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The cell's endogenous antioxidant system is vital to maintain redox homeostasis. Despite its central role in normal and pathophysiology, no noninvasive tools exist to measure this system in patients. The cystine/glutamate antiporter system xc− maintains the balance between intracellular reactive oxygen species and antioxidant production through the provision of cystine, a key precursor in glutathione biosynthesis. Here, we show that tumor cell retention of a system xc−-specific PET radiotracer, (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), decreases in proportion to levels of oxidative stress following treatment with a range of redox-active compounds. The decrease in [18F]FSPG retention correlated with a depletion of intracellular cystine resulting from increased de novo glutathione biosynthesis, shown through [U-13C6, U-15N2]cystine isotopic tracing. In vivo, treatment with the chemotherapeutic doxorubicin decreased [18F]FSPG tumor uptake in a mouse model of ovarian cancer, coinciding with markers of oxidative stress but preceding tumor shrinkage and decreased glucose utilization. Having already been used in pilot clinical trials, [18F]FSPG PET could be rapidly translated to the clinic as an early redox indicator of tumor response to treatment.Significance:[18F]FSPG PET imaging provides a sensitive noninvasive measure of tumor redox status and provides an early marker of tumor response to therapy.See related commentary by Lee et al., p. 701

Supplementary Methods and Figures 1 - 6. Fig. S1 shows effect of TBHP treatment on ROS and [18F]FSPG uptake in ovarian cancer cell lines; Fig. S2 illustrates the relationship between glutamate and cystine concentrations on [18F]FSPG uptake; Fig. S3 quantifies [U-13C6, U-15N2]cystine labeling of M+8 GSSG; Fig. S4 indicates extensive disulfide exchange between cystine and NAC; Fig. S5 depicts doxorubicin-mediated alterations in tumor cell redox state and [18F]FSPG; and Fig. S6 illustrates changes in [18F]FSPG and [18F]FDG tumor retention after doxil treatment of mice.

PDF file - 62K, Age-specific ovarian cancer survival curves by histology, stage and grade

PDF file - 1.5 MB, Fig. S1. Scheme of the c-Myc activation sensor; Fig. S2. Optimal cell plating numbers for HTS format; Fig. S3. Optimal substrate concentration and imaging time for HTS format; Fig. S4. Robustness of the cell assay; Fig. S5. Plot of the occurrence frequency of compound classes for all the clean hits from five libraries; Fig. S6. The percent inhibition at 10�M of all the clean hits from the NCC library and the IC50 of all the clean hits from the Lopac library are plotted; Fig. S7. Plot of IC50 of all the clean hits from combined MS, BMI and BMF libraries; Fig. S8. The top three unexpected potent hits. The chemical structures of the three most potent hits that were not previously described to have an inhibitory effect on c-Myc are shown; Fig. S9A. Western blot analysis of lymphoma and sarcoma cells after NTZ treatment was performed using phospho c-Myc, c-Myc protein and �-actin antibodies; Fig. S9B. Dose dependent inhibition of phospho c-Myc by NTZ in different cancer cell lines; Fig. S10. Effect of NTZ on cell viability; Fig. S11. In vivo efficacy of NTZ in a SK-FST xenograft model; Fig. S12. Plot of the average body weight of NTZ or CMC treated group at indicated days after implantation.

XLS file - 24K, Model behavior using extreme boundry conditions for key input parameters

PDF file - 57K, CA125 Sensitivity Functions Estimated from PLCO and CARET Data

XLS file - 52K, Cost-effectiveness of ovarian cancer screening strategies in populations at increased risk

