Genomic assault in the form of DNA double-strand breaks represents the most efficient mechanism by which radiotherapy mediates killing of cancer cells. In order to preserve cellular integrity and mitigate the adverse effects and complications arising from insult to healthy tissues, a fractionated dosing regimen, where low doses of ionizing radiation are administered over a course of several days, is the principal mode of radiation therapy in clinical settings. Additionally, it has been demonstrated that fractionated dosing has a greater capacity to elicit anti-tumor activity and immune mediated abscopal response, compared to bolus delivery. However, in most preclinical animal models of cancer that employ the use of focal beam radiation, administration of a single bolus dose is the standard practice. To establish a more translationally relevant approach to radiation therapy, we utilized computerized tomography (CT) guided Small Animal Radiation Research Platform (SARRP) in three murine subcutaneous tumor models, MB-49 (bladder), B16F10 (melanoma) and MC38 (colon). Each treatment cohort was subjected to a fractionated dosing regimen, and response was assessed by caliper measurements of tumor volume. Therapy was well tolerated across all models and dosing regimens, as evidenced by no significant body weight loss or presentation of adverse clinical symptoms compared to controls. Fractionated focal radiation at 2Gy delivered over a course of five consecutive days (QDx5) induced a modest anti-tumor response in the MB-49 model, resulting in a 66.7% increase in time to progression (ITP) and a 64.1% median ΔT/ΔC on day 16 post implant. Mice bearing MC38 tumors exhibited meaningful response with incidences of 55% ITIP and a median ΔT/ΔC of 29% on day 26, following treatment with radiation at 2Gy, QDx5. Additionally, fractionated doses at 10Gy and 5Gy both delivered Q5Dx2 produced robust anti-tumor response in the B16F10 model, with incidences of 5% and 21% median ΔT/ΔC on Day 15, respectively. Though notable responses were observed across all treatment cohorts, there were no incidences of tumor free survivors or complete regressions, thus allowing for additional therapeutic intervention and synergistic approaches. Further assessment of fractionated dosing regimens in additional mouse models, as well as potential impact on immunomodulation, are ongoing. Over 50% of cancer patients are treated with radiotherapy, with a significant portion requiring integration with other therapeutic modalities to enhance survival and curative benefits. It is therefore imperative to identify appropriate fractionated dose levels and schedules across preclinical cancer models, in order to more accurately reflect the clinical landscape and provide a more viable framework for interrogating rational combination strategies. Citation Format: Kerry-Ann Bright, Derrik Germain, Erin Trachet, Sheri Barnes. Fractionated dosing: A more clinically relevant approach to radiotherapy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2402.

Lung cancer remains a devastating cancer diagnosis around the world, resulting in 18% of all cancer-related deaths. Lung cancer often metastasizes to the brain, significantly reducing life expectancy. Up to 7% of patients with non-small cell lung cancer (NSCLC) already have brain metastasis when they are first diagnosed, and 20%-40% of patients with NSCLC will develop this complication during disease progression. Currently there are no targeted therapies specific for brain metastases, and the blood-brain barrier can pose a physiologic impediment to many cytotoxic drugs and antibody-based therapies. Here, we will discuss the development and use of xenograft models to address metastatic brain disease via a direct intracranial implant coupled with a subcutaneous “primary” tumor to effectively allow evaluation of the response to treatment at both locations. Treatment with targeted therapies could have significantly different penetration and absorption rates, which will alter pharmacokinetic and/or pharmacodynamic (PK/PD) assessment across either tissue. The dual implant technique can be a powerful tool to assess the simultaneous impact of treatment on established metastatic disease and primary tumor. We have characterized the dual disease induction parameters for two human NSCLC cell lines: NCI-H1975-Luc and PC-9-Luc. Both cell lines have been transfected with luciferase to allow for bioluminescence imaging (BLI) to monitor the intracranial disease progression. Both NCI-H1975-Luc and PC-9-Luc are of interest to the research community due to their unique mutational EGFR T790M status. Consistent with the historical and published data, the H1975-luc and PC-9-luc models behaved as expected, producing reliable tumor progression with minimal intragroup variability. In each case, subcutaneous tumors (primary site) reached an evaluation size of ~1000 mm3 in ~20 days, and intracranial tumors (metastatic site) exhibited a total flux doubling time of ~2 days by BLI. Treatment with Osimertinib, an approved first-line treatment for EGFR+ NSCLC patients, was effective against both the subcutaneous and intracranial tumors. Treatment with Osimertinib at 25 mg/kg, given orally, produced 100% complete regressions (CR) against the subcutaneous tumors, which never re-grew prior to study termination on Day 26 post implant and 100% partial regressions (PR) against the established intracranial tumors, which re-grew following the conclusion of treatment at the same rate as the control brain tumors. These data show a difference between the responsive nature of subcutaneous and intracranial NSCLC tumors representing the difference in responsiveness of primary tumors and brain metastases. Utilizing this in vivo dual implant technique can be effective in comparing and assessing the ability of targeted therapies to inhibit both primary and metastatic diseases. Citation Format: Erin Trachet, Scott Wise. A novel technique to preclinically assess the ability of targeted therapies to inhibit both primary and metastatic non small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 37.

