Bioluminescence Imaging Intensity Research Articles

Tracking Immature Testicular Tissue after Vitrification In Vitro and In Vivo for Pre-Pubertal Fertility Preservation: A Translational Transgenic Mouse Model.

Pediatric cancer survivors experiencing gonadotoxic chemoradiation therapy may encounter subfertility or permanent infertility. However, previous studies of cryopreservation of immature testicular tissue (ITT) have mainly been limited to in vitro studies. In this study, we aim to evaluate in vitro and in vivo bioluminescence imaging (BLI) for solid surface-vitrified (SSV) ITT grafts until adulthood. The donors and recipients were transgenic and wild-type mice, respectively, with fresh ITT grafts used as the control group. In our study, the frozen ITT grafts remained intact as shown in the BLI, scanning electron microscopy (SEM) and immunohistochemistry (IHC) analyses. Graft survival was analyzed by BLI on days 1, 2, 5, 7, and 31 after transplantation. The signals decreased by quantum yield between days 2 and 5 in both groups, but gradually increased afterwards until day 31, which were significantly stronger than day 1 after transplantation (p = 0.008). The differences between the two groups were constantly insignificant, suggesting that both fresh and SSV ITT can survive, accompanied by spermatogenesis, until adulthood. The ITT in both groups presented similar BLI intensity and intact cells and ultrastructures for spermatogenesis. This translational model demonstrates the great potential of SSV for ITT in pre-pubertal male fertility preservation.

Abstract P5-06-06: Hybrid cells generated by Mesenchymal Stem/Stromal Cell Engulfment enhance breast cancer metastasis upon Doxorubicin treatment in mouse model

Abstract Introduction: Our previous findings evidenced that BCCs engulf MSCs in clinical samples of breast cancer metastasis. Among the BCC-engulfing MSCs, we observed that some of them retain some markers of MSCs, resulting in the generation of hybrid cancer cell population. However, the mechanisms of hybrid cancer cell formation and the phenotypic features of hybrid cancer cells are still unclear. Here, we tested the hypothesis that hybrid cancer cells may acquire in vivo a dormant phenotype, with an increased survival advantage and chemoresistant features. Method: MSC labeled with DsRED (DsRED-MSC) were cultured with MDA-MB-231 labeled with GFP (GFP-231) to generate a hybrid cell-enriched co-culture. Live imaging microscopy, flow cytometry, cytokine array and western blot were used to characterize the hybrid cancer cells. GFP-231 were incubated with phRodo labeled MSC and subjected to phagocytosis assay. In the co-culture of DsRED-MSC with GFP-231 treated with Doxorubicin (Doxo) or untreated, we analyzed the percentage of DsRED+/GFP+ hybrid population. Hybrid-enriched co-culture or GFP-231 in single culture labeled with firefly luciferase were intracardially injected in NOD/SCID mice and monitored for metastases by bioluminescence imaging (BLI), upon Doxo treatment. After collecting the tissues at necropsy, metastases were identified by GFP fluorescence microscopy. Results: In co-cultures, DsRED+/GFP+ hybrid cells had a higher percentage of Ki-67 low in G1 and polyploidy compared to GFP-231+ cells. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of senescence-associated secretory phenotype (SASP) factors. Hybrid cancer cell formation occurs through a phagocytosis-like mechanism, which involves WNT5A and MSR1. Doxo treatment increased the percentage of DsRED+/GFP+ hybrid cells, whereas reduced the percentage of GFP-231+ cells. In animal study, we observed a lower qualitative BLI intensity in mice injected with hybrid cell-enriched co-cultures compared to control. Doxo treatment increased the metastatic burden in mice inoculated with hybrid cell-enriched co-cultures compared to untreated Conclusions: MSC engulfment by BCCs results in a hybrid multinucleated cell population. Hybrid cells acquire a dormant phenotype characterized by a higher percentage of Ki-67low cells in G1 and a senescent phenotype compared to controls. Hybrid-cell-enriched co-cultures established less metastasis compared to control in vivo, but became resistant and acquire the ability to form metastasis upon doxo treatment. Citation Format: Giuseppina Augimeri, Maria E. Gonzalez, Daniela Bonofiglio, Sebastiano Andò, Celina G. Kleer. Hybrid cells generated by Mesenchymal Stem/Stromal Cell Engulfment enhance breast cancer metastasis upon Doxorubicin treatment in mouse model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-06.

Development of a Murine Model of Pyogenic Flexor Tenosynovitis.