Ongoing ovarian cancer screening trials are investigating the efficacy of a two-step screening strategy using currently available blood and imaging tests [CA125 and transvaginal sonography (TVS)]. Concurrently, efforts to develop new biomarkers and imaging tests seek to improve screening performance beyond its current limits. This study estimates the mortality reduction, years of life saved, and cost-effectiveness achievable by annual multimodal screening using increasing CA125 to select women for TVS, and predicts improvements achievable by replacing currently available screening tests with hypothetical counterparts with better performance characteristics. An existing stochastic microsimulation model is refined and used to screen a virtual cohort of 1 million women from ages 45 to 85 years. Each woman is assigned a detailed disease course and screening results timeline. The preclinical behavior of CA125 and TVS is simulated using empirical data derived from clinical trials. Simulations in which the disease incidence and performance characteristics of the screening tests are independently varied are conducted to evaluate the impact of these factors on overall screening performance and costs. Our results show that when applied to women at average risk, annual screening using increasing CA125 to select women for TVS achieves modest mortality reduction (∼13%) and meets currently accepted cost-effectiveness guidelines. Screening outcomes are relatively insensitive to second-line test performance and costs. Identification of a first-line test that does substantially better than CA125 and has similar costs is required for screening to reduce ovarian mortality by at least 25% and be reasonably cost-effective. Cancer Prev Res; 5(8); 1015–24. ©2012 AACR.

XLS file - 64K, Impact of screening frequency on mortality reduction and cost-effectiveness of ovarian cancer screening strategies

Deregulation of c-Myc plays a central role in the tumorigenesis of many human cancers. Yet, the development of drugs regulating c-Myc activity has been challenging. To facilitate the identification of c-Myc inhibitors, we developed a molecular imaging sensor–based high-throughput screening (HTS) system. This system uses a cell-based assay to detect c-Myc activation in a HTS format, which is established from a pure clone of a stable breast cancer cell line that constitutively expresses a c-Myc activation sensor. Optimization of the assay performance in the HTS format resulted in uniform and robust signals at the baseline. Using this system, we conducted a quantitative HTS against approximately 5,000 existing bioactive compounds from five different libraries. Thirty-nine potential hits were identified, including currently known c-Myc inhibitors. There are a few among the top potent hits that are not known for anti–c-Myc activity. One of these hits is nitazoxanide, a thiazolide for treating human protozoal infections. Validation of nitazoxanide in different cancer cell lines revealed a high potency for c-Myc inhibition with IC50 ranging between 10 and 500 nmol/L. Oral administration of nitazoxanide in breast cancer xenograft mouse models significantly suppressed tumor growth by inhibition of c-Myc and induction of apoptosis. These findings suggest a potential of nitazoxanide to be repurposed as a new antitumor agent for inhibition of c-Myc–associated neoplasia. Our work also demonstrated the unique advantage of molecular imaging in accelerating discovery of drugs for c-Myc–targeted cancer therapy. Mol Cancer Ther; 12(9); 1896–905. ©2013 AACR.

XLS file - 44K, Variability in model output based on 100 simulations using independent random seeds

PDF file - 105K

PDF file - 91K, Model Overview

PDF file - 17K, Sensitivity analysis around the input parameters for sensitivity of CA125 (first line screen) and TVS (second line screen)

PDF file - 92K, Physicians estimates of survival time in months (median, range and inter-quartile range) in the absence of treatment by histologic type for high (a) and low (b) grade cancer

Supplementary figures

Supplemental Figure S5. Bar graphs showing effect of BAY 87-2243 on RT-PCR expression profiles for (A) SLC16A, (B) ITGB1, (C) IGF-2 and (D) TK-1

PDF file - 300KB, Contains information about the dyes tested and characterization of the imaging agents. Figure S1. Dye Selection. The table lists all the dyes tested with their molecular weight, maximum excitation and emission wavelengths, extinction coefficient and quantum yield. Figure S2. AA3G-740 MALDI spectrum. Figure S3. Cell binding studies. A) Binding of AA3G-740 to PC3 cells was blocked by bombesin in a dose dependent manner. B) LNCaP cells showed minimal binding. At high concentration (30pmol) cell fluorescence was of a similar level as the one corresponding to non-specific binding in PC3 cells Figure S4. Blood lifetime. Very low amount of the agent was at 60 minutes post-injection suggesting short blood lifetime.