Supplementary Fig. S2 from Antitumor activity and pharmacokinetic properties of PF-00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor

Triple immunodeficient mouse models, that lack functional T cells, B cells, and natural killer cells, are necessary when evaluating human cancer cell growth and cell-based therapy activity, allowing for assessment without innate mouse immune system involvement. Due to limited colony sizes at any given vendor, study timelines can sometimes become compromised unless a sufficient alternative is validated. Site location can also be a factor, as licensing restrictions may be an issue for international corporations. In an effort to prove overall similarities across four strains of triple immunodeficient mice, the growth characteristics of BT-474 were compared in female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, NOD.CB17-Prkdcscid IL2rgtm1/BcgenHsd (B-NDG) mice, NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, and NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (CIEA NOG) mice. An immune cell profile was also performed across these four mouse strains and evaluated via flow cytometry to observe any immune cell subset differences in blood, spleen, and tumor samples. Samples analyzed post implant were collected when subcutaneous tumors were ~500 mm3. Model development studies were completed previously to optimize tumor growth kinetics. Historic growth conditions were BT-474 cells implanted at 1.0E+07 cells/implant in the presence of an extracellular matrix into the high right axilla of female NSG mice. Using the same optimized growth conditions, NSG, B-NDG, NCG, and CIEA NOG mice were implanted with BT-474 cells. BT-474 grew well in all the tested mouse strains, producing 100% take rate. Compared to NSG mice, median tumor volume doubling times differed by 1-5 days, median times to an evaluation size of 150 mm3 (typical study enrollment tumor volume) differed by +/- ~4 days and median time to an evaluation size of 1250 mm3 (tumor volume at the end of life) differed by +/- ~10 days. BT-474 did not induce body weight loss in any of the tested mouse strains and all clinical observations were similar. Flow cytometry was performed to quantify lymphocyte and myeloid immune subsets in blood, spleen, and tumor. Overall, compared to NSG mice, the absolute counts were similar for all immune subsets measured except for regulatory T cells (Tregs). B-NDG, NCG, and CIEA NOG mice had reduced Treg numbers in the spleen of tumor-bearing mice. In the tumor, Treg numbers were lower in the B-NDG and CIEA NOG mice. No differences were observed for any immune subset in the blood. Overall, the tumor growth kinetics of BT-474 in B-NDG mice was most similar to historical data using NSG mice. The flow data also confirmed that each strain’s immune cell population generally looked very similar, with limited differences between the four strains of triple immunodeficient mice. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Erin Trachet, Scott Wise. Subcutaneous growth and immune cell profiling of BT-474 human breast carcinoma in four strains of triple immunodeficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 41.

Abstract Preclinical oncology studies aim to model human disease in animal settings. To this effect, the site of tumor implant is thought to impact critical parameters that determine response to treatment. The more frequently used subcutaneous (SC) models have advantages; speed, cost efficiency and ability to monitor growth by caliper measurements but they lack heterogeneous tumor morphology, vessel density and immune cell infiltrates comparable to human disease. Orthotopic (OT) tumor models may allow for tumor modeling in the organ of origin by mimicking many human disease parameters such as vascularization, tumor architecture, metastatic potential, immune profile and tumor microenvironment. OT models may better represent clinical disease but are more challenging to monitor in vivo. Thus, we stably transfected several mouse syngeneic cell lines with luciferase (Luc). The Luc enabled cells facilitate non-invasive monitoring of tumor progression, potential metastasis and response to treatment via bioluminescence imaging (BLI). Here, we focus on pancreas (Pan02-Luc) and lung (LL/2-Luc) models. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Surgical procedures were used with minimal invasion to the respective organs followed by monitoring of tumor take and progression using BLI. Both the LL/2-Luc and Pan02-Luc models showed 90-100% tumor take and successful growth in their respective organs. The OT LL/2-Luc model showed mild response to paclitaxel and cisplatin with an increased time to progression (ITP) of 23% and 46%, respectively, 30% and 46% ITP to checkpoint inhibitors anti-PD-1 and anti-PD-L1 respectively, and 20% ITP to focal radiation (Xstrahl). In comparison, the SC LL/2 model showed 5%, 10%, 4.5% and 1% ITP to cisplatin, paclitaxel, anti-PD-1 and anti-PD-L1, respectively. This demonstrates the potential for the OT model to be more translatable than the SC model. Immune profile of the OT tumors by flow cytometry showed a high percentage of B cells and macrophages in the LL/2-Luc tumors. The OT Pan02-Luc model responded well to gemcitabine treatment with >96% ITP and 62% tumor free survivors while anti-PD-1 treatment resulted in 25% ITP. The SC Pan02 model showed 85% and 9% ITP to gemcitabine and anti-PD1 treatments, respectively. The baseline immune profile of Pan02-Luc tumors showed very few CD4+ and CD8+ T cells and a large myeloid cell population of M-MDSC & M2 TAMs. The poor lymphoid cell infiltration and presence of large numbers of myeloid suppressor cells, characteristic of non-immunogenic tumors corresponds to the lack of robust response to checkpoint inhibitor treatment. The use of OT models may provide an important bridge between the more standard SC models and further clinical approaches. Citation Format: Sumithra Urs, Derrik Germain, Meridith Baugher, Erin Trachet, Maryland Franklin. Characterization of orthotopically implanted syngeneic cell lines as more clinically relevant in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2921.

