Caliper Life Sciences Research Articles

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

MP17-19 RECRUITED T CELLS PROMOTE THE BLADDER CANCER METASTASIS VIA UP-REGULATION OF THE ESTROGEN RECEPTOR β/IL-1/C-MET SIGNALS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-19 RECRUITED T CELLS PROMOTE THE BLADDER CANCER METASTASIS VIA UP-REGULATION OF THE ESTROGEN RECEPTOR β/IL-1/C-MET SIGNALS Le Tao, Jialin Meng*, Edward M. Messing, Jean V. Joseph, Chawnshang Chang, Jianxin Qiu, and ShuYuan Yeh Le TaoLe Tao More articles by this author , Jialin Meng*Jialin Meng* More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Jianxin QiuJianxin Qiu More articles by this author , and ShuYuan YehShuYuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.020AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder cancer (BCa) is the fourth most commonly occurring cancer in men and is the seventh leading cause of death from cancer among men and the eleventh among women in the United States. Previous studies have shown that higher ERβ expression rates in BCa are positively correlated to higher grades and stages, as well as muscle invasiveness, and that both ERα and ERβ signals affect BCa development. Immune cells may infiltrate into different types of cancers and influence cancer progression. An early study demonstrated that CD4+ T cells could promote prostate cancer invasion. In BCa, one report indicated that inflammation elicited from the tumor microenvironment (TME) may be able to influence tumor progression. METHODS: Tissue samples were obtained from 20 patients who were diagnosed with BCa. ERβ (PLKO.1-puro-shERβ) was constructed with the target sequence 5′ GCGAGTAACAAGGGCATGGAA-3′ into lentiviral pLKO.1 vector as ERβ sh-RNA. Chromatin immuno-precipitation (ChIP) assays were used to calculate the binding of ERβ-DNA complex. Female nude mice (6–8 week old) were purchased from NCI and implanted with 5637 cells that were engineered to express a luciferase reporter gene (lentiviral-luciferase). Images were detected 20 min after intraperitoneal injection of luciferin using a fluorescent imager (IVIS Spectrum, Caliper Life Sciences) at 5 different time points (3, 4, 5, 6 and 7 weeks after tumor cells implantation). RESULTS: Results from an in vitroco-culture system found that BCa recruited more CD4+ T cells than did normal bladder cells. The recruiting of T cells to BCa tissues may increase the proliferation and invasion of BCa cells. Mechanistic studies revealed that infiltrating T cells stimulate BCa estrogen receptor beta (ERβ) signaling and consequently increase the expression of the MET proto-oncogene, receptor tyrosine kinase (c-MET), through either direct binding to its promoter or viamodulation of IL-1 expression. Interruption of ERβ/c-MET or ERβ/IL-1/c-MET signaling viaERβ-shRNA, IL-1 antagonist, or the c-MET inhibitor, SU11274, could partially reverse the T cell-enhanced BCa cell invasion and proliferation. Finally, the mouse BCa model with xenografted BCa 5637 cells with T (HH) cells confirmed the results of in vitroco-culture studies. CONCLUSIONS: Infiltrating T cells could promote BCa metastasis viamodulation of the ERβ/c-MET or ERβ/IL-1/c-MET signaling pathways. Source of Funding: This work was partially supported by the University of Rochester CTSI (UL1 TR000042), and the National Natural Science Foundation of China. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e233-e233 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Le Tao More articles by this author Jialin Meng* More articles by this author Edward M. Messing More articles by this author Jean V. Joseph More articles by this author Chawnshang Chang More articles by this author Jianxin Qiu More articles by this author ShuYuan Yeh More articles by this author Expand All Advertisement Loading ...

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in rats.

RationaleTherapeutic agents for memory enhancement in psychiatric disorders, such as schizophrenia, are urgently needed.ObjectiveThe aim of this study is to characterize the preclinical profile of ITI-214, a potent inhibitor of phosphodiesterase 1 (PDE1).MethodsITI-214 was assayed for inhibition of PDE1 versus other PDE enzyme families using recombinant human PDE enzymes and for off-target binding to 70 substrates (General SEP II diversity panel; Caliper Life Sciences). Effects of ITI-214 (0.1–10 mg/kg, po) on memory performance were assayed in rats using the novel object recognition (NOR) paradigm, with drug given at specified time points prior to or following exposure to objects in an open field. ITI-214 was evaluated for potential drug-drug interaction with risperidone in rats using conditioned avoidance response (CAR) and pharmacokinetic assessments.ResultsITI-214 inhibited PDE1A (Ki = 33 pmol) with >1000-fold selectivity for the nearest other PDE family (PDE4D) and displayed minimal off-target binding interactions in a 70-substrate selectivity profile. By using specific timing of oral ITI-214 administration, it was demonstrated in the NOR that ITI-214 is able to enhance acquisition, consolidation, and retrieval memory processes. All memory effects were in the absence of effects on exploratory behavior. ITI-214 did not disrupt the risperidone pharmacokinetic profile or effects in CAR.ConclusionsITI-214 improved the memory processes of acquisition, consolidation, and retrieval across a broad dose range (0.1–10 mg/kg, po) without disrupting the antipsychotic-like activity of a clinical antipsychotic medication, specifically risperidone. Clinical development of ITI-214 is currently in progress.

