Potent suppression of neuroendocrine tumors and gastrointestinal cancers by CDH17CAR T cells without toxicity to normal tissues.

Gastrointestinal cancers (GICs) and neuroendocrine tumors (NETs) are often refractory to therapy after metastasis. Adoptive cell therapy using chimeric antigen receptor (CAR) T cells, though remarkably efficacious for treating leukemia, is yet to be developed for solid tumors such as GICs and NETs. Here we isolated a llama-derived nanobody, VHH1, and found that it bound cell surface adhesion protein CDH17 upregulated in GICs and NETs. VHH1-CAR T cells (CDH17CARTs) killed both human and mouse tumor cells in a CDH17-dependent manner. CDH17CARTs eradicated CDH17-expressing NETs and gastric, pancreatic and colorectal cancers in either tumor xenograft or autochthonous mouse models. Notably, CDH17CARTs do not attack normal intestinal epithelial cells, which also express CDH17, to cause toxicity, likely because CDH17 is localized only at the tight junction between normal intestinal epithelial cells. Thus, CDH17 represents a class of previously unappreciated tumor-associated antigens that is 'masked' in healthy tissues from attack by CAR T cells for developing safer cancer immunotherapy.

Transarterial Embolization Modulates the Immune Response within Target and Nontarget Hepatocellular Carcinomas in a Rat Model.

Background Transarterial embolization (TAE) is the most common treatment for hepatocellular carcinoma (HCC); however, there remain limited data describing the influence of TAE on the tumor immune microenvironment. Purpose To characterize TAE-induced modulation of the tumor immune microenvironment in a rat model of HCC and identify factors that modulate this response. Materials and Methods TAE was performed on autochthonous HCCs induced in rats with use of diethylnitrosamine. CD3, CD4, CD8, and FOXP3 lymphocytes, as well as programmed cell death protein ligand-1 (PD-L1) expression, were examined in three cohorts: tumors from rats that did not undergo embolization (control), embolized tumors (target), and nonembolized tumors from rats that had a different target tumor embolized (nontarget). Differences in immune cell recruitment associated with embolic agent type (tris-acryl gelatin microspheres [TAGM] vs hydrogel embolics) and vascular location were examined in rat and human tissues. A generalized estimating equation model and t, Mann-Whitney U, and χ2 tests were used to compare groups. Results Cirrhosis-induced alterations in CD8, CD4, and CD25/CD4 lymphocytes were partially normalized following TAE (CD8: 38.4%, CD4: 57.6%, and CD25/CD4: 21.1% in embolized liver vs 47.7% [P = .02], 47.0% [P = .01], and 34.9% [P = .03], respectively, in cirrhotic liver [36.1%, 59.6%, and 4.6% in normal liver]). Embolized tumors had a greater number of CD3, CD4, and CD8 tumor-infiltrating lymphocytes relative to controls (191.4 cells/mm2 vs 106.7 cells/mm2 [P = .03]; 127.8 cells/mm2 vs 53.8 cells/mm2 [P < .001]; and 131.4 cells/mm2 vs 78.3 cells/mm2 [P = .01]) as well as a higher PD-L1 expression score (4.1 au vs 1.9 au [P < .001]). A greater number of CD3, CD4, and CD8 lymphocytes were found near TAGM versus hydrogel embolics (4.1 vs 2.0 [P = .003]; 3.7 vs 2.0 [P = .01]; and 2.2 vs 1.1 [P = .03], respectively). The number of lymphocytes adjacent to embolics differed based on vascular location (17.9 extravascular CD68+ peri-TAGM cells vs 7.0 intravascular [P < .001]; 6.4 extravascular CD68+ peri-hydrogel embolic cells vs 3.4 intravascular [P < .001]). Conclusion Transarterial embolization-induced dynamic alterations of the tumor immune microenvironment are influenced by underlying liver disease, embolic agent type, and vascular location. © RSNA, 2022 Online supplemental material is available for this article. See also the editorials by Kennedy et al and by White in this issue.

Variability in biopsy quality informs translational research applications in hepatocellular carcinoma.

