Evaluations of an Early Change in Tumor Pathophysiology in Response to Radiotherapy with Oxygen Enhanced Electron Paramagnetic Resonance Imaging (OE EPRI).

PURPOSE: Electron Paramagnetic Resonance Imaging (EPRI) can image the partial pressure of oxygen (pO2) within in vivo tumor models. We sought to develop Oxygen Enhanced (OE) EPRI that measures tumor pO2 with breathing gases of 21% O2 (pO221%) and 100% O2 (pO2100%), and the differences in pO2 between breathing gases (ΔpO2). We applied OE EPRI to study the early change in tumor pathophysiology in response to radiotherapy in two tumor models of pancreatic cancer. PROCEDURES: We developed a protocol that intraperitoneally administered OX071, a trityl radical contrast agent, and then acquired anatomical MR images to localize the tumor. Subsequently, we acquired two pO221% and two pO2100% maps using the T1 relaxation time of OX071 measured with EPRI and a R1-pO2 calibration of OX071. We studied 4T1 flank tumor model to evaluate the repeatability of OE EPRI. We then applied OE EPRI to study COLO 357 and Su.86.86 flank tumor models treated with 10 Gy radiotherapy. RESULTS: The repeatability of mean pO2 for individual tumors was ± 2.6 Torr between successive scans when breathing 21% O2 or 100% O2, representing a precision of 9.6%. Tumor pO221% and pO2100% decreased after radiotherapy for both models, although the decreases were not significant or only moderately significant, and the effect sizes were modest. For comparison, ΔpO2 showed a large, highly significant decrease after radiotherapy, and the effect size was large. MANOVA and analyses of the HF10 hypoxia fraction provided similar results. CONCLUSIONS: EPRI can evaluate tumor pO2 with outstanding precision relative to other imaging modalities. The change in ΔpO2 before vs. after treatment was the best parameter for measuring the early change in tumor pathophysiology in response to radiotherapy. Our studies have established ΔpO2 from OE EPRI as a new parameter, and have established that OE EPRI is a valuable new methodology for molecular imaging.

Evaluations of the performances of PET and MRI in a simultaneous PET/MRI instrument for pre-clinical imaging.

BACKGROUND: PET/MRI is an attractive imaging modality due to the complementary nature of MRI and PET. Obtaining high quality small animal PET/MRI results is key for the translation of novel PET/MRI agents and techniques to the radiology clinic. To obtain high quality imaging results, a hybrid PET/MRI system requires additional considerations beyond the standard issues with separate PET and MRI systems. In particular, researchers must understand how their PET system affects the MR acquisitions and vice versa. Depending on the application, some of these effects may substantially influence image quality. Therefore, the goal of this report is to provide guidance, recommendations, and practical experiments for implementing and using a small animal PET/MRI instrument. RESULTS: Various PET and MR image quality parameters were tested with their respective modality alone and in the presence of both systems to determine how the combination of PET/MRI affects image quality. Corrections and calibrations were developed for many of these effects. While not all image characteristics were affected, some characteristics such as PET quantification, PET SNR, PET spatial resolution, PET partial volume effects, and MRI SNR were altered by the presence of both systems. CONCLUSIONS: A full exploration of a new PET/MRI system before performing small animal PET/MRI studies is beneficial and necessary to ensure that the new instrument can produce highly accurate and precise PET/MR images.

Radiometal-Based PET/MRI Contrast Agents for Sensing Tumor Extracellular pH.

Acidosis is a useful biomarker for tumor diagnoses and for evaluating early response to anti-cancer treatments. Despite these useful applications, there are few methods for non-invasively measuring tumor extracellular pH, and none are routinely used in clinics. Responsive MRI contrast agents have been developed, and they undergo a change in MRI signal with pH. However, these signal changes are concentration-dependent, and it is difficult to accurately measure the concentration of an MRI contrast agent in vivo. PET/MRI provides a unique opportunity to overcome this concentration dependence issue by using the PET component to report on the concentration of the pH-responsive MRI agent. Herein, we synthesized PET/MRI co-agents based on the design of a pH-dependent MRI agent, and we have correlated pH with the r1 relaxivity of the MRI co-agent. We have also developed a procedure that uses PET radioactivity measurements and MRI R1 relaxation rate measurements to determine the r1 relaxivity of the MRI co-agent, which can then be used to estimate pH. This simultaneous PET/MRI procedure accurately measured pH in solution, with a precision that depended on the concentration of the MRI co-agent. We used our procedure to measure extracellular pH in a subcutaneous flank model of MIA PaCa-2 pancreatic cancer. Although the PET co-agents were stable in serum, post-imaging studies showed evidence that the PET co-agents were degraded in vivo. These results showed that tumor acidosis can be evaluated with simultaneous PET/MRI, although improvements are needed to more precisely measure MRI R1 relaxation rates, and ensure the in vivo stability of the agents.

