Anthony R. Guastella

Tryptophan PET Imaging of the Kynurenine Pathway in Patient-Derived Xenograft Models of Glioblastoma:

Increasing evidence demonstrates the immunosuppressive kynurenine pathway’s (KP) role in the pathophysiology of human gliomas. To study the KP in vivo, we used the noninvasive molecular imaging tracer α-[11C]-methyl-l-tryptophan (AMT). The AMT-positron emission tomography (PET) has shown high uptake in high-grade gliomas and predicted survival in patients with recurrent glioblastoma (GBM). We generated patient-derived xenograft (PDX) models from dissociated cells, or tumor fragments, from 5 patients with GBM. Mice bearing subcutaneous tumors were imaged with AMT-PET, and tumors were analyzed to detect the KP enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, tryptophan 2,3-dioxygenase, kynureninase, and kynurenine 3-monooxygenase. Overall, PET imaging showed robust tumoral AMT uptake in PDX mice with prolonged tracer accumulation over 60 minutes, consistent with AMT trapping seen in humans. Immunostained tumor tissues demonstrated positive detection of multiple KP enzymes. Furthermore, intracranial implantation of GBM cells was performed with imaging at both 9 and 14 days postimplant, with a marked increase in AMT uptake at 14 days and a corresponding high level of tissue immunostaining for KP enzymes. These results indicate that our PDX mouse models recapitulate human GBM, including aberrant tryptophan metabolism, and offer an in vivo system for development of targeted therapeutics for patients with GBM.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

BackgroundThere is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches.MethodsA primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed.ResultsHistopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64–66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma.ConclusionsAlthough derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.

Assessment of tryptophan uptake and kinetics using 1-(2-[18F]fluoroethyl)-L-tryptophan and α-[11C]-methyl-L-tryptophan PET imaging in mice implanted with patient-derived brain tumor xenografts.

Abnormal tryptophan metabolism via the kynurenine pathway is involved in the pathophysiology of a variety of human diseases including cancers. α-11C-methyl-l-tryptophan (11C-AMT) PET imaging demonstrated increased tryptophan uptake and trapping in epileptic foci and brain tumors, but the short half-life of 11C limits its widespread clinical application. Recent in vitro studies suggested that the novel radiotracer 1-(2-18F-fluoroethyl)-l-tryptophan (18F-FETrp) may be useful to assess tryptophan metabolism via the kynurenine pathway. In this study, we tested in vivo organ and tumor uptake and kinetics of 18F-FETrp in patient-derived xenograft mouse models and compared them with 11C-AMT uptake. Methods: Xenograft mouse models of glioblastoma and metastatic brain tumors (from lung and breast cancer) were developed by subcutaneous implantation of patient tumor fragments. Dynamic PET scans with 18F-FETrp and 11C-AMT were obtained for mice bearing human brain tumors 1–7 d apart. The biodistribution and tumoral SUVs for both tracers were compared. Results: 18F-FETrp showed prominent uptake in the pancreas and no bone uptake, whereas 11C-AMT showed higher uptake in the kidneys. Both tracers showed uptake in the xenograft tumors, with a plateau of approximately 30 min after injection; however, 18F-FETrp showed higher tumoral SUV than 11C-AMT in all 3 tumor types tested. The radiation dosimetry for 18F-FETrp determined from the mouse data compared favorably with the clinical 18F-FDG PET tracer. Conclusion: 18F-FETrp tumoral uptake, biodistribution, and radiation dosimetry data provide strong preclinical evidence that this new radiotracer warrants further studies that may lead to a broadly applicable molecular imaging tool to examine abnormal tryptophan metabolism in human tumors.

Role of Kynurenine Pathway in Neuro-oncology

Despite decades of research, both primary and metastatic brain tumors remain intractable clinical problems. While the kynurenine (KYN) pathway of tryptophan metabolism has been well explored in other cancer types, there are few studies of human brain tumors. The rate-limiting enzymes of the conversion of tryptophan to kynurenine (both forms of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO2)) have been studied predominantly in gliomas with in vitro methods, in vivo animal studies, and ex vivo tissue studies of surgically resected specimens. In these studies, IDO1 has been shown regulated by interferon-γ (as in other cancer types) and TDO2 by the glucocorticoid receptor. IDO and TDO2 have also been positively correlated with tumor grade and negatively correlated with patient survival in glioma. One seminal study also identified KYN as an activator of the aryl hydrocarbon receptor (not previously shown in any other cancer types). Therefore, the KYN pathway may represent new opportunities for treatment strategies for brain tumors.

Investigation of the aryl hydrocarbon receptor and the intrinsic tumoral component of the kynurenine pathway of tryptophan metabolism in primary brain tumors

IntroductionThere is mounting evidence supporting the role of tryptophan metabolism via the kynurenine pathway (KP) in the pathogenesis of primary brain tumors. Under normal physiological conditions, the KP is the major catabolic pathway for the essential amino acid tryptophan. However, in cancer cells, the KP becomes dysregulated, depletes local tryptophan, and contributes to an immunosuppressive tumor microenvironment.MethodsWe examined the protein expression levels (in 73 gliomas and 48 meningiomas) of the KP rate-limiting enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, and tryptophan 2,3-dioxygenase (TDO2), as well as, the aryl hydrocarbon receptor (AhR), a carcinogenic transcription factor activated by KP metabolites. In addition, we utilized commercially available small-molecules to pharmacologically modulate IDO1, IDO2, TDO2, and AhR in patient-derived glioma and meningioma cell lines (n = 9 each).ResultsWe observed a positive trend between the grade of the tumor and the average immunohistochemical staining score for IDO1, IDO2, and TDO2, with TDO2 displaying the strongest immunostaining. AhR immunostaining was present in all grades of gliomas and meningiomas, with the greatest staining intensity noted in glioblastomas. Immunocytochemical staining showed a positive trend between nuclear localization of AhR and histologic grade in both gliomas and meningiomas, suggesting increased AhR activation with higher tumor grade. Unlike enzyme inhibition, AhR antagonism markedly diminished patient-derived tumor cell viability, regardless of tumor type or grade, following in vitro drug treatments.ConclusionsCollectively, these results suggest that AhR may offer a novel and robust therapeutic target for a patient population with highly limited treatment options.

Overview of the Kynurenine Pathway of Tryptophan Metabolism

Tryptophan (TRP) is an essential amino acid that plays a critical role in synthesis of a host of modulatory biomolecules including serotonin, melatonin, tryptamine, and kynurenine (KYN). TRP can either be incorporated into proteins, converted to the neurotransmitter serotonin (5-hydroxytryptamine), or metabolized to kynurenine. The majority of dietary TRP is metabolized via the kynurenine pathway (KP). The initial and rate-limiting step in the KP involves one of three enzymes, namely, the two isoforms of indoleamine 2-3-dioxygenase (IDO1 and IDO2) and tryptophan 2,3-dioxygenase (TDO). In this chapter, we provide a broad overview of the KP and explore the gene regulation of the key enzymes involved.

Experimental Models to Study the Kynurenine Pathway

The kynurenine pathway is the major pathway that degrades tryptophan that has not been incorporated into proteins. The metabolites that make up the pathway have roles in a broad range of disease states spanning from neurodegenerative diseases, such as Huntington’s disease, to infections to cancer. The studying of this pathway is crucial to further our understanding of the many diseases it is incorporated in and furthermore presents the opportunity to discover novel therapeutics. In this chapter, we hope to shed light on the transgenic animal models available (Ido1, Ido2, Tdo2, Kmo, and KatII) and the diseases they have currently been used to study, as well as give a glance at some of the inhibitors available.