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Ibrutinib Significantly Prolonged Survival in a Human Burkitt Lymphoma (BL) Xenograft NSG Mouse Model: Ibrutinib May be a Potential Adjuvant Agent in the Treatment of BL

BACKGROUND: Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH 2012). Children with relapsed or progressive BL develop chemoimmunotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO 2012). Bruton's tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young/Staudt et al, Nat Rev Drug Dicov 2013). Preclinical studies of ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) suggest that its inhibitory effect on cell proliferation is in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk Res 2013), which is higher than the typical plasma concentration range in patients at the recommended dose of 560mg (<0.6uM) (Advani et al, JCO 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was initially approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 and CLL in February 2014, who have received at least one prior therapy. BL, however, is associated with tonic vs chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. These findings suggest that the antitumor activity of ibrutinib in BL, which is associated with tonic BCR signaling, may be less pronounced than in Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), CLL or MCL.OBJECTIVES: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NSG mice.METHODS: Raji Burkitt lymphoma (BL) cells (ATCC) were exposed to ibrutinib (0-10uM, generously provided by Janssen R&D LLC) alone and in combination with dexamethasone (1uM) for 5 days and then MTS cell proliferation assay (Promega) was performed. The IC50 values were determined with CompuSynTM software (Chou and Martin, ComboSyn, 2005) based on data derived from MTS assay. Raji BL cells were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, 6-8 weeks old) from Jackson laboratory were irradiated (2.5 Gy) and then mice were subcutaneously injected with one million ffluc-zeo tumor cells. Tumor progression and tumor burden were monitored by Bioluminescent Imaging system using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with either vehicle or ibrutinib (12.5mg/kg) for 10 days (once a day) at post-tumor cells injection. Analysis of survival rates were determined by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: Ibrutinib compared to control induced a significant dose-dependent decrease in cell proliferation in Raji BL cells (5uM, 0.561 ± 0.170, p<0.03, IC50=5.20uM) following 120 hours (5 days) of ibrutinib treatment. Interestingly, we observed a significant decrease in cell proliferation in Raji BL cells (5uM, 0.213 ± 0.078, p<0.002, IC=0.50 ± 0.374uM) following ibrutinib and 1uM dexamethasone combination treatment following 120 hours (5 days) of treatment. We observed a significant decrease of tumor progression by tumor luminescence signal intensity following ibrutinib treated Raji BL xenografted NSG mice at day 20 (12.5mg/kg, p<0.001 and 25mg/kg, p<0.05) and day 25 (12.5mg/kg, p<0.05 and 25mg/kg, p<0.05) compared to control (Figure 1A).Ibrutinib (12.5 mg/kg) treated mice (n=39) significantly prolonged survival compared to vehicle controls (n=28) (p<0.0001) (Figure 1B).CONCLUSIONS: Ibrutinib (12.5mg/kg) significantly decreased tumor progression and significantly increased survival in Raji BL xenografted NSG mice, suggesting that ibrutinib may be a potential adjuvant agent for therapy in the treatment of BL. Future studies will investigate the efficacy of combination therapy with ibrutinib on survival in BL xenografted NOD/SCID mice. [Display omitted] DisclosuresGalardy:Mission Therapeutics: Research Funding.

Ruxolitinib Significantly Prolongs Survival in Both a Primary Mediastinal B-Cell Lymphoma (PMBL) and Hodgkin Lymphoma (HL) Xenograft NSG Mouse Model: Ruxolitinib May be a Potential Adjuvant Agent in the Treatment of PMBL and of HL

BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL) are two of the most common malignancies among adolescents and young adults (AYA). PMBL and HL share similar molecular features by gene expression profiling and pathogenesis (Rosenwald et al., J Exp Med., 2003). HL represents only approximately 4-5% of all cancers in children younger than 15 years of age, but is the most common cancer in the 15-35 yrs AYA group. While the prognosis is excellent with AYA HL, there are significant late effects secondary to chemoradiotherapy and therefore new targeted agents are needed to avoid these morbid late effects in HL (Hochberg/Cairo et al., Br. J. Haem., 2009). Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity (Bentz et al., Genes Chromosomes Cancer, 2001), suggesting aberrant activity of JAK2 and Signal Transducer and Activator of Transcription (STAT) pathways, which may in part play an important role in the pathogenesis of HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor against myeloproliferative neoplasms (MPNs) that consistently exhibits dysregulation of the JAK1/JAK2 pathway, including those MPNs with a JAK2V617F mutation. Ruxolitinb also inhibits JAK2/STAT5 signaling in vitro and in a murine model of MPN (Quintas-Cardama et al., Blood, 2010).OBJECTIVES: We hypothesize that ruxolitinib may potentially function as targeted therapeutic agent for both PMBL and HL and therefore, investigated the efficacy of ruxolitinib in PMBL and HL cells xenografted into NSG mice.METHODS: Cell proliferation and apoptosis analysis were assessed using MTS and Caspase-3/7 assay (Promega), respectively. The expression of protein was examined by immunoblotting and statistical significance was determined by Student t-test. Karpas-1106P PMBL cell line and L-428 HL cell line were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. The six to eight weeks old Female NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were from Jackson laboratory. The ffluc-zeo NSG mice were irradiated (2.5 Gy) and then were subcutaneously injected with 1x106 ffluc-zeo Karpas-1106P or L-428 tumor cells. Tumor burden and tumor progression were monitored by bioluminescence imaging (BLI) using the Xenogen IVIS-200 (Caliper Life Sciences) for up to 60 days. Mice were orally gavaged with either vehicle or ruxolitinib (45.0mg/kg or 90.0mg/kg) (generously provided by Incyte Corporation, Wilmington, DE, USA) for 21 days. Survival rates were analyzed by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: We observed that ruxolitinib significantly decreased the phosphorylation of STAT3, -5 and -6 in Karpas-1106P PMBL and L-428 HL cells. The reduction in cell proliferation by 10uM ruxolitinib are 41% in Karpas-1106P (p<0.04) and 19% in L-428 (p<0.03), respectively. In addition, ruxolitinib significantly induced Caspase-3/7 activity (p<0.05) and increased cleavage of PARP and caspase-3 fragments (p<0.001). In PMBL and HL xenografted mice models, we observed a significant decrease in tumor luminescence intensity following ruxolitinib treated PMBL (45.0mg/kg and 90.0mg/kg, p<0.05) and HL xenografted NSG mice at day 21 and 28 (45.0mg/kg, p<0.05) compared to control mice (Figure 1A). Ruxolitinib (45.0mg/kg) treated xenograft NSG mice had a significantly prolonged survival in PMBL (p<0.0001) and HL (p=0.0001) compared to control mice (Figure 1B).CONCLUSIONS: Ruxolitinib significantly inhibited JAK2 enzymatic activity by the abrogation of the phosphorylation of STAT3, STAT5 and STAT6, and ruxolitinib showed significant anti-proliferative effects. Ruxolitinib (45mg/kg) significantly inhibited tumor progression and significantly prolonged survival in PMBL and HL xenografted NSG mice compared to control mice. Ruxolitinib may be a potential adjuvant agent in the treatment of PMBL and HL. Future studies will focus on whether the addition of ruxolitinib to chemoimmunotherapy improves the survival in a NSG PMBL and HL xenograft mouse models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Downregulation of DLEU1 Significantly Shortened Survival in a Rituximab-Treated DLEU1 Knockout Human Burkitt Lymphoma (BL) Xenograft NSG Mouse Model: DLEU1 May Act As a Tumor Suppressor Gene in Pediatric BL

BACKGROUND: Burkitt lymphoma (BL) is the most common histological subtype of non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo, BJH, 2012). We have previously identified secondary chromosomal aberrations in 70% of pediatric BL patients (PBL) with a C-MYC gene rearrangement (Poirel/Cairo et al., Leukemia, 2009). Specifically, we identified significantly inferior event free survival (EFS) and overall survival in children and adolescents with a specific loss of the 13q14.3 locus (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., Br. J. Haematol., 2009). In a multivariate analysis controlling for stage, lactate dehydrogenase levels, country of treatment, and group classification, children with BL who had a 13q deletion had significantly poorer EFS compared to the remainder of patients treated with the same French-American-British (FAB) chemotherapy regimen (Poirel/Cairo et al., Leukemia, 2009). Deleted in lymphocytic leukemia 1 (DLEU1) is a BL classifier gene in the 13q14.3 region (Dave et al., NEJM, 2006) and interacts with C-MYC, Tubulin beta-2C chain (TUBB2C), E3 ubiquitin-protein ligase (UBR1), cellular tumor antigen p53, and Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) (Stelzl et al., Cell, 2005). The sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) have been developed for targeted genome editing in in vitro and in vivo experiments with high efficiency as a new therapeutic tool (Sander et al., Nat. Biotechnol., 2011). We have previously reported a down-regulated DLEU1 mRNA expression significantly associated with an increase in BL proliferation in vitro (Lee/Cairo et al., AACR, 2015). We hypothesize that DLEU1 may have function as a tumor suppressor gene; however, the role of DLEU1 regulating programmed cell death in BL are poorly understood.OBJECTIVES: We hypothesize that DLEU1 may act as a tumor suppressor gene in BL and whether down-regulation of DLEU1 expression results in changes in BL survival following targeted immunotherapy.METHODS: TALENs induced DLEU1 knockout Raji cells were previously generated (Lee/Cairo et al., ASH, 2013) and DLEU1 knockout cells were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. Four- to six- week-old female NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice from The Jackson Laboratory were irradiated (2.5 Gy), and then mice were subcutaneously injected with 1x106 ffluc-zeo tumor cells. Tumor burden and tumor progression were monitored by bioluminescence imaging system using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were treated with either PBS, IgG isotype control, rituximab (30 mg/kg) or cyclophosphamide (25 mg/kg) and in combination by intraperitoneal (i.p) injection at 7 day intervals. Survival rates were analyzed by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: There were significant increases of luciferase signal intensity in DLEU1 knockout mice (DLEU1-KO) compared to that in wild type (WT) mice on day 31 of rituximab (p<0.05), cyclophosphamide (p<0.05) or rituximab and cyclophosphamide (p<0.01) combined treatment. Consistent with these initial findings in tumor growth, we found that rituximab-treated DLEU1-KO mice (n=12 per group) had a significantly shortened survival time with a median of 42 days compared to that of WT mice (n=12 per group) (52 days, p<0.005) (Figure 1A). For the rituximab and cyclophosphamide combination treated group, WT mice (n=12 per group) had a significantly extended survival time compared to the DLEU1-KO group (55.5 days vs 48 days, p<0.05) (Figure 1B). There were no significant differences in survival between WT and DLEU1-KO mice with PBS and IgG isotope treatment.CONCLUSIONS: The down-regulation of DLEU1 expression significantly decreased the survival rate in DLEU1-KO xenografted NSG mice following rituximab and in combination with cyclophosphamide treatment. Therefore, the down-regulation of DLEU1 expression in BL may in part result in immunotherapy resistance and may result in a consideration of alternative therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Platform Technologies for Directly Reconstructing 3D Living Biomaterials.