In the era of precision medicine, biopsies are playing an increasingly central role in cancer research and treatment paradigms; however, patient outcomes and analyses of biopsy quality, as well as impact on downstream clinical and research applications, remain underreported. Herein, we report biopsy safety and quality outcomes for percutaneous core biopsies of hepatocellular carcinoma (HCC) performed as part of a prospective clinical trial. Patients with a clinical diagnosis of HCC were enrolled in a prospective cohort study for the genetic, proteomic, and metabolomic profiling of HCC at two academic medical centers from April 2016 to July 2020. Under image guidance, 18G core biopsies were obtained using coaxial technique at the time of locoregional therapy. The primary outcome was biopsy quality, defined as tumor fraction in the core biopsy. 56 HCC lesions from 50 patients underwent 60 biopsy events with a median of 8 core biopsies per procedure (interquartile range, IQR, 7-10). Malignancy was identified in 45/56 (80.4%, 4 without pathology) biopsy events, including HCC (40/56, 71.4%) and cholangiocarcinoma (CCA) or combined HCC-CCA (5/56, 8.9%). Biopsy quality was highly variable with a median of 40% tumor in each biopsy core (IQR 10-75). Only 43/56 (76.8%) and 23/56 (41.1%) samples met quality thresholds for genomic or metabolomic/proteomic profiling, respectively, requiring expansion of the clinical trial. Overall and major complication rates were 5/60 (8.3%) and 3/60 (5.0%), respectively. Despite uniform biopsy protocol, biopsy quality varied widely with up to 59% of samples to be inadequate for intended purpose. This finding has important consequences for clinical trial design and highlights the need for quality control prior to applications in which the presence of benign cell types may substantially alter findings.

Radial Flow Perfusion Enables Real-Time Profiling of Cellular Metabolism at Low Oxygen Levels with Hyperpolarized 13C NMR Spectroscopy.

In this study, we describe new methods for studying cancer cell metabolism with hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS) that will enable quantitative studies at low oxygen concentrations. Cultured hepatocellular carcinoma cells were grown on the surfaces of non-porous microcarriers inside an NMR spectrometer. They were perfused radially from a central distributer in a modified NMR tube (bioreactor). The oxygen level of the perfusate was continuously monitored and controlled externally. Hyperpolarized substrates were injected continuously into the perfusate stream with a newly designed system that prevented oxygen and temperature perturbations in the bioreactor. Computational and experimental results demonstrated that cell mass oxygen profiles with radial flow were much more uniform than with conventional axial flow. Further, the metabolism of HP [1-13C]pyruvate was markedly different between the two flow configurations, demonstrating the importance of avoiding large oxygen gradients in cell perfusion experiments.

Pharmacodynamics and pharmacokinetics of hyperpolarized [1-13 C]-pyruvate in a translational oncologic model.

This study investigates the in vivo pharmacokinetics and pharmacodynamics of hyperpolarized [1-13 C]-pyruvate in a translational cancer model in order to inform the application of dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopic imaging (MRSI) as a tool for imaging liver cancer. Intratumoral metabolism within autochthonous hepatocellular carcinomas in male Wistar rats was analyzed by MRSI following hyperpolarized [1-13 C]-pyruvate injections with 80 mM (low dose [LD]) or 160 mM (high dose [HD]) pyruvate. Rats received (i) LD followed by HD injection, (ii) sequential LD injections with or without an interposed lactate dehydrogenase inhibitor (LDHi) injection, or (iii) a single LD injection. A subset of rats in (ii) were sacrificed immediately after imaging, permitting measurement of active LDH concentrations in tumor extracts. Urine and serum were collected before and after injections for rats in (iii). Comparison of LD and HD injections confirmed concentration-dependent variation of intratumoral metabolite fractions and intermetabolite ratios. In addition, quantification of the lactate-to-pyruvate ratio was sensitive to pharmacologic inhibition with intermetabolite ratios correlating with active LDH concentrations in tumor extracts. Finally, comparison of pre- and post-DNP urine collections revealed that pyruvate and the radical source are renally excreted after injection. These data demonstrate that DNP-MRSI facilitates real-time quantification of intratumoral metabolism that is repeatable and reflective of intracellular processes. A translational model system confirmed that interpretation requires consideration of probe dose, administration frequency and excretion.