Abstract Many advances in the treatment of breast cancer have been driven by the development of targeted therapies that inhibit signal transduction pathways, as well as the development of therapies that activate a patient's immune system to unleash antitumor immunity. The choice of an animal model that mimic aspects of human breast cancer is crucial for evaluating new immunotherapeutic combination strategies. An excellent syngeneic model of breast cancer is 4T1. 4T1-luc2 is poorly immunogenic and shares many characteristics with human breast cancer. To understand the potential benefit from combining a targeted therapy with an immune modulator we utilized the orthotopic 4T1-luc2 model. The mTOR pathway plays an important role in metabolism, cell growth and survival. Targeting mTOR has been an active area of oncology drug discovery and clinical development for breast cancer and other malignancies. In addition, clinical success through blockade of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) has resulted in a paradigm shift for drug development within the oncology community. To this end, we designed a series of experiments to evaluate rapamycin, a first generation mTOR inhibitor, in combination with the immune checkpoint inhibitor antibody against CTLA-4. We find that 4T1-luc2 tumors have very few T cells but a significant MDSC (myeloid derived suppressive cells) population. Treatment with anti-CTLA-4 (10mg/kg) produced a slight increase in T cells and a slight decrease in MDSCs. However, these changes in the immune profile did not manifest into meaningful changes in tumor burden. Treatment with low dose (8, 5 or 3mg/kg) rapamycin was well tolerated and produced a modest tumor growth inhibition (~50%) and modulation on the immune profile. The overall impact on the immune profile was like what we've seen with anti-CTLA-4 treatment, with a ~5% decrease in MDCSs but a relatively untouched T cell population. This made the paring of these agents appealing as we hypothesized that the combination may result in further reduction of MDSCs which could make the tumor microenvironment more susceptible to therapeutic intervention. To test this, we looked at rapamycin (8mg/kg) given either simultaneously with, or 1 week prior to, anti-CTLA4. Single agent activity was as expected with %T/C on day 22 at 63% with rapamycin and 84% with anti-CTLA-4. Combination therapy was well tolerated but we found that neither dosing schedule resulted in improved anti-tumor activity over monotherapies with 52% T/C following simultaneous treatment and 53% following sequential treatment. A study to evaluate the impact of the drug combinations on the immune profile is ongoing and will be presented. While both rapamycin and anti-CTLA-4 were capable of modulating the immune profile, improvements in efficacy were not observed. This model provides a system with which preclinical hypotheses can be efficiently tested. Citation Format: Erin Trachet, Chris Elders, Sumithra Urs, Sarah Krueger, Alden Wong, Maryland Rosenfeld Franklin. Combination of rapamycin with immune checkpoint blockade in a syngeneic breast carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2741.

Multiple myeloma is a clonal B-cell malignancy characterized by the accumulation of terminally differentiated, antibody-producing plasma cells in the bone marrow. Genetic mutations within the myeloma cells and their interaction with various cytokines and growth factors contribute to the invasiveness of the disease and enhanced drug resistance. Patients are generally asymptomatic until very late-state disease. However, once disease is present, localization in the bones, especially the spine, is common. There are few preclinical models that recapitulate human disease. In this work, we have evaluated the human MM.1S model and the murine 5TGM-1 model. MM.1S was derived from a 42-year-old African American woman and has been documented to express CD25, CD38, CD52, and CD59. It also expresses the glucocorticoid receptor and is dexamethasone sensitive. To more effectively monitor in vivo disease progression, we transfected the MM.1S line with luciferase (MM.1S-pMMP-LucNeo). With bioluminescence imaging (BLI) we track and monitor disseminated disease progression over time and find reproducible in vivo growth in SCID beige mice. The median tumor volume doubling time is ~2 days and median time to morbidity/mortality endpoint is 35-45 days. MM.1S-pMMP-LucNeo was also evaluated in NSG mice which show more aggressive disease onset (staging 7 days post implant vs. 15 days in SCID mice). In NSG mice the tumor volume doubling time is ~1.8 days and median time to endpoint is ~21 days, roughly half the overall survival time observed in the SCID mouse. In all mouse strains we have investigated, the animals are relatively asymptomatic throughout the lifetime of the study and only begin to present symptoms at late-stage disease, similar to how the disease manifests itself in humans. Common, late-stage clinical signs in the mice include lethargy and paralysis. BLI on these animals shows localization of the signal in the spine, long bones, and mandible. As in the clinic, bortezomib (Velcade®) has limited effect on disease progression in this model. Expression of CD138 is a hallmark of plasma cells and multiple myeloma cells. In the NSG study we determined, by flow cytometry, that >85% of the CD45- gate in the bone marrow was CD138+ 21 days post-implant, suggesting significant engraftment of the tumor cells within the bone marrow. 5TGM-1 is a syngeneic model generated from the C57BL/KaLwRij mouse which is predisposed to develop several monoclonal B-cell proliferative disorders, including a low percentage (0.5%) of the mice developing multiple myeloma. From this strain, several cell clones were isolated and called the 5TMM series, which included the 5T33MM clone. 5TGM-1 is a sub-clone of 5T33MM cells and we have used 5TGM-1-luc to monitor disease progression and localization in vivo. The 5TGM-1-luc model is aggressive and BLI has shown localization of disease in the long bones. We have compared this model in both the syngeneic C57BL/KaLwRij strain and the immune-deficient NIH III (triple deficient) strain. Disease progression behaves similarly in both strains of mice, with significant tumor cells localizing in the spine and the long bones. The model is sensitive to traditional chemotherapies like doxorubicin and cyclophosphamide, but bortezomib (Velcade) only produces modest activity and carfilzomib has shown little activity at the dose and schedule tested. A greater understanding of the preclinical behavior of these models, including response to standard of care and molecular profiling, will provide critical insight into designing more clinically relevant preclinical experiments. Citation Format: Erin Trachet, Maryland Rosenfeld Franklin. Preclinical models of multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 36.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