Bio-electrospraying and cell electrospinning is explored for reconstructing living biomaterials for regenerative biology and medicine. The investigations carried out in this study demonstrate these approaches as platform biotechnologies for tissue reconstruction for repair, replacement, and rejuvenation of damaged and/or ageing tissues and/or organs.

Abstract 2608: Ibrutinib significantly improves survival in a human Burkitt lymphoma (BL) xenograft NSG mouse model: Ibrutinib may be a potential adjuvant agent in the treatment of BL

Abstract BACKGROUND: Burkitt lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH, 2012). Children with relapsed or progressive BL develop chemotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO, 2012). Bruton's tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young et al, Nat Rev, 2013). Ibrutinib has been highly effective in the treatment of refractory patients with CLL and MCL and has been approved by the FDA for patients with CLL or MCL who have received at least one prior therapy (USPI) (Byrd et al, NEJM, 2013 and Wang et al, NEJM, 2013). BL, however, is associated with tonic or possibly chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. We have previously demonstrated ibrutinib significantly decrease BL proliferation and viability in vitro (Lee/Cairo et al, ASH, 2014) OBJECTIVE: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NOD/SCID (NSG) mice. METHODS: The luciferase expression plasmid (ffluc-Zeo), kindly provided by L. Cooper, MD) was transfected into Raji cells. Cells were subcutaneously injected into NSG mice (6-8wks old, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson lab). Tumor cells engraftment and progression were examined by Bioluminescent Imaging using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with vehicle control or Ibrutinib (1.25, 12.5 and 25mg/kg/day, generously provided by Janssen R&amp;D, LLC) for 10 days. Statistical probability of survival and curve were determined by Kaplan Meier method. RESULTS: We observed a significant decrease in tumor luminescence intensity following ibrutinib treated BL xenografted NSG mice at day 20 (12.5mg/kg, p&amp;lt;0.001 and 25mg/kg, p&amp;lt;0.05) and at day 25 (12.5mg/kg, p&amp;lt;0.05 and 25mg/kg, p&amp;lt;0.05) compared to vehicle control. Furthermore, ibrutinib (12.5 mg/kg) treated mice significantly prolonged survival compared to vehicle control mice (p&amp;lt;0.05). The median survival of mice following ibrutinib treatment (1.25, 12.5 and 25 mg/kg) were 31.5, 37.5 and 22.5 days, respectively, compared to control (24 days). CONCLUSIONS: Ibrutinib significantly decreased tumor progression and significantly increased the survival in BL xenografted NSG mice following 12.5mg/kg ibrutinib treatment. These results indicate that ibrutinib may be a potential adjuvant agent therapy in the treatment of BL. Future directions will be investigated the efficacy of ibrutinib in combination with dexamethasone in BL xenografted NSG mice. Citation Format: Sanghoon Lee, Changhong Yin, Timmy O'Connell, Matthew Barth, Janet Ayello, Lauren Harrison, Carmella van de Ven, Rodney Miles, Paul Galardy, Stanton C. Goldman, Megan Lim, Michelle Hermiston, Linda McAllister-Lucas, Lisa G. Roth, Sherrie L. Perkins, Mitchell S. Cairo. Ibrutinib significantly improves survival in a human Burkitt lymphoma (BL) xenograft NSG mouse model: Ibrutinib may be a potential adjuvant agent in the treatment of BL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2608. doi:10.1158/1538-7445.AM2015-2608

Albumin anchored docetaxel lipid nanoemulsion for improved targeting efficiency – preparation, characterization, cytotoxic, antitumor and in vivo imaging studies

The aim was to develop albumin anchored docetaxel lipid nanoemulsion (ALNE) for improving tumor targeted delivery. The O/W lipid nanoemulsion, LNEs were prepared by homogenization and ultrasonication processes. The size of globules and zeta potential were measured by Malvern Zetasizer. Albumin was coupled to stearylamine containing lipid nanoemulsion (SALNE) globules using water soluble EDC reaction. The drug content and entrapment efficiencies for the LNEs were determined by the high-performance liquid chromatography. The in vitro cytotoxic studies of the delivery systems were performed on MCF-7 and Hela cells. The IC 50 values of ALNE on both the cell lines were statistically significant. The in vivo antitumor activity was tested on solid tumors induced in C57BL/6 mice. This study revealed that the percentage tumor inhibition for the groups treated with DLNE, SALNE and ALNE when compared with untreated control was found to be 55.62 ± 5.41%, 54.27 ± 4.85% and 80.01 ± 2.74%, respectively. Furthermore, in vivo distribution studies were carried out in breast cancer MDA-MB231 xenografted Balb/c mice. The LNEs were loaded with fluorescent DiD oil and the distribution in different organs after 6 h was tracked using Caliper life sciences in vivo imaging system. The studies revealed that ALNE was superior in tumor targeting activity when compared with DLNE and SALNE by 3.04 and 2.26 folds, respectively. The average radiance values of ALNE on the tumor tissue were statistically significant when compared with DLNE, SALNE at p < 0.01. In addition, this strategy can become a platform technology for other lipophilic drugs to target tumors.

