line HepG2. Using flow cytometry and biolayer interferometry (BLI), we compared the binding of the TJ12P1 and L5 peptide variants to the binding of corresponding scrambled peptides having the same amino acid composition, but in random sequence. Results: While both peptides bound to G1 and HepG2, they also bound to A431. The corresponding scrambled peptides demonstrated greater apparent binding to both G1 and A431 than their specific counterparts. BLI confirmed lack of binding at 0.5-1 µM for both peptides. Conclusions: We conclude that neither TJ12P1 nor L5 variant demonstrates selectivity for GPC3 at concentrations near the reported KD, and that the peptides lack potency or are nonspecific, making them inadequate for use as imaging agents.

Integrating Small Animal Irradiators withFunctional Imaging for Advanced Preclinical Radiotherapy Research.

Translational research aims to provide direct support for advancing novel treatment approaches in oncology towards improving patient outcomes. Preclinical studies have a central role in this process and the ability to accurately model biological and physical aspects of the clinical scenario in radiation oncology is critical to translational success. The use of small animal irradiators with disease relevant mouse models and advanced in vivo imaging approaches offers unique possibilities to interrogate the radiotherapy response of tumors and normal tissues with high potential to translate to improvements in clinical outcomes. The present review highlights the current technology and applications of small animal irradiators, and explores how these can be combined with molecular and functional imaging in advanced preclinical radiotherapy research.

High Single Doses of Radiation May Induce Elevated Levels of Hypoxia in Early-Stage Non-Small Cell Lung Cancer Tumors.

PURPOSE: Tumor hypoxia correlates with treatment failure in patients undergoing conventional radiation therapy. However, no published studies have investigated tumor hypoxia in patients undergoing stereotactic body radiation therapy (SBRT). We aimed to noninvasively quantify the tumor hypoxic volume (HV) in non-small cell lung cancer (NSCLC) tumors to elucidate the potential role of tumor vascular response and reoxygenation at high single doses. METHODS AND MATERIALS: Six SBRT-eligible patients with NSCLC tumors >1 cm were prospectively enrolled in an institutional review board-approved study. Dynamic positron emission tomography images were acquired at 0 to 120 minutes, 150 to 180 minutes, and 210 to 240 minutes after injection of 18F-fluoromisonidazole. Serial imaging was performed prior to delivery of 18 Gy and at approximately 48 hours and approximately 96 hours after SBRT. Tumor HVs were quantified using the tumor-to-blood ratio (>1.2) and rate of tracer influx (>0.0015 mL·min·cm-3). RESULTS: An elevated and in some cases persistent level of tumor hypoxia was observed in 3 of 6 patients. Two patients exhibited no detectable baseline tumor hypoxia, and 1 patient with high baseline hypoxia only completed 1 imaging session. On the basis of the tumor-to-blood ratio, in the remaining 3 patients, tumor HVs increased on day 2 after 18 Gy and then showed variable responses on day 4. In the 3 of 6 patients with detectable hypoxia at baseline, baseline tumor HVs ranged between 17% and 24% (mean, 21%), and HVs on days 2 and 4 ranged between 33% and 45% (mean, 40%) and between 18% and 42% (mean, 28%), respectively. CONCLUSIONS: High single doses of radiation delivered as part of SBRT may induce an elevated and in some cases persistent state of tumor hypoxia in NSCLC tumors. Hypoxia imaging with 18F-fluoromisonidazole positron emission tomography should be used in a larger cohort of NSCLC patients to determine whether elevated tumor hypoxia is predictive of treatment failure in SBRT.

Quantification of Tumor Hypoxic Fractions Using Positron Emission Tomography with [18F]Fluoromisonidazole ([18F]FMISO) Kinetic Analysis and Invasive Oxygen Measurements.

PURPOSE: The purpose of this study is to use dynamic [18F]fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) to compare estimates of tumor hypoxic fractions (HFs) derived by tracer kinetic modeling, tissue-to-blood ratios (TBR), and independent oxygen (pO2) measurements. PROCEDURES: BALB/c mice with EMT6 subcutaneous tumors were selected for PET imaging and invasive pO2 measurements. Data from 120-min dynamic [18F]FMISO scans were fit to two-compartment irreversible three rate constant (K 1, k 2, k 3) and Patlak models (K i). Tumor HFs were calculated and compared using K i, k 3, TBR, and pO2 values. The clinical impact of each method was evaluated on [18F]FMISO scans for three non-small cell lung cancer (NSCLC) radiotherapy patients. RESULTS: HFs defined by TBR (≥1.2, ≥1.3, and ≥1.4) ranged from 2 to 85 % of absolute tumor volume. HFs defined by K i (>0.004 ml min cm-3) and k 3 (>0.008 min-1) varied from 9 to 85 %. HF quantification was highly dependent on metric (TBR, k 3, or K i) and threshold. HFs quantified on human [18F]FMISO scans varied from 38 to 67, 0 to 14, and 0.1 to 27 %, for each patient, respectively, using TBR, k 3, and K i metrics. CONCLUSIONS: [18F]FMISO PET imaging metric choice and threshold impacts hypoxia quantification reliability. Our results suggest that tracer kinetic modeling has the potential to improve hypoxia quantification clinically as it may provide a stronger correlation with direct pO2 measurements.

Event-by-Event Continuous Respiratory Motion Correction for Dynamic PET Imaging.