Abstract Background: Multimodality imaging presents a unique opportunity to image the efficacy of candidate compounds against various models of cancer. The challenge is in integrating the complimentary data that these modalities can provide. The purpose of this study was to evaluate the feasibility of quintuple modality image in order to optimize workflows for maximal efficacy testing of candidate compounds using PET, SPECT, CT, MRI, and Bioluminescence imaging (BLI). Materials and Methods: Male nude mice were injected into the left ventricle with 3x106 PC-3M-luc-C6 cells. BLI was performed to track disease progression and ensure that animals exhibited established metastatic disease prior to initiation of multimodality imaging. Animals were injected with 500µCi Tc99m-MDP and allowed a 1h uptake period prior to imaging. The animals were then placed into an Animal Handling System (AHS, ASI Instruments) that coupled to multiple integrated Positioning Receiver Assemblies (PRA's, ASI Instruments) designed for the PET, SPECT, CT, and MRI scanners, and a 15min, multi-pinhole SPECT scan was acquired. After completion of the SPECT scan, 200µCi of 18F-FDG was administered under anesthesia. MicroCT and MRI anatomical images were acquired during the FDG uptake period. Body temperature was maintained via the integrated warm water circulation in the AHS to ensure adequate FDG uptake and clearance during the procedures. Whole body CT images were acquired at 512 projections, 75kVp, and 220µA. A series of T2-weighted and T1-weighted gadolinium-enhanced images were acquired using a 7T MRI system. At 1h post-FDG administration, a 10min static PET emission scan was acquired and reconstructed using a 3DOSEM/MAP algorithm. Results: Animals were maintained under anesthesia for a total of approximately 2 hours in order to acquire data from all the modalities. Data was able to be co-registered in various forms in order to verify the location and extent of disease, as well as the usefulness of 18F-FDG PET and Tc99m-MDP to serve as biomarker probes for tumor cell metabolism, and osteolytic and osteoblastic activity in bone lesions. Conclusions: Animals tolerated the length of anesthesia well. The feasibility of this imaging was only made possible through the use of the multimodality AHS bed and docking system that permitted reproducible imaging across the different platforms. In order to further optimize workflows, multiple AHS bed systems should be employed so that data can be acquired on the various modalities in parallel, instead of sequentially, thereby greatly increasing animal throughputs. Citation Format: John L. Chunta, Deanne Lister, Chris Bull, Deepa Balagurunathan, Erin Trachet, Chris Chiodo, Scott Wise, Dick Leopold, Patrick McConville. Evaluating the feasibility and throughput of quintuple modality imaging in a prostate cancer bone metastasis model with PET, SPECT, CT, MRI, and bioluminescence imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2014-4945

Abstract BACKGROUND: Intraocular melanomas in the West represents 70% of all primary eye cancers with a mortality rate greater than 50%. Current treatment options include enucleation, plaque radiotherapy, and proton beam radiotherapy. While enucleation is a highly invasive surgical procedure that removes the eye, the side effects of the two different radiotherapies include cataracts, retinopathy, cystoid macular edema, and secondary retinal detachment. Due to the relatively protected nature of the eye within the orbit, most topical treatment applications are precluded and systemic treatments increases the chance and incidence of off-target side effects. Up and coming targeted therapies that may not be constrained by such limitations require a non-invasive, imaging-relevant model in order to evaluate their efficacy. Therefore, the aim of this study was to characterize the imaging of orthotopic luc-enabled OCM-1 human choroidal melanoma tumors using bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) for future use in anti-cancer drug efficacy testing. MATERIALS & METHODS: Human choroidal melanoma cells were modified to express luciferase. 8-12 week old female NIH-Foxn1rnu rats were implanted in the suprachoroidal space with OCM-1-luc tumor spheroids on Day 0. BLI and MRI imaging was performed every 5-7 days starting at Day 7 after implantation of the OCM-1-luc spheroids until animals reached a moribund state to monitor disease progression. For BLI, animals were injected with 150mg/kg luciferin, anesthetized with isoflurane in air and imaged at 10 minutes following luciferin administration. Anatomical MRI was performed using a gradient-echo pulse sequence on a 7T MRI system. After euthanasia, eyes were removed and preserved in formalin for histological analysis. RESULTS & CONCLUSIONS: Animals tolerated the ocular tumor implant well. As a result of these efforts, we have successfully characterized the growth of orthotopic luc-enabled OCM-1 choroidal melanomas using both bioluminescence and magnetic resonance imaging. This provides a simple approach with which to monitor tumor growth and treatment response in a highly efficient way which should allow a high daily animal throughput. These promising results will serve as a solid foundation with which dose routes and levels of anti-cancer drugs can be optimized and their efficacy evaluated with noninvasive imaging. Citation Format: John L. Chunta, Meridith Baugher, Deanne Lister, Erin Trachet, Kevin P. Guley, Chris Bull, Scott Wise, Wilbur R. Leopold, Patrick McConville. Orthotopic human choroidal melanoma model characterization with bioluminescence and magnetic resonance imaging for therapeutic efficacy evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2014-3929