Ablative Fractional Radiofrequency Combined with Sonophoresis Increases Skin Penetration of Indocyanine Green.

Dear Editor: Indocyanine green (ICG) is a water-soluble tricarbocyanine dye with peak spectral absorption at 780 nm. Its intravenous application has been approved by the US Food and Drug Administration since 1959 for the determination of cardiac output, liver function diagnostics, and ophthalmic angiography1,2,3.There are various reports of the use of ICG in dermatology, including contrast enhancement for in vivo epidermal and dermal structures visualized by fluorescence confocal microscopy4, the treatment of mild to moderate acne5, augmentation of therapeutic effect of diode lasers in port-wine stains, and photodynamic therapy for actinic keratosis6. Compared to conventional photosensitizers such as aminolevulinic acid (ALA) and methyl aminolevulinic acid (MAL), ICG is considered a good alternative because of its minimal side effects and fair therapeutic efficacy. Previous studies report the effect of ablative fractional laser for facilitating skin penetration of MAL and ALA according to the detection of increased porphyrin fluorescence7,8,9. Using ICG as a test drug, we measured immediate drug absorption assisted by ablative fractional radiofrequency (RF) combined with sonophoresis ex vivo. Two male domestic Yorkshire swine (6~10 weeks old, 8~12 kg) were used. Skin tissue without subcutaneous fat was obtained from the flank area. For thawing, 3×3-cm2 skin tissue cryosections were kept at 40℃ for 1 hour and washed with saline. The experimental protocols were approved by the Kyung Hee University Animal institutional review board (KHMC-IACUC 11-028). Fractional ablative with the RF Pixel handpiece of the Legato system (Alma Lasers, Caesarea, Israel) was performed at 50 or 100 W, 40.68 MHz, and 15.4 ms pulse duration. A single-pass procedure was conducted without overlapping by using 6×50-pixel matrix on the tip. ICG cream (0.2%) was prepared by mixing ICG (Dongindang Pharm, Siheung, Korea) with petroleum jelly. The cream was applied at 0.3 g per 3×3 cm2 skin to an approximate thickness of 1 mm. The ICG cream-treated areas were covered and occluded with aluminum foil to avoid light exposure. After ICG application, sonophoresis using the IMPACT Pixel handpiece of the Legato system (Alma Lasers) was performed with power of 50 Hz for 30 seconds. To detect and image ICG fluorescence, the Maestro system (Caliper Life Sciences Inc., Hopkinton, MA, USA) with a near-infrared 740:10:950 filter was used. The average fluorescence index was measured by the Maestro system immediately after treatment. Higher fluorescence intensity indicates greater percutaneous ICG uptake8. The results are summarized in Table 1. Fluorescence intensity was higher after fractional and sonophoresis without ICG application than that in the untreated control. It should be noted that when the dermal connective tissue is exposed after ablative treatment, the autofluorescence derived from collagen and elastin could increase the fluorescence intensity. Table 1 Skin surface fluorescence intensities ICG-induced fluorescence was significantly higher on fractional and sonophoresis-pretreated skin than the untreated area (p<0.0001). Higher treatment energy tended to enhance fluorescence to a greater extent than lower energy, although the difference between energies was not significant (Fig. 1). Fig. 1 Representative images showing skin surface fluorescence as a result of different treatments. (A) Without pretreatment, (B) pretreatment with fractional radiofrequency (RF)+sonophoresis at 50 W, (C) pretreatment with fractional RF+sonophoresis at 100 W. ... Fang et al.7 report that among Er:YAG laser, microdermabrasion, iontophoresis, and electroporation, Er:YAG laser resulted in the greatest enhancement of ALA permeation. In addition, the addition of iontophoresis or electroporation toresurfacing techniques caused a profound synergistic effect on ALA permeation. Haedersdal et al.8 report that ablative fractional laser treatment facilitates the delivery of topical MAL deep into the skin. Our results suggest ablative followed by sonophoresis can immediately double ICG fluorescence. Ablative using the Legato system uses energy to produce micro-sparks transmitted between the skin surface and electrode, producing micro-channels. Acoustic pressure ultrasound between the skin and sonotrode can enhance the delivery of ICG fluorescence via these micro-channels through a hammer-like push-and-pull effect. In a recent in vivo study using human skin, Issa et al.10 demonstrated that pretreatment with ablative fractional associated with acoustic pressure improves the efficacy of steroids in hypertrophic scar treatment. Hence, it can be assumed that fractional laser and sonophoresis can be applied to enhance the absorption of intralesionally administered hydrophilic drugs. The major limitation of the current study is that the test method was not compared with a previous method such as ablative laser or sonophoresis alone. In conclusion, fractional combined with sonophoresis pretreatment can facilitate the skin penetration of ICG. This pretreatment process can shorten the incubation period of ICG and maximize the treatment effect.