Functional Genetic Screening Enables Theranostic Molecular Imaging in Cancer.

PURPOSE: Targeted therapies for cancer have accelerated the need for functional imaging strategies that inform therapeutic efficacy. This study assesses the potential of functional genetic screening to integrate therapeutic target identification with imaging probe selection through a proof-of-principle characterization of a therapy-probe pair using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopic imaging (MRSI). EXPERIMENTAL DESIGN: CRISPR-negative selection screens from a public dataset were used to identify the relative dependence of 625 cancer cell lines on 18,333 genes. Follow-up screening was performed in hepatocellular carcinoma with a focused CRISPR library targeting imaging-related genes. Hyperpolarized [1-13C]-pyruvate was injected before and after lactate dehydrogenase inhibitor (LDHi) administration in male Wistar rats with autochthonous hepatocellular carcinoma. MRSI evaluated intratumoral pyruvate metabolism, while T2-weighted segmentations quantified tumor growth. RESULTS: Genetic screening data identified differential metabolic vulnerabilities in 17 unique cancer types that could be imaged with existing probes. Among these, hepatocellular carcinoma required lactate dehydrogenase (LDH) for growth more than the 29 other cancer types in this database. LDH inhibition led to a decrease in lactate generation (P < 0.001) and precipitated dose-dependent growth inhibition (P < 0.01 overall, P < 0.05 for dose dependence). Intratumoral alanine production after inhibition predicted the degree of growth reduction (P < 0.001). CONCLUSIONS: These findings demonstrate that DNP-MRSI of LDH activity using hyperpolarized [1-13C]-pyruvate is a theranostic strategy for hepatocellular carcinoma, enabling quantification of intratumoral LDHi pharmacodynamics and therapeutic efficacy prediction. This work lays the foundation for a novel theranostic platform wherein functional genetic screening informs imaging probe selection to quantify therapeutic efficacy on a cancer-by-cancer basis.

Targeting glutamine metabolism slows soft tissue sarcoma growth.

Tumour cells frequently utilize glutamine to meet bioenergetic and biosynthetic demands of rapid cell growth. However, glutamine dependence can be highly variable between in vitro and in vivo settings, based on surrounding microenvironments and complex adaptive responses to glutamine deprivation. Soft tissue sarcomas (STSs) are mesenchymal tumours where cytotoxic chemotherapy remains the primary approach for metastatic or unresectable disease. Therefore, it is critical to identify alternate therapies to improve patient outcomes. Using autochthonous STS murine models and unbiased metabolomics, we demonstrate that glutamine metabolism supports sarcomagenesis. STS subtypes expressing elevated glutaminase (GLS) levels are highly sensitive to glutamine starvation. In contrast to previous studies, treatment of autochthonous tumour-bearing animals with Telaglenastat (CB-839), an orally bioavailable GLS inhibitor, successfully inhibits undifferentiated pleomorphic sarcoma (UPS) tumour growth. We reveal glutamine metabolism as critical for sarcomagenesis, with CB-839 exhibiting potent therapeutic potential.

Hyperpolarized Metabolic Imaging Detects Latent Hepatocellular Carcinoma Domains Surviving Locoregional Therapy.

BACKGROUND AND AIMS: Advances in cancer treatment have improved survival; however, local recurrence and metastatic disease-the principal causes of cancer mortality-have limited the ability to achieve durable remissions. Local recurrences arise from latent tumor cells that survive therapy and are often not detectable by conventional clinical imaging techniques. Local recurrence after transarterial embolization (TAE) of hepatocellular carcinoma (HCC) provides a compelling clinical correlate of this phenomenon. In response to TAE-induced ischemia, HCC cells adapt their growth program to effect a latent phenotype that precedes local recurrence. APPROACH AND RESULTS: In this study, we characterized and leveraged the metabolic reprogramming demonstrated by latent HCC cells in response to TAE-induced ischemia to enable their detection in vivo using dynamic nuclear polarization (DNP) magnetic resonance spectroscopic imaging (MRSI) of 13 carbon-labeled substrates. Under TAE-induced ischemia, latent HCC cells demonstrated reduced metabolism and developed a dependence on glycolytic flux to lactate. Despite the hypometabolic state of these cells, DNP-MRSI of 1-13 C-pyruvate and its downstream metabolites, 1-13 C-lactate and 1-13 C-alanine, predicted histological viability. CONCLUSIONS: These studies provide a paradigm for imaging latent, treatment-refractory cancer cells, suggesting that DNP-MRSI provides a technology for this application.

