Glucose and glutamine are the two principal nutrients that cancer cells use to proliferate and survive. Many cancers show altered glucose metabolism, which constitutes the basis for in vivo positron ...emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)F-FDG). However, (18)F-FDG is ineffective in evaluating gliomas because of high background uptake in the brain. Glutamine metabolism is also altered in many cancers, and we demonstrate that PET imaging in vivo with the glutamine analog 4-(18)F-(2S,4R)-fluoroglutamine ((18)F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced (18)F-FGln tumor avidity, corresponding with decreased tumor burden. (18)F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where (18)F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that (18)F-FGln is avidly taken up by gliomas, can be used to assess metabolic nutrient uptake in gliomas in vivo, and may serve as a valuable tool in the clinical management of gliomas.
The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of ...radioisotopes with long physical half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels-Alder click reaction between tetrazine and transcyclooctene.
A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and (64)Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with (64)Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen-expressing, SW1222 colorectal cancer xenografts.
The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the (64)Cu-NOTA-labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9-10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with (64)Cu-NOTA-labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either (64)Cu or (89)Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the (64)Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with (89)Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively).
The high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.
The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a ...first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed
Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the
status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with
Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models.
.
The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the ...diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal 89Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with 89Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid 89Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.
Multiparametric imaging of tumor perfusion and hypoxia with dynamic 18F-fluoromisonidazole (18F-FMISO) PET may allow for an improved response assessment to antiangiogenic therapies. Cediranib ...(AZD2171) is a potent inhibitor of tyrosine kinase activity associated with vascular endothelial growth factor receptors 1, 2, and 3, currently in phase II/III clinical trials. Serial dynamic 18F-FMISO PET was performed to investigate changes in tumor biomarkers of perfusion and hypoxia after cediranib treatment. Methods: Twenty-one rats bearing HT29 colorectal xenograft tumors were randomized into a vehicle-treated control group (0.5% methylcellulose daily for 2 d 5 rats or 7 d 4 rats) and a cediranib-treated test group (3 mg/kg daily for 2 or 7 d; 6 rats in both groups). All rats were imaged before and after treatment, using a 90-min dynamic PET acquisition after administration of 42.1 ± 3.9 MBq of 18F-FMISO by tail vein injection. Tumor volumes were delineated manually, and the input function was image-derived (abdominal aorta). Kinetic modeling was performed using an irreversible 1-plasma 2-tissue compartmental model to estimate the kinetic rate constants K1, K1/k2, and k3--surrogates for perfusion, 18F-FMISO distribution volume, and hypoxia-mediated entrapment, respectively. Tumor-to-blood ratios (TBRs) were calculated on the last dynamic frame (80-90 min). Tumors were assessed ex vivo by digital autoradiography and immunofluorescence for microscopic visualization of perfusion (pimonidazole) and hypoxia (Hoechst 33342). Results: Cediranib treatment resulted in significant reduction of mean voxelwise 18F-FMISO TBR, K1, and K1/k2 in both the 2-d and the 7-d groups (P < 0.05). The k3 parameter was increased in both groups but reached significance only in the 2-d group. In the vehicle-treated groups, no significant change in TBR, K1, K1/k2, or k3 was observed (P > 0.2). Ex vivo tumor analysis confirmed the presence of hypoxic tumor regions that nevertheless exhibited relatively lower 18F-FMISO uptake. Conclusion: 18F-FMISO kinetic modeling reveals a more detailed response to antiangiogenic treatment than a single static image is able to reveal. The reduced mean K1 reflects a reduction in tumor vascular perfusion, whereas the increased k3 reflects a rise in hypoxia-mediated entrapment of the radiotracer. However, if only late static images are analyzed, the observed reduction in 18F-FMISO uptake after treatment with cediranib may be mistakenly interpreted as a global decrease, rather than an increase, in tumor hypoxia. These findings support the use of 18F-FMISO kinetic modeling to more accurately characterize the response to treatments that have a direct effect on tumor vascularization and perfusion.
Alpha-emitters can be pharmacologically delivered for irradiation of single cancer cells, but cellular lethality could be further enhanced by targeting alpha-emitters directly to the nucleus. PARP-1 ...is a druggable protein in the nucleus that is overexpressed in neuroblastoma compared with normal tissues and is associated with decreased survival in high-risk patients. To exploit this, we have functionalized a PARP inhibitor (PARPi) with an alpha-emitter astatine-211. This approach offers enhanced cytotoxicity from conventional PARPis by not requiring enzymatic inhibition of PARP-1 to elicit DNA damage; instead, the alpha-particle directly induces multiple double-strand DNA breaks across the particle track. Here, we explored the efficacy of
AtMM4 in multiple cancers and found neuroblastoma to be highly sensitive
and
Furthermore, alpha-particles delivered to neuroblastoma show antitumor effects and durable responses in a neuroblastoma xenograft model, especially when administered in a fractionated regimen. This work provides the preclinical proof of concept for an alpha-emitting drug conjugate that directly targets cancer chromatin as a therapeutic approach for neuroblastoma and perhaps other cancers.
pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP ...peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or (18)F-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.