Abstract 3795: Cabozantinib affects multiple signaling pathways in glioblastoma and is effective in a subset of xenograft tumors

Abstract Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor characterized by poor prognosis and high recurrence rate. Several new targeted agents are being considered for the adjuvant treatment of GBMs. Cabozantinib, a multi-kinase currently in clinical trials for several malignancies, including glioblastoma, targets c-Met and VEGFR2 receptors, as well as Ret, Kit, Flt-1/3/4, Tie2, and AXL. Our goal is to first evaluate sensitivity and molecular response of patient-derived GBM cancer stem cells (CSCs) to XL184 in vitro. Anti-tumor efficacy of cabozantinib as a single agent and in combination with the DNA-alkylating agent temozolomide (TMZ) in orthotopic xenografts was then evaluated. Methods: Neurospheres enriched in CSCs were cultured from resected GBM tumors. Sensitivity to cabozantinib was determined in vitro. Cells were treated (IC40) in triplicate, and cell lysates were analyzed by reverse phase protein microarrays. GBM CSCs were implanted intracranially into nude mice. Cabozantinib was administered by oral gavage at a dose of 60mg/kg for 4 weeks (5 days/week) as a single agent or in combination with 40mg/kg TMZ. Tumor growth and response to treatment were monitored by non-invasive in vivo biolumiescent imaging (BLI) using the Xenogen IVIS System (Caliper Life Sciences), and overall survival. Results: Sensitivity to cabozantinib treatment varied for the different GBM CSCs. From 70 proteins and phosphoproteins measured, 29 distributed among several signaling pathways were significantly altered after treatment in both resistant and sensitive GBM CSCs. Cabozantinib single agent treatment reduced GBM tumor growth and increased mouse survival in two xenograft lines. Cabozantinib monotherapy reduced tumor size, as measured by BLI, but had no significant effect on overall survival for another xenograft line, however, the combination treatment resulted in sensitization of these xenografts to TMZ treatment. Conclusion: The use of multi-kinase inhibitors is a promising strategy for GBMs, and given the range of responses, identifying predictive biomarkers, though challenging, will be invaluable for patient stratification. Citation Format: Ana C. deCarvalho, Kimberly Arnold, Claudius Mueller, Emanuel F. Petricoin, Laila M. Poisson, Tom Mikkelsen. Cabozantinib affects multiple signaling pathways in glioblastoma and is effective in a subset of xenograft tumors. [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 3795. doi:10.1158/1538-7445.AM2014-3795

CABOZANTINIB IS EFFECTIVE IN A SUBSET OF XENOGRAFT GBM TUMORS AND AFFECTS MULTIPLE SIGNALING PATHWAYS

BACKGROUND: (blind field). METHODS: Neurospheres enriched in CSCs were cultured from resected GBM tumors. Sensitivity to cabozantinib was determined in vitro. Cells were treated (IC40) in triplicate, and cell lysates were analyzed by reverse phase protein microarrays (RPPAs). GBM CSCs were implanted intracranially into nude mice. Cabozantinib was administered by oral gavage at a dose of 60 mg/kg for 4 weeks (5 days/week) as a single agent or in combination with 40 mg/kg TMZ. Tumor growth and response to treatment were monitored by non-invasive in vivo bioluminescence imaging (BLI) using the Xenogen IVIS System (Caliper Life Sciences), and overall survival. RESULTS: Sensitivity to cabozantinib treatment varied for the different GBM CSCs. From 70 proteins and phosphoproteins measured, 29 distributed among several signaling pathways were significantly altered after treatment in both resistant and sensitive GBM CSCs, including Met, Ret, AKT, MAPK/ERK. Cabozantinib single agent treatment reduced GBM tumor growth and increased mouse survival in two xenograft lines. Cabozantinib monotherapy reduced tumor size, as measured by BLI, but had no significant effect on overall survival for another xenograft line, however, the combination treatment resulted in sensitization of these xenografts to TMZ treatment. RPPA confirmed downregulation of the described targets for XL184, including activated Met, VEGFR2 and Ret (in vitro). CONCLUSIONS: Consistent with the clinical experience, both sensitive and resistant GBMs are represented in our CSC xenografts. More extensive evaluation will likely identify baseline biomarkers which might be valuable in identifying potentially sensitive sub-populations for subsequent clinical trials. RPPA and next-gen sequencing (NGS) on terminal tumors is underway. SECONDARY CATEGORY: Tumor Biology.