Ischemia Induces Quiescence and Autophagy Dependence in Hepatocellular Carcinoma.

Purpose To characterize hepatocellular carcinoma (HCC) cells surviving ischemia with respect to cell cycle kinetics, chemosensitivity, and molecular dependencies that may be exploited to potentiate treatment with transarterial embolization (TAE). Materials and Methods Animal studies were performed according to institutionally approved protocols. The growth kinetics of HCC cells were studied in standard and ischemic conditions. Viability and cell cycle kinetics were measured by using flow cytometry. Cytotoxicity profiling was performed by using a colorimetric cell proliferation assay. Analyses of the Cancer Genome Atlas HCC RNA-sequencing data were performed by using Ingenuity Pathway Analysis software. Activation of molecular mediators of autophagy was measured with Western blot analysis and fluorescence microscopy. In vivo TAE was performed in a rat model of HCC with (n = 5) and without (n = 5) the autophagy inhibitor Lys05. Statistical analyses were performed by using GraphPad software. Results HCC cells survived ischemia with an up to 43% increase in the fraction of quiescent cells as compared with cells grown in standard conditions (P < .004). Neither doxorubicin nor mitomycin C potentiated the cytotoxic effects of ischemia. Gene-set analysis revealed an increase in mRNA expression of the mediators of autophagy (eg, CDKN2A, PPP2R2C, and TRAF2) in HCC as compared with normal liver. Cells surviving ischemia were autophagy dependent. Combination therapy coupling autophagy inhibition and TAE in a rat model of HCC resulted in a 21% increase in tumor necrosis compared with TAE alone (P = .044). Conclusion Ischemia induces quiescence in surviving HCC cells, resulting in a dependence on autophagy, providing a potential therapeutic target for combination therapy with TAE. © RSNA, 2017 Online supplemental material is available for this article.

Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth.

In soft tissue sarcomas (STS), low intratumoural O2 (hypoxia) is a poor prognostic indicator. HIF-1α mediates key transcriptional responses to hypoxia, and promotes STS metastasis; however, the role of the related HIF-2α protein is unknown. Surprisingly, here we show that HIF-2α inhibits high-grade STS cell growth in vivo, as loss of HIF-2α promotes sarcoma proliferation and increases calcium and mTORC1 signalling in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. We find that most human STS have lower levels of EPAS1 (the gene encoding HIF-2α) expression relative to normal tissue. Many cancers, including STS, contain altered epigenetics, and our findings define an epigenetic mechanism whereby EPAS1 is silenced during sarcoma progression. The clinically approved HDAC inhibitor Vorinostat specifically increases HIF-2α, but not HIF-1α, accumulation in multiple STS subtypes. Vorinostat inhibits STS tumour growth, an effect ameliorated by HIF-2α deletion, implicating HIF-2α as a biomarker for Vorinostat efficacy in STS.

Segmental Transarterial Embolization in a Translational Rat Model of Hepatocellular Carcinoma.