Abstract Introduction: Brain edema is a prominent feature of brain cancer and contributes to neurologic dysfunction and impaired quality of life. Efficacious treatments with minimal side effects are sought after in this indication. Quantitative, non-invasive methods for detecting and measuring brain tumor associated edema are needed to facilitate therapeutic discovery efforts. In this work, an intracranial U251-luc human glioma model was characterized for edema incidence and progression. Multiple MRI scan protocols were used to detect tumor, edema and fluid and quantify the volume of each. Apparent diffusion coefficient (ADC) based quantification was compared with a combined T2-weighted/T1-contrast-enhanced method. Methods: Female nude mice were implanted intracranially with 1x106 U251-luc (Luc-mCherry) glioma cells. A multi-modal MRI scan approach consisting of T2-weighted (T2w), T1-weighted (T1w) contrast-enhanced and ADC scans were used to detect and delineate tumor, fluid, edema and normal brain tissue. These scans yielded: V(T2): volume defined by hyper intense T2 - assumed to be volume[tumor + edema + fluid] V(CE): contrast enhancing volume - assumed to be volume[tumor] V(ADC_high): volume with ADC > threshold value that delineated fluid - assumed to be volume[fluid] A combinatorial approach to yield the volume of edema, V(edema) was used with: V(edema) = V(T2) - V(CE) - V(ADC_high) This approach was then used to examine the ADC and T2 distributions within each of these volumes to determine the sensitivity of T2 and ADC alone for distinguishing tumor, fluid and edema and quantifying edema volume. Results: The edema incidence was 100% based on T2w and T1w CE scans in orthotopically implanted U251-luc (Luc-mCherry) gliomas. Edema volume could be quantified using the multi-modal MRI scan protocol. Edema progression occurred with increasing tumor volume over time. Regions of edema exhibited greater T2 and ADC values compared to tumor tissue regions (that also had a greater T2 signal than normal tissue). This is consistent with edematous tissue water content, compared with tumor tissue and normal brain. The use of T2 and ADC alone provided good delineation of fluid from other tissues, and reasonable delineation of edema from tumor (based on the initial compartmentalization analysis). However, the combined T2w/ T1w-CE/ADC protocol was considered more precise. Conclusion: These results support the use of intracranial U251-luc (Luc-mCherry) as a reliable model for studying the effects of therapies targeting tumor-related cerebral edema. The application of multi-parametric MRI was an effective method for quantifying cerebral edema longitudinally in vivo. Citation Format: Deanne Lister, Deepa Balagurunathan, Meridith Baugher, Athena Flecha, Erin Trachet, Scott Wise, W.R. Leopold, Patrick McConville. Comparison of ADC MRI, T2-weighted MRI and combined T2-weighted/T1-contrast-enhanced/ADC MRI quantification of cerebral edema in an intracranial glioma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2066. doi:10.1158/1538-7445.AM2014-2066

Abstract Pegylated recombinant human hyaluronidase PH20 (PEGPH20) is an investigational therapeutic agent under clinical development for use alone or in combination with other cancer therapies for the treatment of patients with non-hematologic malignancies that may accumulate hyaluronan (HA), a glycosaminoglycan that is a significant component of the extracellular matrix of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, decreases tumor interstitial pressure, increases vascular area, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. As dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a clinically translatable MRI perfusion protocol, we used DCE-MRI imaging to evaluate the effects of PEGPH20 treatment on tumor vascular permeability, vascular surface area and/or blood flow. In mice, prostate cancer PC3 cells were implanted adjacent to the right tibial periosteum. When tumors reached ∼500 mg, animals were staged into vehicle or PEGPH20 (4.5 mg/kg) treatment groups. Utilizing gadopentetate dimeglumine (Gd-DTPA) as a contrast agent, pre-treatment baseline DCE scans of the tumors were acquired. Animals were subsequently IV dosed with test article and post-treatment scans were acquired at 24 h. Initial area under the curve (IAUC) and Ktrans were calculated voxel-by-voxel using a 2-compartment PK model. Although vehicle alone did not increase or decrease Ktrans or IAUC, PEGPH20 treatment increased Ktrans (1/sec) and IAUC (mmol.s) by 74% and 78.7%, respectively. Concurrent to preclinical studies, DCE-MRI imaging was conducted pre- and post- PEGPH20 in 9 patients in a Phase 1 dose-escalation, safety, tolerability, PK/PD study of PEGPH20 treatment in patients with advanced solid tumors. Patients were treated with 1.6, 3 or 5 μg/kg PEGPH20 1-2/weekly. Baseline, day 1, day 2-5 and end-of-cycle (EOC) scans were acquired (w/ Gd-DTPA) in 3 patients. Images were fit on a pixel-by-pixel basis to a 2-compartment, 3-parameter, PK model and Ktrans and extracellular volume (Ve) calculated. Results suggest an early and rapid increase in tumor perfusion in target lesions, compared to baseline (mean ΔKtrans = 428%; N=3; Day 2-5 scans), as well as an expansion of the extracellular space with a reduction of these parameters towards baseline by EOC (mean ΔKtrans = 28% above baseline). Taken together, these studies suggest that PEGPH20 increases vessel permeability, blood flow and/or vessel surface area in HA-rich solid tumors. Additional preclinical and clinical studies, complete with DCE-MRI imaging, are ongoing to further evaluate PEGPH20 in the treatment HA-rich malignancies. Citation Format: Curtis B. Thompson, Patrick McConville, Ron Korn, Jenifer Baranski, Meridith Baugher, Deanne Lister, Erin Trachet, Jacob Y. Hesterman, Jack Hoppin, Joy Zhu, Daniel C. Maneval. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) increases tumor perfusion in mouse xenografts and phase 1 cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4955. doi:10.1158/1538-7445.AM2013-4955