Multiparametric imaging of tumor perfusion and hypoxia with dynamic
F-fluoromisonidazole (
F-FMISO) PET may allow for an improved response assessment to antiangiogenic therapies. Cediranib (AZD2171) ...is a potent inhibitor of tyrosine kinase activity associated with vascular endothelial growth factor receptors 1, 2, and 3, currently in phase II/III clinical trials. Serial dynamic
F-FMISO PET was performed to investigate changes in tumor biomarkers of perfusion and hypoxia after cediranib treatment.
Twenty-one rats bearing HT29 colorectal xenograft tumors were randomized into a vehicle-treated control group (0.5% methylcellulose daily for 2 d 5 rats or 7 d 4 rats) and a cediranib-treated test group (3 mg/kg daily for 2 or 7 d; 6 rats in both groups). All rats were imaged before and after treatment, using a 90-min dynamic PET acquisition after administration of 42.1 ± 3.9 MBq of
F-FMISO by tail vein injection. Tumor volumes were delineated manually, and the input function was image-derived (abdominal aorta). Kinetic modeling was performed using an irreversible 1-plasma 2-tissue compartmental model to estimate the kinetic rate constants
,
/
, and
-surrogates for perfusion,
F-FMISO distribution volume, and hypoxia-mediated entrapment, respectively. Tumor-to-blood ratios (TBRs) were calculated on the last dynamic frame (80-90 min). Tumors were assessed ex vivo by digital autoradiography and immunofluorescence for microscopic visualization of perfusion (pimonidazole) and hypoxia (Hoechst 33342).
Cediranib treatment resulted in significant reduction of mean voxelwise
F-FMISO TBR,
, and
/
in both the 2-d and the 7-d groups (
< 0.05). The
parameter was increased in both groups but reached significance only in the 2-d group. In the vehicle-treated groups, no significant change in TBR,
,
/
, or
was observed (
> 0.2). Ex vivo tumor analysis confirmed the presence of hypoxic tumor regions that nevertheless exhibited relatively lower
F-FMISO uptake.
F-FMISO kinetic modeling reveals a more detailed response to antiangiogenic treatment than a single static image is able to reveal. The reduced mean
reflects a reduction in tumor vascular perfusion, whereas the increased
reflects a rise in hypoxia-mediated entrapment of the radiotracer. However, if only late static images are analyzed, the observed reduction in
F-FMISO uptake after treatment with cediranib may be mistakenly interpreted as a global decrease, rather than an increase, in tumor hypoxia. These findings support the use of
F-FMISO kinetic modeling to more accurately characterize the response to treatments that have a direct effect on tumor vascularization and perfusion.
Generally, solid tumors (>400 mm ³) are inherently acidic, with more aggressive growth producing greater acidity. If the acidity could be targeted as a biomarker, it would provide a means to gauge ...the pace of tumor growth and degree of invasiveness, as well as providing a basis for predicting responses to pH-dependent chemotherapies. We have developed a ⁶⁴Cu pH (low) insertion peptide (pHLIP) for targeting, imaging, and quantifying acidic tumors by PET, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acid-extruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity, such as hypoxia, lactate dehydrogenase A, and CAIX. We find that the probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.
Purpose
The current study presents
18
FPARPi-FL as a bimodal fluorescent/positron emission tomography (PET) agent for PARP1 imaging.
Procedures
18
FPARPi-FL was obtained by
19
F/
18
F isotopic ...exchange and PET experiments, biodistribution studies, surface fluorescence imaging, and autoradiography carried out in a U87 MG glioblastoma mouse model.
Results
18
FPARPi-FL showed high tumor uptake
in vivo
and
ex vivo
in small xenografts (< 2 mm) with both PET and optical imaging technologies. Uptake of
18
FPARPi-FL in blocked U87 MG tumors was reduced by 84 % (0.12 ± 0.02 %injected dose/gram (%ID/g)), showing high specificity of the binding. PET imaging showed accumulation in the tumor (1 h p.i.), which was confirmed by
ex vivo
phosphor autoradiography.
Conclusions
The fluorescent component of
18
FPARPi-FL enables cellular resolution optical imaging, while the radiolabeled component of
18
FPARPi-FL allows whole-body deep-tissue imaging of malignant growth.
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