Pyogenic flexor tenosynovitis is a debilitating infection of the hand flexor tendon sheath with high morbidity despite standard treatments of empiric antibiotics with irrigation and debridement. In vivo studies in the available literature have used avian models, but these models are difficult to scale and maintain. The purpose of this study was to demonstrate the plausibility of a murine model of pyogenic flexor tenosynovitis utilizing bioluminescence imaging and tissue analysis at harvest. A 2-μL inoculate of bioluminescent Xen29 Staphylococcus aureus or sterile phosphate-buffered saline solution (sPBS) was administered to the tendon sheath of 36 male C57BL/6J mice. The infectious course was monitored by bioluminescence imaging (BLI) via an in vivo imaging system, gross anatomic deformity, and weight change. The infected hind paws were harvested at 4 time points: 24 hours, 72 hours, 1 week, and 2 weeks for histological analysis using Alcian blue, hematoxylin, and Orange-G staining. Two-way analysis of variance with the Sidak multiple comparison test was used to assess differences in bioluminescence and weight at each time point. The infected cohort displayed significantly elevated bioluminescence values, had reductions in weight, and exhibited swelling of the infected digit throughout the course of infection. By day 4, most infected mice saw a substantial decrease in BLI signal intensity; however, 2 infected mice exhibited persistent BLI intensity through day 14. Histological analysis of the infected cohort showed tissue disorganization and the presence of a cellular infiltrate in and around the flexor tendon sheath. A murine model of pyogenic flexor tenosynovitis is possible and can serve as an experimental platform for further investigation of the pathophysiology of pyogenic flexor tenosynovitis. This animal model can be utilized in elucidating the basic molecular and/or cellular mechanisms of pyogenic flexor tenosynovitis while simultaneously evaluating novel therapeutic strategies.

Non-invasive Bioluminescence Monitoring of Hepatocellular Carcinoma Therapy in an HCR Mouse Model.

Animal models play crucial roles in the development of anticancer therapeutics. The ability to quickly assess the localized primary hepatocellular carcinoma (HCC) status in a non-invasive manner would significantly improve the effectiveness of anti-HCC therapeutic studies. However, to date, animal models with this advantage are extremely scarce. In this study, we developed a novel animal model for the fast assessment of drug efficacy against primary HCC in vivo. HCC was induced in immunocompetent hepatocarcinogenesis reporter (HCR) mice by diethylnitrosamine (DEN) injection and confirmed by histopathological staining. Using the bioluminescence imaging (BLI) technique, HCC progression was longitudinally visualized and monitored in a non-invasive way. Tests of two clinical drugs showed that both sorafenib and oxaliplatin significantly inhibited the BLI signal in mouse liver in a dose-dependent manner. The in vivo intensity of BLI signals was highly consistent with the final tumor burden status in mouse liver after drug treatment. The inhibitory effect of anti-HCC drugs was accurately evaluated through in vivo BLI intensity detection. Our study successfully established a bioluminescence mouse model for non-invasive real-time monitoring of HCC therapy, and this HCR mouse model would be a useful tool for potential anti-HCC drug screening and new therapeutic strategy development.

Enhancement of PSMA-Directed CAR Adoptive Immunotherapy by PD-1/PD-L1 Blockade

Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb). PDL-1 expression was detected in Myc-CaP murine prostate tumors growing in immune competent FVB/N and immune-deficient SCID mice. Endogenous CD3+ T cells were restricted from the centers of Myc-CaP tumor nodules growing in FVB/N mice. Following anti-programmed cell death protein 1 (PD-1) treatment, the restriction of CD3+ T cells was reversed, and a tumor-treatment response was observed. Adoptive hPSMA-CAR T cell immunotherapy was enhanced when combined with PD-1 blockade, but the treatment response was of comparatively short duration, suggesting other immune modulation mechanisms exist and restrict CAR T cell targeting, function, and persistence in hPSMA expressing Myc-CaP tumors. Interestingly, an “inverse pattern” of CAR T cell BLI intensity was observed in control and test tumors, which suggests CAR T cells undergo changes leading to a loss of signal and/or number following hPSMA-specific activation. The lower BLI signal intensity in the hPSMA test tumors (compared with controls) is due in part to a decrease in T cell mitochondrial function following T cell activation, which may limit the intensity of the ATP-dependent Luciferin-luciferase bioluminescence signal.

Complementary use of bioluminescence imaging and contrast-enhanced micro-computed tomography in an orthotopic brain tumor model.