Abstract Breast cancer is the most frequently diagnosed form of cancer and the second leading cause of death in women. Death and most of the complications associated with breast cancer are due to the metastasis of the primary tumor to the lung, as well as other tissues. To visualize the metastatic process in real time, the genetically engineered mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) mouse model was employed along with in vivo µCT (computed tomography) imaging to evaluate and characterize the size and incidence of lung nodules. µCT images are able to detect lung nodules as small as approximately 0.5mm3. The MMTV-PyMT model is widely used to evaluate metastatic breast cancer; the model produces lung metastases (80-90% incidence rate) in a more clinically relevant manner in a syngeneic mouse with intact immune system. In this study female MMTV-PyMT mice were allowed to develop primary mammary fat pad tumors (>3grams) which were then excised and processed into single cell suspension. Syngeneic female mice (FVB/J) were implanted with the cell suspension into mammary fat pad #4 and the resulting tumors were resected once the primary tumor burden surpassed 750mg (late stage). Standard twice weekly intravenous therapy with eribulin at 1mg/kg was initiated following resection and continued the duration of the experiment. µCT images were used to assess the presence and size of lung metastases in vivo starting at 3 weeks post-primary tumor resection. Mice were monitored daily via cage side observations and imaged a second time once labored breathing was apparent. Treatment with eribulin (1mg/kg, IV) not only decreased the incidence of lung metastases by 75% compared to the vehicle control group but also inhibited the ability of the primary tumor to regrow. Coupling the MMTV-PyMT model with µCT imaging allowed for the longitudinal evaluation of therapeutic efficacy on progressive lung metastases. Visualizing lung metastases in real time with in vivo imaging is advantageous since traditional pharmacology endpoints of the MMTV-PyMT model can be extremely long (20-40 weeks). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B145. Citation Format: Mary Anne Meade, Deepa Balagurunathan, Chris Bull, Deanne Lister, Erin Trachet, Bryan Smith, Daniel Flynn, Scott C. Wise. Use of μCT imaging in the PyMT breast cancer model to monitor lung metastasis development and determine therapeutic benefit in real time. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B145.

Abstract Background: Multimodality imaging presents a unique opportunity to image the efficacy of candidate compounds against various models of cancer. The challenge is in integrating the complimentary data that these modalities can provide. The purpose of this study was to evaluate the feasibility of quintuple modality imaging in order to optimize workflows for maximal efficacy testing of candidate compounds using PET, CT, MRI, Bioluminescence imaging (BLI), and Fluorescence Molecular Tomography (FMT). Materials and Methods: Female SCID-Beige mice were implanted IV with 5×10∧6 MM1s-luc cells and female NIH III mice were implanted with 5×10∧6 5TGM-1-luc cells. The presence and extent of disease was confirmed with BLI concurrently or before multimodality imaging was initiated 28-35 days post-implant. The day prior to imaging, mice were injected IV with 2nmol of Integrisense 750 (PerkinElmer) to allow for 24 hours of uptake prior to FMT imaging. The next day, animals were anesthetized with 2.0% isoflurane, were injected with 200µCi 18F-FDG, and were immediately positioned in an FMT cassette for imaging. Whole body FMT data was acquired with excitation/emission at 755nm/775nm. MicroCT data was then acquired by placing the FMT cassette into a custom Animal Handling System (AHS) dock that coupled the FMT cassette to an integrated bed holder (ASI Instruments) for the remaining modalities. Whole body CT images were acquired at 512 projections, 75kVp, and 220µA. A ten minute static PET data set was then acquired with a Seimen's Inveon system after a 1 hour tracer uptake period, and reconstructed with a 3D-OSEM algorithm. MicroPET imaging was followed by a series of T1-weighted, gadolinium-enhanced, and T2-weighted anatomical MR images using an Agilent 7 Tesla MRI. Results: Animals were maintained under anesthesia for a total of approximately 2 hours in order to acquire data from all the modalities. Data was able to be co-registered in various forms in order to verify the location and extent of disease, as well as the usefulness of 18F-FDG PET and Integrisense to serve as biomarker probes. Conclusions: Animals tolerated the length of anesthesia well. Initial difficulties were found in performing the BLI on the same day as the remaining modalities, as anesthesia administration to animals inside the FMT cassette while acquiring BLI perturbed animal positioning. When BLI was acquired the day prior to multimodality imaging, the reliability of positioning was increased, permitting more facile fusion of the various modalities. The feasibility of this imaging was only made possible through the use of the AHS bed and dock that permitted reproducible imaging across the different platforms. In order to further optimize workflows, multiple AHS bed systems should be employed so that data can be acquired on the various modalities in parallel, instead of sequentially, thereby greatly increasing animal throughputs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B142. Citation Format: John L. Chunta, Deanne Lister, Chris Bull, Deepa Balagurunathan, Erin Trachet, Chris Chiodo, Scott Wise, Wilbur R. Leopold, Patrick McConville. Evaluating the feasibility and throughput of quintuple modality imaging in disseminated models of cancer with PET, CT, MRI, bioluminescence, and fluorescence molecular tomography. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B142.