PURPOSE: To develop a clinically relevant, minimally invasive technique for transarterial embolization in a translational rat model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Oral diethylnitrosamine was administered to 53 male Wistar rats ad libitum for 12 weeks. Tumor induction was monitored using magnetic resonance imaging. Minimally invasive lobar or segmental transarterial embolization was performed through a left common carotid artery approach. Necropsy was performed to evaluate periprocedural mortality. Histologic analysis of tumors that received embolization was performed to assess percent tumor necrosis. RESULTS: Severe cirrhosis and autochthonous HCCs were characterized in a cohort of rats composed of two groups of rats identically treated with diethylnitrosamine with median survival times of 101 days and 105 days (n = 10/group). A second cohort was used to develop minimally invasive transarterial embolization of HCCs (n = 10). In a third cohort, lobar embolization was successfully performed in 9 of 10 rats and demonstrated a high rate of periprocedural mortality (n = 5). Necropsy performed for periprocedural mortality after lobar embolization demonstrated extensive tissue necrosis within the liver (n = 3) and lungs (n = 2), indicating nontarget embolization as the likely cause of mortality. In a fourth cohort of rats, a segmental embolization technique was successfully applied in 10 of 13 rats. Segmental embolization resulted in a reduction in periprocedural mortality (P = .06) relative to selective embolization and a 19% increase in average tumor necrosis (P = .04). CONCLUSIONS: Minimally invasive, segmental embolization mimicking the currently applied clinical approach is feasible in a translational rat model of HCC and offers the critical advantage of reduced nontarget embolization relative to lobar embolization.

Fructose-1,6-bisphosphatase opposes renal carcinoma progression.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel-Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.

Imaging intratumoral convection: pressure-dependent enhancement in chemotherapeutic delivery to solid tumors.

PURPOSE: Low-molecular weight (LMW) chemotherapeutics are believed to reach tumors through diffusion across capillary beds as well as membrane transporters. Unexpectedly, the delivery of these agents seems to be augmented by reductions in tumor interstitial fluid pressure, an effect typically associated with high-molecular weight molecules that reach tumors principally through convection. We investigated the hypothesis that improved intratumoral convection can alter tumor metabolism and enhance the delivery of a LMW chemotherapeutic agent to solid tumors. EXPERIMENTAL DESIGN: For this purpose, we applied 31P/19F magnetic resonance spectroscopy (MRS) and magnetic resonance spectroscopic imaging (MRSI) to examine the influence of type I collagenase on tumor bioenergetics and the delivery of 5-fluorouracil (5FU) to HT29 human colorectal tumors grown s.c. in mice. RESULTS: Collagenase effected a 34% reduction in tumor interstitial fluid pressure with an attendant disintegration of intratumoral collagen. Neither mice-administered collagenase nor controls receiving PBS showed changes in (31)phosphorus MRS-measured tumor bioenergetics; however, a time-dependent increase in the content of extracellular inorganic phosphate (Pi(e)) was observed in tumors of collagenase-treated animals. (31)Phosphorus MRSI showed that this increase underscored a more homogeneous distribution of Pi(e) in tumors of experimental mice. (19)Fluorine MRS showed that these changes were associated with a 50% increase in 5FU uptake in tumors of experimental versus control animals; however, this increase resulted in an increase in 5FU catabolites rather than fluoronucleotide intermediates that are required for subsequent cytotoxicity. CONCLUSIONS: These data indicate that the modulation of convective flow within tumors can improve the delivery of (LMW) chemotherapeutics and show the potential role for noninvasive imaging of this process in vivo.

Imaging transgene activity in vivo.

The successful translation of gene therapy for clinical application will require the assessment of transgene activity as a measure of the biological function of a therapeutic transgene. Although current imaging permits the noninvasive detection of transgene expression, the critical need for quantitative imaging of the action of the expressed transgene has not been met. In vivo magnetic resonance spectroscopic imaging (MRSI) was applied to quantitatively delineate both the concentration and activity of a cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) fusion enzyme expressed from a transgene. MRSI enabled the generation of anatomically accurate maps of the intratumoral heterogeneity in fusion enzyme activity. We observed an excellent association between the CD-UPRT concentration and activity and the percentage of CD-UPRT(+) cells. Moreover, the regional levels of UPRT activity, as measured by imaging, correlated well with the biological affect of the enzyme. This study presents a translational imaging paradigm for precise, in vivo measurements of transgene activity with potential applications in both preclinical and clinical settings.

Targeted elimination of prostate cancer by genetically directed human T lymphocytes.

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3zeta chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.

High-resolution imaging of bone precursor cells within the intact bone marrow cavity of living mice.