Physical exercise attenuates inflammation via suppression of systemic macrophage activation

Physical exercise attenuates inflammation via suppression of systemic macrophage activation

Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Significantly Mediate Burkitt Lymphoma (BL) Regression and Improve Survival In Human BL Xenografted NSG Mice

BackgroundBurkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma that occurs in children and adolescents (Miles/Cairo, BJH, 2012). The 5-year event-free survival (EFS) of children and adolescents with newly diagnosed BL has been significantly improved but for patients with relapsed/refractory disease, the prognosis is dismal due to chemotherapy resistance (Cairo, et al, J Clin Oncol, 2012; Cairo, et al Blood 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. We have previously demonstrated that CD20 is expressing in ≥98% of pediatric BL (Perkins/Cairo, Clin Adv Hematol Oncol. 2003) and also reported that expanded Peripheral Blood Natural Killer (exPBNK) Cells electroporated with anti-CD20 Chimeric Antigen Receptor (CAR) mRNA have significant cytotoxicity against CD20+ BL in vitro (Chu/Cairo, et al, ASH, 2012). ObjectiveTo examine the anti-tumor effect of anti-CD20 chimeric antigen receptor (CAR+) modified expanded PBNK cells against disseminated CD20+ Burkitt Lymphoma in vivo using human CD20+ BL xenografted NSG mice. MethodsPBMC were expanded with lethally irradiated K562-mbIL15-41BBL cells for 14 days as we previously reported. CD56+CD3- expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit. CAR mRNA was nucleofected and CAR expression was detected using anti-mouse IgG, F(ab')2 fragment-specific antibody (Chu & Cairo, et al, ASH, 2012).5x105 Raji cells expressing luciferase (Raji-Luc) were intraperitoneally (i.p.) injected into the NSG mice (6 weeks, Jackson Labs). Raji engraftment and progression was evaluated using the Xenogen IVIS-200 system (Caliper Life Sciences) after i.p. injection of 150mg D-luciferin/kg/mouse. Photons emitted from luciferase-expression cells were quantified using the Living Image software. After Raji-Luc engraftment was verified in mice at day 7, 5x106 anti-CD20 CAR exPBNK or MOCK exPBNK (no anti-CD20 CAR mRNA electroporation) were i.p. injected to each mouse once a week (day 9,16, 23). Control mice received culture medium instead of NK cells. The cumulative luciferase signals were measured weekly to indicate the tumor growth, dissemination and progression.Statistical probability of survival and comparison of survival curves were determined by Kaplan Meier Method and the log-rank test. The results with a P value < 0.05 were deemed statistically significant. ResultsWe found that luciferase signals from Raji-luc were not significantly different between the untreated, mock exPBNK treated, and CAR exPBNK treated groups (p=0.0538) after the first NK injection. However, the luciferase signals measured in the CAR exPBNK treated group were significantly different than that in the control mice (two tailed P= 0.0013) and the mock exPBNK treated mice (two tailed P= 0.0229) after the second NK injection. After the third NK injection, the luciferase signals measured in the CAR exPBNK treated group were also significantly different than that in the control mice (two tailed P= 0.0173) and the mock exPBNK treated mice (two tailed P= 0.0256) (Fig. 1A). But luciferase signals were not significantly different between the untreated mice and the mock exPBNK treated mice (two tailed P= 0.0729).Consistent with the reduced luciferase signals, the CAR exPBNK treated mice had significantly extended survival time with median 40 days compared to the untreated mice (29 days, P<0.001) and the mock exPBNK treated mice (30 days, P<0.001) (Fig. 1B). There was no significant difference of survival between the untreated mice and the mock exPBNK treated mice. ConclusionMultiple injections of anti-CD20 CAR mRNA electroporated exPBNK cells can significantly mediate disseminated Raji tumor regression in Raji xenografted NSG mice compared to untreated and mock exPBNK treated mice. Furthermore, anti-CD20 CAR exPBNK significantly increased the survival of Raji xenografted mice. These results indicate therapeutic potential of multiple injections of anti-CD20 CAR mRNA modified exPBNK cells against BL in patients with chemo-immunotherapy resistance. Future directions include examining the anti-tumor activity of CAR+ exPBNK against rituximab/chemotherapy resistant BL in xenografted NSG mice. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Immortalized human fetal bone marrow-derived mesenchymal stromal cell expressing suicide gene for anti-tumor therapy in vitro and in vivo

Background aimsCancer is one of the greatest health challenges facing the world today with >10 million new cases of cancer every year. The self-renewal, tumor-homing ability and low immunogenicity of mesenchymal stromal cells (MSCs) make them potential delivery candidates for suicide genes for anti-tumor therapy. However, unstable supply and short life span of adult MSCs in vitro have limited this therapeutic potential. In this study, we aimed to evaluate if immortalization of human fetal bone marrow-derived mesenchymal stromal cells by simian virus 40 (SV40-hfBMSCs) could be a stable source of MSCs for clinical application of suicide gene therapy. Methods and ResultsTransduction of SV40 and herpes simplex virus thymidine kinase-IRES-green fluorescent protein (TK-GFP) did not cause significant change in the stem cell properties of hfBMSCs. The anti-tumor effect of SV40-TK-hfBMSCs in the presence of the prodrug ganciclovir was demonstrated in vitro and in nude mice bearing human prostate cancer cells, DU145 and PC3, which had been transduced with luciferase and GFP for imaging evaluation by an in vivo live imaging system (IVIS 200 imaging system; Caliper Life Sciences). Repeated injection of low doses (1 × 106 cells/kg) of SV40-TK-hfBMSCs was as effective as previously reported and did not cause observable harmful side effects in multiple organs. Mixed lymphocyte reaction showed that SV40-TK-hfBMSCs did not induce significant proliferation of lymphocytes isolated from healthy adults. ConclusionsTaken together, immortalized hfBMSCs represent a reliable and safe source of MSCs for further clinical translational study.