Introduction: Brain edema is a prominent feature of brain cancer and contributes to neurologic dysfunction and impaired quality of life. While corticosteroids are the standard of care, improved treatments that are more efficacious and reduce side effects are sought after. In discovery of novel therapies for edema, animal brain tumor models that show relevant, reproducible and high incidence of tumor and cerebral edema are needed. Additionally, quantitative, non-invasive methods for detecting and measuring brain tumor associated edema are needed to facilitate therapeutic discovery efforts. In this work, an intracranial U251-luc human glioma model was characterized for edema incidence and progression. MRI-based relaxivity and contrast-enhancement protocols for edema detection and delineation from tumor tissue, were tested and characterized in the model. Methods: Female nude mice were implanted intracranially with 1×10∧6 U251-luc (Luc-mCherry) glioma cells. Tumor volume assessment was performed by manual segmentation of T1-weighted, gadolinium-enhanced anatomical brain images based on the assumption of tumor enhancement due to leaky vessels or compromised blood-brain barrier. Edema assessment was performed by manual segmentation of T2-weighted anatomical brain images based on the assumption that enhancing regions include tumor tissue in addition to regions of brain edema. The difference in T2-weighted and T1-weighted volumes was assumed to be edema. T2 maps were also generated over the whole brain to measure T2 values over the respective regions of interest, and confirm the differentiation between tumor tissue and edema. Results: Edema volume was successfully distinguished from contrast enhancing tumor tissue and quantified in orthotopically implanted U251-luc (Luc-mCherry) glioma. Incidence was 100%. Edema progression occurred with increasing tumor volume over time. Regions of edema exhibited greater T2 values compared to tumor tissue regions (that also had greater T2 than normal tissue). This is consistent with edematous tissue water content, compared with tumor tissue and normal brain. These results support the use of intracranial U251-luc (Luc-mCherry) as a reliable model for studying the effects of therapies targeting tumor-related cerebral edema, and the application of MR imaging for quantifying those effects real-time in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B152. Citation Format: Deanne R. Lister, Deepa Balagurunathan, Meridith Baugher, Erin Trachet, Patrick McConville, Scott Wise, W.R. Leopold. MR-based assessment and quantification of cerebral edema in an orthotopic mouse glioma model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B152.

Abstract BACKGROUND: Gliobastomas (GBM) are highly aggressive tumors. When patients are treated with the standard of care of combined radiation therapy and temozolomide (TMZ), median survival is less than 15 months. An earlier prediction of treatment response could allow alternative salvage therapies to be applied sooner if efficacy is not evident. Non-invasive medical imaging provides the opportunity to link imaging-based biomarkers to treatment response. It was hypothesized that a multimodality imaging protocol (heretofore lacking) could allow more accurate prediction of treatment response than conventional means. MATERIALS & METHODS: 1x106 U87MG-luc cells were intracranially injected into 6-8 week old female Nude mice using a stereotactic surgical apparatus. Animals were staged for treatment using a T2-weighted MRI sequence and treatment groups were populated with a tumor volume of 10-20mm3. Treatment was delivered for 5 days as follows 1) Control and 2) TMZ. DCE MRI images were acquired with a gadolinium (Gd) contrast agent and dynamic 18F-FDG PET imaging was acquired at baseline and days 7 and 14 post-treatment. Initial AUC (at 30, 60 and 90s) and Ktrans were determined by fitting the Gd time course data to a generalized 2 compartment kinetic model (Tofts-Kermode approach) and calculated on a voxel-by-voxel basis. PET data was analyzed with traditional kinetic modeling (Ki) using Patlak analysis from 12-70 min and for the SUVmean and SUVmax. Unified metrics that combined Ktrans, Ki and/or SUV were evaluated for predictive power, as compared to each parameter alone. RESULTS: Animals treated with TMZ had a significant increase in median lifespan of 87 days, as compared to controls (59 days; p=0.002). Comparisons of the influx constant, Ki, showed no significant differences between control and treated groups, regardless of whether mean or maximum values were used. Similarly, there were no significant differences between groups in the SUV mean and SUVmax. DCE MRI data is currently undergoing analysis. CONCLUSIONS: Control and TMZ-treated groups produced expected median lifespan results as compared to historical data. Static and dynamic analysis of 18F-FDG PET data did not demonstrate differences between treated and untreated groups. It is possible that the time interval to differentiate the two groups was too proximate to extrapolate response from the derived parameters or that 18F-FDG is not the optimal tracer for this type analysis. It is therefore proposed that future studies utilize later time points in evaluating 18F-FDG-derived biomarkers and/or evaluate the utility of alternate radiotracers (e.g. 18F-FLT). Additionally, it is expected that combining radiation therapy with TMZ will produce more measurable differences in these biomarkers at early time points. A hybrid metric that combines PET and DCE MRI outputs is under development as an enhanced predictor of response. Citation Format: Erin Trachet, John Chunta, Chris Bull, Jeni Baranski, Tracey Woolliscroft, Deanne Lister, Patrick McConville, Dick Leopold. Combined Dynamic 18F-FDG PET Imaging and DCE MRI prediction of treatment response in an orthotopic model of glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2681. doi:10.1158/1538-7445.AM2013-2681