Bone precursor cells (BPCs) play a critical role in bone maintenance and regeneration. Currently, no tool exists to study BPCs or other bone marrow cell types directly within their complex microenvironment. Here, we describe in vivo magnetic resonance imaging (MRI) of anatomical structures inside the medullary cavity of the mouse femur. We demonstrate that BPCs passively labeled with iron oxide-containing particles can be monitored by MRI within the intact bone marrow at an in-plane resolution of 43x25 microm. Anatomical detail provided by MRI is complemented by functional optical imaging of reporter gene expression. Single-cell dual iron oxide-reporter gene labeling has potential for combined cell tracking and cell biology studies. In summary, we describe a versatile platform suitable for studying the biology of many bone marrow cell types in living bone.

An 19F magnetic resonance-based in vivo assay of solid tumor methotrexate resistance: proof of principle.

PURPOSE: Studies in oncology have implicated multiple molecular mechanisms as contributors to intrinsic and acquired tumor resistance to antifolate therapy. Here we show the utility of an (19)F-labeled methotrexate (FMTX) with (19)F magnetic resonance to differentiate between sensitive and resistant tumors in vivo and thus predict therapeutic response. EXPERIMENTAL DESIGN: Human sarcoma xenografts in nude mice were used in this study. The sarcoma cell lines chosen for this study (HT-1080, HS-16, and M-805) are well characterized in terms of their methotrexate sensitivity and molecular mechanisms of resistance. The pharmacokinetics of tumor uptake/washout of FMTX were monitored via in vivo (19)F magnetic resonance spectroscopy (pulse/acquire with surface coil localization) following an i.v. bolus injection. Response post-therapy, following leucovorin rescue, was monitored via tumor growth. RESULTS: The three tumor models show differences in both the peak concentrations of tumor FMTX and the dynamics of uptake/retention. These differences are most pronounced for time points late in the magnetic resonance observation period (225-279 minutes post-injection). A statistically significant linear correlation between tumor tissue concentrations of FMTX at these late time points and therapeutic response in the days/weeks post-treatment is shown (R = 0.81, F = 9.27, P < 0.001). Interestingly, a 400 mg/kg i.v. bolus injection of FMTX is a more potent cytotoxic agent in vivo against methotrexate-sensitive tumors than is the parent compound (P = 0.011). CONCLUSIONS: In principle, the assay method described herein could be implemented in the clinic as a diagnostic tool to make decisions regarding therapeutic protocol for the treatment of osteosarcoma on a case-by-case basis.

In vivo 5-fluorouracil and fluoronucleotide T1 relaxation time measurements using the variable nutation angle method.

19Fluorine NMRS has the potential to enable noninvasive predictions of tumor response to 5-fluorouracil (5FU) therapy based on tumor pharmacokinetics. Knowledge of the T1's of 5FU and its fluoronucleotide anabolites (FNuc) is required for quantitative spectral analysis and selection of optimal pulse parameters. We used the variable nutation angle (VNA) method to determine T1's of 5FU and FNuc in subcutaneous Walker 256 rat mammary carcinosarcoma tumors transfected with a cytosine deaminase/uracil phosphoribosyltransferase fusion gene. We calibrated in vivo NAs using methoxydifluoroacetate to ensure the accuracy of these measurements. The T1's were calculated based on signal intensities acquired with NAs of 20 degrees, 35 degrees, 45 degrees, 60 degrees, and 75 degrees. The acquisition order of these NAs was shuffled to reduce the effect of signal variations. The determined T1's for 5FU and FNuc (2.3 +/- 0.1 s and 1.3 +/- 0.1 s, respectively) represent the first reported in vivo measurements for these metabolites in tumor.

MRI of mouse models for gliomas shows similarities to humans and can be used to identify mice for preclinical trials.

Magnetic resonance imaging (MRI) has been utilized for screening and detecting brain tumors in mice based upon their imaging characteristics appearance and their pattern of enhancement. Imaging of these tumors reveals many similarities to those observed in humans with identical pathology. Specifically, high-grade murine gliomas have histologic characteristics of glioblastoma multiforme (GBM) with contrast enhancement after intravenous administration of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), implying disruption of the blood-brain barrier in these tumors. In contrast, low-grade murine oligodendrogliomas do not reveal contrast enhancement, similar to human tumors. MRI can be used to identify mice with brain neoplasms as inclusion criteria in preclinical trials.