Development of a Nanoscale Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agent That Measures pH.

AcidoCEST MRI can measure the extracellular pH (pHe) of the tumor microenvironment in mouse models of human cancers and in patients who have cancer. However, chemical exchange saturation transfer (CEST) is an insensitive magnetic resonance imaging (MRI) contrast mechanism, requiring a high concentration of small-molecule agent to be delivered to the tumor. Herein, we developed a nanoscale CEST agent that can measure pH using acidoCEST MRI, which may decrease the requirement for high delivery concentrations of agent. We also developed a monomer agent for comparison to the polymer. After optimizing CEST experimental conditions, we determined that the polymer agent could be used during acidoCEST MRI studies at 125-fold and 488-fold lower concentration than the monomer agent and iopamidol, respectively. We also determined that both agents can measure pH with negligible dependence on temperature. However, pH measurements with both agents were dependent on concentration, which may be due to concentration-dependent changes in hydrogen bonding and/or steric hindrance. We performed in vivo acidoCEST MRI studies using the three agents to study a xenograft MDA-MB-231 model of mammary carcinoma. The tumor pHe measurements were 6.33 ± 0.12, 6.70 ± 0.15, and 6.85 ± 0.15 units with iopamidol, the monomer agent, and polymer agent, respectively. The higher pHe measurements with the monomer and polymer agents were attributed to the concentration dependence of these agents. This study demonstrated that nanoscale agents have merit for CEST MRI studies, but consideration should be given to the dependence of CEST contrast on the concentration of these agents.

Multimodal imaging of the tumor microenvironment and biological responses to immune therapy.

Beyond heterogeneous cancer cells, the tumor microenvironment includes stromal and immune cells, blood vessels, extracellular matrix and biologically active molecules. Abnormal signaling, uncontrolled proliferation and high interstitial pressure all contribute to a chaotic, non-hierarchical vascular organization. Using an immune competent 4T1 breast adenocarcinoma murine model, this study fully characterizes the architecture and immunocyte milieu of the tumor microenvironment. Heterogeneous vessel distribution, chaotic connectivity, limited perfusion, cancer cell density, immune phenotype, and biological responses to immune therapy are presented. Cancer cell density mirrored the distribution of large, perfusable vessels, both predominately in the tumor periphery. Intratumoral administration of the proinflammatory cytokine IL-12 led to an increase in CD45+ leukocytes, with a specific increase in CD4+ and CD8+ T cells, and a decrease in the percentage of Gr-llo myeloid-derived suppressor cells. Concomitantly, serum G-CSF, IL-10 and VEGF decreased, while CXCR9 and interferon gamma increased. The distribution pattern of infiltrating monocytes/macrophages, visualized using a fluorescent perfluorocarbon emulsion, indicated that macrophages predominately localize in the vicinity of large blood vessels. Electron microscopy supports the presence of dense tumor cell masses throughout the tumor, with the largest vessels present in the surrounding mammary fat pad. Overall, large vessels in the 4T1 tumor periphery support high, localized vascular perfusion and myeloid accumulation. The pro-inflammatory cytokine IL-12 stimulated a transition towards T helper 1 cytokines in serum, supporting suppression of tumor growth and angiostatic conditions.

Feasibility of multianimal hyperpolarized (13) C MRS.

PURPOSE: There is great potential for real-time investigation of metabolism with MRS and hyperpolarized (HP) (13) C agents. Unfortunately, HP technology has high associated costs and efficiency limitations that may constrain in vivo studies involving many animals. To improve the throughput of preclinical investigations, we evaluate the feasibility of performing HP MRS on multiple animals simultaneously. METHODS: Simulations helped assess the viability of a dual-coil strategy for spatially localized multivolume MRS. A dual-mouse system was assembled and characterized with bench- and scanner-based experiments. Enzyme phantoms mixed with HP [1-(13) C] pyruvate emulated real-time metabolism and offered a controlled mechanism for evaluating system performance. Finally, a normal mouse and a mouse bearing a subcutaneous xenograft of colon cancer were simultaneously scanned in vivo using an agent containing HP [1-(13) C] pyruvate. RESULTS: Geometric separation/rotation, active decoupling, and use of low input impedance preamplifiers permitted an encode-by-channel approach for spatially localized MRS. A precalibrated shim allowed straightforward metabolite differentiation in enzyme phantom and in vivo experiments at 7 Tesla, with performance similar to conventional acquisitions. CONCLUSION: The initial feasibility of multi-animal HP (13) C MRS was established. Throughput scales with the number of simultaneously scanned animals, demonstrating the potential for significant improvements in study efficiency.