Supplementary document with figures 1-5.

Supplemental Fig. S1. 89Zr-DFO-OX40 mAb PET in different time points post vaccination. (A) To explore the optimal timing to perform 89Zr-DFO-OX40 mAb PET post vaccination, early time point PET studies were conducted (The schedule in the main manuscript was described as late time point here, in which PET imaging was performed on day 3 post vaccination/day 1 post tracer injection). 89Zr-DFO-OX40 mAb was injected on the following day of vaccine treatment, and PET scanning was performed on day 1, 2, and 4. (B) Representative images of vaccinated and control mice in early and late time point imaging. Lymphatic organs were not visualized in both vaccinated and control groups in early time point imaging. (C) No significant difference in 89Zr-DFO-OX40 mAb uptake was seen through day 4 between vaccinated and control groups. Ax, axillary lymph node; Cer, cervical lymph node. Data are shown with mean {plus minus} SEM.

Immunotherapy is innovating clinical cancer management. Nevertheless, only a small fraction of patient's benefit from current immunotherapies. To improve clinical management of cancer immunotherapy, it is critical to develop strategies for response monitoring and prediction. In this study, we describe inducible T-cell costimulator (ICOS) as a conserved mediator of immune response across multiple therapy strategies. ICOS expression was evaluated by flow cytometry, 89Zr-DFO-ICOS mAb PET/CT imaging was performed on Lewis lung cancer models treated with different immunotherapy strategies, and the change in tumor volume was used as a read-out for therapeutic response. ImmunoPET imaging of ICOS enabled sensitive and specific detection of activated T cells and early benchmarking of immune response. A STING (stimulator of interferon genes) agonist was identified as a promising therapeutic approach in this manner. The STING agonist generated significantly stronger immune responses as measured by ICOS ImmunoPET and delayed tumor growth compared with programmed death-1 checkpoint blockade. More importantly, ICOS ImmunoPET enabled early and robust prediction of therapeutic response across multiple treatment regimens. These data show that ICOS is an indicator of T-cell–mediated immune response and suggests ICOS ImmunoPET as a promising strategy for monitoring, comparing, and predicting immunotherapy success in cancer.Significance:ICOS ImmunoPET is a promising strategy to noninvasively predict and monitor immunotherapy response.See related commentary by Choyke, p. 2975

Purpose: Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin αvβ6, a cell surface receptor being evaluated as a novel clinical biomarker.Experimental Design: To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3–6 nmol/L) to integrin αvβ6. The binders do not cross-react with related integrin αvβ5, integrin α5β1, or tumor-angiogenesis–associated integrin, αvβ3.Results: Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin αvβ6. Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting αvβ6-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3).Conclusions: We have engineered highly stable cystine knot peptides with potent and specific integrin αvβ6-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging. Clin Cancer Res; 18(3); 839–49. ©2011 AACR.

Figure S2 shows characterization of B7H3-sr39tk CAR phenotypic subsets

Zr-OX40 DFO-OX40 mAb PET Control, day 5

Day4_Control

Video S1: Combo group Day 2 PET image

Figure S2: Day7 biodistribution study of Combo, STING i.t, PD-1 i.t and PBS i.t groups (n{greater than or equal to}3): the quantification of TDLN was expressed as %ID, other organs were expressed as %ID/g. All values represent the mean {plus minus} SD unless otherwise specified. Two-way ANOVA and unpaired 2-tailed student's t-test were used for analysis. (*: P

Video S5: PD-1 i.p group Day 2 PET image

Day7_Control

Video S4: PBS i.t group Day 2 PET image

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities.Significance:This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells.