Small animal models are crucial to link molecular discoveries and implementation of clinically relevant therapeutics in oncology. Using these models requires noninvasive imaging techniques to monitor disease progression and therapy response. Micro-computed tomography (CT) is less studied for the in vivo monitoring of murine intracranial tumors and traditionally suffers from poor soft tissue contrast, whereas bioluminescence imaging (BLI) is known for its sensitivity but is not frequently employed for quantifying tumor volume. A widely used orthotopic glioblastoma multiforme (GBM) tumor model was applied in nude mice, and tumor growth was evaluated by BLI and contrast-enhanced microCT imaging. A strong correlation was observed between CT volume and BLI-integrated intensity (Pearson coefficient (r) = .85, p = .0002). Repeated contouring of contrast-enhanced microCT-delineated tumor volumes achieved an intraobserver average pairwise overlap ratio of 0.84 and an average tumor volume coefficient of variance of 0.11. MicroCT-delineated tumor size was found to correlate with tumor size obtained via histologic analysis (Pearson coefficient (r) = .88, p = .005). We conclude that BLI intensity can be used to derive tumor volume but that the use of both contrast-enhanced microCT and BLI provides complementary tumor growth information, which is particularly useful for modern small animal irradiation devices that make use of microCT and BLI for treatment planning, targeting, and monitoring.

Longitudinal Bioluminescence Imaging of the Dynamics of Doxorubicin Induced Apoptosis

Objectives: Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene. Methods: Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining. Results: Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and GloTM luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil. Conclusions: BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Comparison of Optical and Power Doppler Ultrasound Imaging for Non-Invasive Evaluation of Arsenic Trioxide as a Vascular Disrupting Agent in Tumors

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.

Effects of lazaroid U-74389G liposomes in a glioblastoma mouse model.

2098 Background: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effects against radiation-induced lipid peroxidation. Also in vitro antiproliferative effects have been reported against glioblastoma cell lines. The aim of this study was to use a liposomal formulation to evaluate LAZ radioprotective and antiproliferative effects in a glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed at the University of Houston, College of Pharmacy. Glioblastoma cell line U87-expressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. There were 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation 2Gy weekly (M+R), Lipo G at 5 mg/kg dose IP twice per week (M+L) and radiation with Lipo G (M+R+L). Treatment lasted three weeks. Tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse. Mice were sacrificed after 3 weeks. Brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. Survival was evaluated using Kaplan Meier analysis at P= 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to control . For the radiation treated groups (M+R and M+R+L), there was no significant difference in tumor size compared to control group. MDA brain concentration in M+L and M+R+L groups was significantly less than in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. Mean survival of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group Conclusions: LAZ liposomal formulations administered at 5 mg/kg dose reduced tumor growth by 65%. LAZ also protected brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration by 50%. These provocative data warrant further investigation of LAZ as a radiation protectant and chemotherapeutic agent.

ATP-Binding Cassette Transporters Modulate Both Coelenterazine- and D-Luciferin-Based Bioluminescence Imaging

Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC) transporters on BLI readout, we generated click beetle (cLuc), firefly (fLuc), Renilla (rLuc), and Gaussia (gLuc) luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2). In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type-independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.

Abstract 5460: Dual efficacy of Lazaroid U-74389G liposomes in glioblastoma mouse model

Abstract Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively). Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2011-5460

Response of Human Glioma U87 Xenografted on Mice to Non Thermal Plasma Treatment

Non thermal atmospheric plasma is a new promising candidate in anticancer therapy. We have already reported the absence of skin damage induced by our plasma treatment. Preliminary results suggested that this treatment could also induce an antitumor effect on U87 malignant glioma xenografts, and we conducted this work to evaluate the antitumor efficacy of plasma in this model. Antitumor effects were assessed by tumor volume measurement and bioluminescence imaging (BLI). Plasma treatment was applied during five consecutive days in open air with a μs-duration pulsed floating electrode dielectric barrier discharge (FE-DBD), which delivered about 0.75 W at 200 Hz on the mouse skin. Our results showed a significant tumor volume decrease of 56% for treated mice at the end of the treatment, with a concomitant decrease of BLI intensity. Moreover, this tumor volume reduction translated into an increase of mouse life span of 60%, median survival being 9.5 and 15.0 days for control and plasma-treated mice, respectively. In conclusion, our study demonstrates a marked antitumor effect of plasma treatment in U87 glioma xenografts. These results, obtained in both a radio and chemoresistant model, are very promising and highlight the potential of plasma treatment as an anticancer treatment with little or no toxic side effects.

Firefly luciferase-based dynamic bioluminescence imaging: a noninvasive technique to assess tumor angiogenesis.