A randomized controlled trial of celecoxib to prevent recurrence of nonmuscle-invasive bladder cancer.

Significant morbidity and expense result from frequent recurrences of nonmuscle-invasive bladder cancer (NMIBC) after standard treatment, and carcinoma in situ (Tis) is a poor prognostic factor. Predicated on observational and preclinical data strongly supporting cyclooxygenase-2 (COX-2) in the pathogenesis, and the activity of COX-2 inhibitors, in bladder cancer, we conducted a randomized, double-blind, placebo-controlled trial to determine whether celecoxib could reduce the time-to-recurrence (TTR) in NMIBC patients at high risk for recurrence. A total of 146 patients were randomized to celecoxib (200 mg) or placebo orally twice daily for at least 12 months. The average treatment duration was 1.25 years. Primary intent-to-treat analysis revealed celecoxib did not statistically significantly prolong TTR compared with placebo (P = 0.17, log rank) with a median follow-up of 2.49 years. The recurrence-free rate at 12 months with celecoxib was 88% (95% CI: 0.81-0.96) versus 78% (95% CI: 0.69-0.89) with placebo. After controlling for covariates with Cox regression analysis, recurrence rates did not differ between the two study arms (HR = 0.69; 95% CI: 0.37-1.29). However, celecoxib had a marginally significant effect on reducing metachronous recurrences (vs. placebo) with HR of 0.56 (95% CI: 0.3-1.06; P = 0.075). Celecoxib was well tolerated, with similar adverse events and quality-of-life in both arms. Our clinical trial results do not show a clinical benefit for celecoxib in preventing NMIBC recurrence but further investigation of COX-2 inhibitors in this setting is warranted.

Analysis of the expression of biomarkers in urinary bladder cancer using a tissue microarray.

Dysregulation of Akt, PTEN, Drg-1, Cx-26, and L-plastin expression appear to be important in the progression of various cancers. Their expression in bladder cancer has not been well characterized. To assess the expression of these genes and their relationship to the outcome of bladder cancer, we used a bladder cancer tissue microarray (TMA) of 251 transitional cell carcinomas. We quantitated immunohistochemical staining of each protein using both automated and manual methods and correlated the expression levels with the clinicopathologic characteristics of the tumor and patient survival. Overall, the results from both automated and manual analyses were similar. We found a significant correlation between the expression of PTEN, Cx-26 and L-plastin with known clinically important pathologic features of bladder cancer (tumor grade, stage, and growth pattern). Aberrant localization patterns of Cx-26 and Drg-1 were observed in bladder tumors. There was also a significant correlation in expression among pAkt, PTEN, and L-plastin. Although the expression of these genes correlated with factors known to be associated with patient outcome, none of them was an independent predictor of progression-free or overall survival.

Noninvasive detection of bladder cancer in an orthotopic murine model with green fluorescence protein cytology.

PURPOSE: Orthotopic models of bladder cancer mimic the normal microenvironment and provide an opportunity to study new therapies for superficial bladder cancer. The use of green fluorescent protein (GFP) transduced cells provides a sensitive way of monitoring this disease. We investigated whether examining voided urine for GFP expressing cells would indicate the presence of GFP producing tumors in an orthotopic bladder tumor model in nude mice. MATERIALS AND METHODS: The human bladder cancer cell lines KU-7, UM-UC-3 and UM-UC-14 were used. GFP transductants were generated after transfection with pEGFP-N3, followed by G418 selection. After the cells were inoculated in an orthotopic model of superficial bladder cancer voided urine was collected on slides weekly for 3 weeks and observed for GFP expressing cells by fluorescence microscopy. Bladder tumor imaging for GFP was performed in surgically exposed bladders to determine the tumor incidence. RESULTS: KU-7 GFP cells produced tumors in all 16 mice on whole bladder GFP imaging. UM-UC-3 and UM-UC-14 GFP cells produced tumors in 8 of 12 (67%) and 18 of 25 (72%) mice, respectively. The rate of GFP positive cells in spontaneously voided urine varied by cell line and increased with time but it was generally less than the rate of detection by whole bladder GFP imaging. All mice with GFP expressing cells in the urine had GFP expressing bladder tumors. CONCLUSIONS: Examining urine for GFP expressing cells is less sensitive than imaging surgically exposed bladders but it is 100% specific.