Multiple myeloma is a cancer of the plasma cells that is characterized by multiple localized lesions in the marrow, particularly of the spine, skull, and pelvis, although soft tissue lesions also occur. It is the second most common blood cancer, affecting approximately 45,000 people in the US. Most preclinical modeling of myeloma employs SC xenografts that mimic the less common plasmacytoma form of the disease. Systemic (IV) implants are also used, but studies typically are based on a single survival endpoint, limiting knowledge about the progression and response of the disease under treatment. In order to more quantitatively monitor disseminated disease progression and response to treatment, we have characterized two human (JJN3 and MM1S) and one murine (5TGM1) myeloma models that have been modified to express luciferase. All models were characterized by 100% tumor take rate and focal dissemination of the disease to the spine and skull that mimic clinical experience. These models showed individual and reproducible patterns of spread to other sites, and differed in their sensitivities to standards of care. Analysis of tumor doubling times, tumor titrations, luminescence-based growth delay, and survival all indicated that the bioluminescence signal was a reliable quantitative indicator of viable tumor burden, even under treatment with clinical standard of care agents. Luciferase labeling and the tight correlation between luminescence signal and viable tumor burden raises the possibility of differential real-time tracking of tumor progression and response at individual sites. Citation Format: Meridith Baugher, Jenifer Baranski, Deepa Balagurunathan, Christopher Bull, Noah Winchell, Darren Shaw, Erin Trachet, Deanne Lister, Patrick McConville, Wilbur R. Leopold. Characterization of imaging-enhanced models of disseminated multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2013-3859

Abstract Glioblastoma multiforme (GBM) is the most common and most aggressive form of malignant primary brain tumors, affecting nearly 35,000 people in the United States. Most preclinical studies in glioma utilize survival as the primary endpoint to study, which provides limited information about disease progression, tumor burden response to treatment (a primary clinical end point). We have characterized two human glioma cell lines, Gli36 and LN827 that were modified to express luciferase in order to enable in vivo monitoring of disease progression and response to treatment using bioluminescence imaging (BLI). Anatomical magnetic resonance imaging (MRI) was also performed to directly correlate bioluminescence signal with tumor volume. Both models exhibited >90% tumor take-rate and responded to treatment with temozolomide, a clinical standard of care. Analysis of lifespan, tumor volume doubling times, and tumor growth delay all indicated that BLI is a reliable indicator of disease progression and response to treatment. BLI-based endpoints also showed good correlation with MR-based endpoints. These results support the use of in vivo imaging in these modified cell lines for longitudinal monitoring of tumor progression and response to therapy. Imaging was not only a reliable method for quantifying tumor burden, but enabled clinically relevant end points that could not be accessed through non-imaging means or lifespan determination alone. Further utility can be driven in these models through the use of other functional imaging end points. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B15. Citation Format: Deanne Lister, Mary Anne Meade, Tracey Woolliscroft, Deepa Balagurunathan, Erin Trachet, Wilbur Leopold, Patrick McConville. Increased clinical relevance of orthotopic glioma models through bioluminescence and MR imaging. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B15.

Abstract Introduction: Spontaneous metastasis models are sought after in order to assess the potential of therapies aimed at prevention or treatment of primary tumor metastases. While some human tumor xenograft mouse models show spontaneous metastasis, few show high tissue specific incidence. Commonly the growth of the inherently more advanced primary tumor also limits the ability to characterize far less advanced metastases. Bioluminescence imaging provides a quantitative and convenient means for assessing location, incidence and progression of metastases using metastatic luciferase expressing tumor lines. However, substantially greater light signal from the primary tumor inhibits the ability to find and track much smaller metastasis signals. Several approaches to optimizing metastasis incidence and quantification, and controlling primary tumor growth were used and compared in a MDA-MB-231-luc-D3H2LN xenograft model. Methods: To assess both mouse strain dependence and implant site dependence of primary tumor metastasis, MDA-MB-231-luc-D3H2LN cells (2x106) were implanted in either the mammary fat pad, the sacral region or the quadriceps in several mouse strains, including outbred nu/nu, SHO and nd/bg/xid. To control primary tumor growth in order to facilitate metastasis quantification, both surgical excision and radiation based growth inhibition of of the primary tumors were used. In all cases, whole body bioluminescence imaging was used to quantify metastasis incidence and growth. Shileding of the primary tumor was used in order to characterize smaller metastases. Results and Discussion: While the MDA-MB-231-luc-D3H2LN mammary fat pad implant model has been shown the metastasize spontaneously, tissue specific incidence is generally < 100% and the ability to quantify metastases severely limited by the substantially greater light signal overwhelming much smaller metastasis signals, even when primary tumor shielding is used. In this study, both surgical excision and radiation based primary tumor growth inhibition enhanced the ability to detect metastases in a variety of tissues, including lymph nodes and lungs. The mammary fat pad implant site and the SCID Beige strain provided the highest spontaneous metastasis incidence. Future work will test radiation timing and dose to further optimize metastasis quantification in this and other spontaneous metastasis models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2450. doi:1538-7445.AM2012-2450