Bioluminescence imaging (BLI) is emerging as a cost-effective, high-throughput, noninvasive, and sensitive imaging modality to monitor cell growth and trafficking. We describe the use of dynamic BLI as a noninvasive method of assessing vessel permeability during brain tumor growth. With the use of stereotactic technique, 10 firefly luciferase-transfected GL26 mouse glioblastoma multiforme cells were injected into the brains of C57BL/6 mice (n = 80). After intraperitoneal injection of D-luciferin (150 mg/kg), serial dynamic BLI was performed at 1-minute intervals (30 seconds exposure) every 2 to 3 days until death of the animals. The maximum intensity was used as an indirect measurement of tumor growth. The adjusted slope of initial intensity (I90/Im) was used as a proxy to monitor the flow rate of blood into the vascular tree. Using a modified Evans blue perfusion protocol, we calculated the relative permeability of the vascular tree at various time points. Daily maximum intensity correlated strongly with tumor volume. At postinjection day 23, histology and BLI demonstrated an exponential growth of the tumor mass. Slopes were calculated to reflect the flow in the vessels feeding the tumor (adjusted slope = I90/Im). The increase in BLI intensity was correlated with a decrease in adjusted slope, reflecting a decrease in the rate of blood flow as tumor volume increased (y = 93.8e-0.49, R2 = 0.63). Examination of calculated slopes revealed a peak in permeability around postinjection day 20 (n = 42, P < .02 by 1-way analysis of variance) and showed a downward trend in relation to both postinjection day and maximum intensity observed; as angiogenesis progressed, tumor vessel caliber increased dramatically, resulting in sluggish but increased flow. This trend was correlated with Evans blue histology, revealing an increase in Evans blue dye uptake into the tumor, as slope calculated by BLI increases. Dynamic BLI is a practical, noninvasive technique that can semiquantitatively monitor changes in vascular permeability and therefore facilitate the study of tumor angiogenesis in animal models of disease.

Abstract B27: A preclinical mouse model of orthotopic pleural cancer that facilitates noninvasive quantitative monitoring of tumor progression and therapy response

Abstract Purpose: The purpose of our study is to (a) establish a reproducible mouse model of orthotopic pleural cancer that recapitulates the pathobiology of human pleural malignancy (including primary malignant pleural mesothelioma, lung and metastatic breast cancers), and (b) validate the use of bioluminescence imaging (BLI) and serum biomarker assessment to noninvasively monitor tumor progression and therapy response in preclinical therapeutic trials. Experimental Design and Results: Human mesothelioma and lung cancer cell lines with variable expression levels of the tumor-associated antigen, mesothelin, were retrovirally transduced to stably express GFP-Firefly Luciferase, to facilitate BLI. Orthotopic xenograft mice were established by direct tumor cell inoculation into the pleural space with &amp;gt; 95% engraftment in both athymic nude and SCID-beige mice. The pathobiologic resemblance of the xenograft tumors to human disease was confirmed by demonstrating: (a) visceral and parietal pleural tumor growth 58 pattern, mediastinal lymph node involvement, and diaphragmatic invasion by magnetic resonance imaging (MRI) and histology, (b) strong, sustained expression of tumor markers at advanced stage of disease by WT-1, Calretinin, Mesothelin, and TTF-1 immunohistochemistry, and (c) extensive tumor lymphangiogenesis by CD-34 and LYVE-1 immunofluorescence. Tumor burden correlated with BLI signal (total flux photons/s) and soluble mesothelin-related peptide, SMRP (Mesomark® ELISA assay) both in vitro and in vivo (Pearson's correlation r=0.998, p&amp;lt;0.0001 and r=0.89, p=0.04, respectively). We determined the accuracy of BLI and SMRP (range 0.4–23nM) in monitoring tumor progression by serial assessment of xenograft mice (n=36, with initial tumor burden of 1×103–1×106 cells). In the orthotopic pleural xenografts, BLI signal correlated with SMRP (r=0.56, p=0.0001); no such correlation was observed in subcutaneous flank or intraperitoneal xenografts. To further demonstrate the reliability of BLI and SMRP to noninvasively monitor therapeutic response, groups of pleural mesothelioma xenografts (n=28) were treated with either: (a) Cisplatin (4mg/kg IP weekly), (b) thoracic radiation (20Gy total in 5 fractions), (c) combination chemoradiation, or (d) no treatment. BLI, SMRP, and survival were followed serially. Therapy response was demonstrated by decreased BLI intensity in mice treated with chemoradiation compared to controls (3.7×107 vs 2.0×108 photons/s, respectively, p=0.04) as well as decreased SMRP (p=0.03) which correlated with improved survival by Kaplan-Meier analysis (64 vs 34.5 days, p=0.0001). Conclusions: Pleural involvement by solid tumors remains a significant problem in clinical oncology affecting nearly 250,000 patients annually in the U.S. with no validated preclinical model for therapeutic investigations. We have validated a clinically relevant, reproducible orthotopic model of pleural malignancy that recapitulates human disease and allows for noninvasive quantitative monitoring of pleural tumor burden, progression and therapeutic response. Our xenograft model provides a novel platform to investigate targeted therapies for pleural malignancies. Citation Information: Cancer Res 2009;69(23 Suppl):B27.