PET/CT video

Supplementary Tables - PDF file 86K, Supplementary Table 1. Overall results of 2nd experiment shown as percent change from baseline by experimental group: Control, Low Dose, and High Dose. Supplementary Table 2. Mitotic activity and Ki-67 labeling in the 3 animals from each group that had their brain excised for pathology. Mitotic activity was assessed, by counting mitotic figures in ten high powered (400x) fields, in each of the animals in the most proliferative areas of tumor. Ki-67 labeling was assessed semi-quantitatively

Video S2: STING i.t group Day 2 PET image

Figure S3: Ex-vivo biodistribution study of PD-1 i.p and PBS i.p groups (n{greater than or equal to}5) on Day 8 post anti PD-1 treatment: the quantification of TDLN was expressed as %ID, other organs were expressed as %ID/g. All values represent the mean {plus minus} SD unless otherwise specified. Unpaired 2-tailed student's t-test were used for analysis. (*: P

Supplementary movie 1. Boundary selection software

Video S6: PBS i.p group Day 2 PET image

Supplementary Figures - PDF file 110K, Supplementary Figure 1. Dose titration curves of ficlatuzumab. The numbers in parenthesis are the median survival in days. Supplementary Figure 2. combination therapy of ficlatuzumab and temozolomide studied in an intracranial U87 MG xenograft mouse model. The numbers in parenthesis are the median survival in days. Supplementary Figure 3. Comparison of U87 MG parental and U87 MG-Luc2 cells showing similarity in in vitro (a) and in vivo (b) characteristics. Supplementary Figure 4. Change in weight (mean � standard deviation) over the course of the experiment in the control, low-dose, and high-dose groups. Results shown are normalized to the baseline value. The p-values shown are the results of the ANOVA for each group. In this case, there were no significant changes in weight

Supplemental Materials and Methods. A detailed description of the radiosynthesis of each radiotracer, and detailed PET and SPECT imaging protocols.

Detailed overview of 510(k) cleared imaging devices by the FDA.

Supplemental Fig. S2 Calculation of Total Lesion Immune response (TLIR). When 3D volume of interest (VOI) was placed to include a targeted lesion (green circle), Osirix MD software automatically calculates the volume above 42% of the maximum uptake within the VOI (purple area). The threshold 42% was applied following the majority of previous clinical PET studies. TLIR was defined as mean tracer uptake within the targeted VOI multiplied by volume above 42% of maximum uptake.

Movie S1

FIgure S3 shows B7H3 CAR expression of remaining viable B7H3-sr39tk CAR T-cells after GCV treatment

Purpose: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of 18F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3–triggered nanoaggregation.Experimental Design: Here, we compared the preclinical utility of 18F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, 18F-FDG, 99mTc-Annexin V, and 18F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo.Results: In drug-treated lymphoma cells, 18F-FDG, 99mTc-Annexin V, and 18F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R2 > 0.8; P < 0.001), with no correlation measured for 18F-ML-10 (R2 = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of 99mTc-Annexin V and 18F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although 99mTc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either 18F-FDG or 18F-ML-10.Conclusions: We have demonstrated here that 18F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with 18F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896–905. ©2015 AACR.

Figure S1 shows mass spectometry results of the targeting agent (A), and control peptide (B). It also shows photostability of agent. Figure S2 shows characterisation of the different cell lines used. Figure S3 shows the difference in fluorescence between PDAC and peritumoral inflammation Figure S4 shows results of the preliminary toxicity study in mice

Supplemental Video M3. 18FML-10 uptake in naive tumors

PDF file - 1.3MB, Figures, Tables, Methods.

Supplementary Figure 3. MALDI-MS of TIGITAb and DFO-TIGITAb demonstrating successful bioconjugation.

Table S1: Characterization of 89Zr-DFO-ICOS mAb, n=4

Supplementary Figure 4. A) 64Cu-TIGITAb (red dotted line) iTLC shows a pure product, with free 64Cu (green solid line) is shown for comparison. B) 89Zr-TIGITAb (red dotted line) iTLC shows a pure product, with free 89Zr (green solid line) is shown for comparison. C) SE HPLC shows UV-Vis abs (280 nm, red line) and radio-signal (blue dotted line) at the same retention time, demonstrating the intact TIGITAb was successfully radiolabeled with 64Cu