Abstract 1270: Mimicking infection for immunotherapy against breast cancer - fooling the immune system.

Abstract Short description: Immunotherapy represents a potential and innovative means to combat cancer. It essentially harnesses the body's immune system to fight against cancer. Previous literature suggests that cancer vaccines designed against a specific tumor antigen have been efficiently utilized to trigger immune responses against tumor cells. Despite the preliminary evidence in animal models, low immunogenicity is one of the major hurdles in the development of vaccines in humans. In order to surmount this obstacle, several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored. Purpose: The purpose of this study was to develop “bacteriomimetic nanoparticles” to enhance adaptive cell-mediated immune responses (CD4+ and CD8+ T cell responses) against tumor antigen as a therapeutic option for cancer treatment. Materials and Methods: NPs were prepared by modified solid/oil/water solvent evaporation method using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany). We used membrane preparations of the 4T1 mouse mammary cancer cell line as a tumor antigen and CpG ODN's as a “bactriomimetic” stimulant. Fourteen days before tumor challenge BALB/c female mice (6-8 weeks) were pre-immunized with CpG followed by secondary immunization using respective NPs encapsulated with the membrane antigen preparation. Subsequently, mice (n=4) were challenged with 105 tumor cells intravenously (IV). Mice were sacrificed and tumors were harvested at days 3, 7 and 14 respectively. CD4+ and CD8+ T cell responses were measured in lower respiratory node and spleen using flow cytometry. In another experimental set, following the same immunization schedule as mentioned above, mice (n=5) were challenged subcutaneously (SC) with 105 tumor cells. Primary tumor size was monitored using vernier caliper and bioluminiscence imaging (Caliper Life Sciences Inc., MA, USA). Mice were sacrificed on day sixteen after tumor challenge; spleen cells were used for flow cytometric analysis and primary tumor tissue was used to evaluate CD4+ and CD8+ T cell via immunohistochemistry. Results: We found significant reduction in progression of tumor growth in mice immunized with CpG coated NPs containing tumor antigen (CpG-NP-Tag). Cytometry analysis demonstrated increased CD4+ (helper) and CD8+ (cytotoxic) T cell response emphasizing enhanced immunogenicity against cancer cells. IHC data indicated greater CD4+ T cell infiltration of the tumor tissue for the animals immunized with CpG-NP-Tag. Conclusions: Primary tumor size, IHC and flow cytometry analysis indicate that CpG-NP-Tag NPs were successfully employed to boost the immune response against tumor cells. Citation Format: Rutika Kokate, Sanjay Thamake, Harlan Jones, Jamboor Vishwanatha, Brittany Mott. Mimicking infection for immunotherapy against breast cancer - fooling the immune system. [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 1270. doi:10.1158/1538-7445.AM2013-1270

Abstract 3915: Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc.

Abstract A new investigational therapeutic agent, pegylated recombinant human hyaluronidase PH20 (PEGPH20) is a currently under clinical development for the treatment of tumors that accumulate hyaluronan (HA), a nonsulfated glycosaminoglycan and a major constituent of the extracellular matrix (ECM) of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival in multiple malignancies. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. To facilitate non-invasive clinical selection of patients with HA-rich malignancies, and confirm PEGPH20 enzymatic targeting of these malignancies, we have developed a soluble recombinant HA binding protein by fusing a tumor necrosis factor-stimulated gene-6 protein (TSG6) fragment, mutated at the heparin-binding site, with human IgG1 Fc (TSG6dHep-Fc). In preclinical proof-of-concept studies, recombinant TSG6dHep-Fc was labeled with the near-infrared fluorophore DyLight755 (TSG6dHep-FcDL755) and administered IV to the following xenograft peritibial athymic mouse models: HA-rich pancreatic BxPC3, HA-poor prostate Du145 and HA-rich prostate Du145/HAS2. TSG6dHep-FcDL755 HA binding was imaged via fluorescence (IVIS Lumina II, Caliper Life Sciences, Inc.). Systemic/dermal HA binding was weakly present, but easily removed via background subtraction to allow tumor intensity quantification. In the HA-rich xenograft models, pancreatic BxPC3 and prostate Du145/HAS2, TSG6dHep-FcDL755 strongly labeled the peritibial tumors, peaking at 2 days post administration and was completely absent at day 10 post administration. In contrast, the HA-poor prostate Du145 tumors were very weakly labeled, making background subtraction of systemic/dermal HA binding difficult. In separate experiments, mice were treated with a single dose of PEGPH20 (4.5 mg/kg, IV) or vehicle prior to TSG6dHep-FcDL755 imaging. In all groups dosed with PEGPH20, TSG6dHep-FcDL755 signal intensity was extremely low and appeared to be limited to non-specific, whole body labeling. These results suggest that imaging with labeled TSG6dHep-Fc could be a useful tool for selecting patients with HA-rich malignancies, thereby enabling clinicians to noninvasively identify patients who might benefit from therapies that target HA and the ECM. Citation Format: Xiaoming Li, Lei Huang, Ge Wei, Ryan J. Osgood, Qiping Zhao, Ping Jiang, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc. [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 3915. doi:10.1158/1538-7445.AM2013-3915