UNLABELLED: Existing respiratory motion-correction methods are applied only to static PET imaging. We have previously developed an event-by-event respiratory motion-correction method with correlations between internal organ motion and external respiratory signals (INTEX). This method is uniquely appropriate for dynamic imaging because it corrects motion for each time point. In this study, we applied INTEX to human dynamic PET studies with various tracers and investigated the impact on kinetic parameter estimation. METHODS: The use of 3 tracers-a myocardial perfusion tracer, (82)Rb (n = 7); a pancreatic ß-cell tracer, (18)F-FP(+)DTBZ (n = 4); and a tumor hypoxia tracer, (18)F-fluoromisonidazole ((18)F-FMISO) (n = 1)-was investigated in a study of 12 human subjects. Both rest and stress studies were performed for (82)Rb. The Anzai belt system was used to record respiratory motion. Three-dimensional internal organ motion in high temporal resolution was calculated by INTEX to guide event-by-event respiratory motion correction of target organs in each dynamic frame. Time-activity curves of regions of interest drawn based on end-expiration PET images were obtained. For (82)Rb studies, K1 was obtained with a 1-tissue model using a left-ventricle input function. Rest-stress myocardial blood flow (MBF) and coronary flow reserve (CFR) were determined. For (18)F-FP(+)DTBZ studies, the total volume of distribution was estimated with arterial input functions using the multilinear analysis 1 method. For the (18)F-FMISO study, the net uptake rate Ki was obtained with a 2-tissue irreversible model using a left-ventricle input function. All parameters were compared with the values derived without motion correction. RESULTS: With INTEX, K1 and MBF increased by 10% ± 12% and 15% ± 19%, respectively, for (82)Rb stress studies. CFR increased by 19% ± 21%. For studies with motion amplitudes greater than 8 mm (n = 3), K1, MBF, and CFR increased by 20% ± 12%, 30% ± 20%, and 34% ± 23%, respectively. For (82)Rb rest studies, INTEX had minimal effect on parameter estimation. The total volume of distribution of (18)F-FP(+)DTBZ and Ki of (18)F-FMISO increased by 17% ± 6% and 20%, respectively. CONCLUSION: Respiratory motion can have a substantial impact on dynamic PET in the thorax and abdomen. The INTEX method using continuous external motion data substantially changed parameters in kinetic modeling. More accurate estimation is expected with INTEX.

Synthesis of [(18)F]FMISO in a flow-through microfluidic reactor: Development and clinical application.

INTRODUCTION: The PET radiotracer [(18)F]FMISO has been used in the clinic to image hypoxia in tumors. The aim of the present study was to optimize the radiochemical parameters for the preparation of [(18)F]FMISO using a microfluidic reaction system. The main parameters evaluated were (1) precursor concentration, (2) reaction temperature, and (3) flow rate through the microfluidic reactor. Optimized conditions were then applied to the batch production of [(18)F]FMISO for clinical research use. METHODS: For the determination of optimal reaction conditions within a flow-through microreactor synthesizer, 5-400 µL the precursor and dried [(18)F]fluoride solutions in acetonitrile were simultaneously pushed through the temperature-controlled reactor (60-180 °C) with defined flow rates (20-120 µL/min). Radiochemical incorporation yields to form the intermediate species were determined using radio-TLC. Hydrolysis to remove the protecting group was performed following standard vial chemistry to afford [(18)F]FMISO. RESULTS: Optimum reaction parameters for the microfluidic set-up were determined as follows: 4 mg/mL of precursor, 170 °C, and 100 µL/min pump rate per reactant (200 µL/min reaction overall flow rate) to prepare the radiolabeled intermediate. The optimum hydrolysis condition was determined to be 2N HCl for 5 min at 100 °C. Large-scale batch production using the optimized conditions gave the final, ready for human injection [(18)F]FMISO product in 28.4 ± 3.0% radiochemical yield, specific activity of 119 ± 26 GBq/µmol, and >99% radiochemical and chemical purity at the end of synthesis (n = 4). CONCLUSION: By using the NanoTek microfluidic synthesis system, [(18)F]FMISO was successfully prepared with good specific activity and high radiochemical purity for human use. The product generated from large-scale batch production using flow chemistry is currently being used in clinical research.

Molecular imaging of tumor hypoxia with positron emission tomography.

The problem of tumor hypoxia has been recognized and studied by the oncology community for over 60 years. From radiation and chemotherapy resistance to the increased risk of metastasis, low oxygen concentrations in tumors have caused patients with many types of tumors to respond poorly to conventional cancer therapies. It is clear that patients with high levels of tumor hypoxia have a poorer overall treatment response and that the magnitude of hypoxia is an important prognostic factor. As a result, the development of methods to measure tumor hypoxia using invasive and noninvasive techniques has become desirable to the clinical oncology community. A variety of imaging modalities have been established to visualize hypoxia in vivo. Positron emission tomography (PET) imaging, in particular, has played a key role for imaging tumor hypoxia because of the development of hypoxia-specific radiolabelled agents. Consequently, this technique is increasingly used in the clinic for a wide variety of cancer types. Following a broad overview of the complexity of tumor hypoxia and measurement techniques to date, this article will focus specifically on the accuracy and reproducibility of PET imaging to quantify tumor hypoxia. Despite numerous advances in the field of PET imaging for hypoxia, we continue to search for the ideal hypoxia tracer to both qualitatively and quantitatively define the tumor hypoxic volume in a clinical setting to optimize treatments and predict response in cancer patients.