Tumor Bioluminescence Research Articles

Immune Effects of M51R Vesicular Stomatitis Virus Treatment of Carcinomatosis From Colon Cancer

BackgroundThe purpose of this study was to analyze the oncolytic and immunomodulatory functions of an M protein mutant of vesicular stomatitis virus (M51R VSV) in a murine model of peritoneal surface dissemination from colon cancer (PSD from CRC). MethodsLuciferase-expressing CT26 peritoneal tumors were established in Balb/c mice to evaluate the impact of M51R VSV treatment on intraperitoneal tumor growth and overall survival. The mice were treated with either intraperitoneal phosphate buffered saline (n = 10) or 5 × 106 PFU M51R VSV (n = 10) at 5 d after tumor implantation. Tumor bioluminescence was measured every 3 d during the 60-day study period. The immunomodulatory effect of M51R VSV treatment was evaluated in mice treated with either intraperitoneal phosphate buffered saline (n = 21) or M51R VSV (n = 21). Peritoneal lavages were collected at days 1, 3, and 7 after M51R VSV treatment for flow cytometry and multiplex cytokine bead analysis. ResultsA single, intraperitoneal treatment with M51R VSV inhibited the growth of PSD from CRC as evidenced by decreased bioluminescence and improved survival. This treatment approach also resulted in significantly higher frequencies of peritoneal CD4+ T (10.95 ± 1.17 versus 6.19 ± 0.44, P = 0.004) and B1b cells (5.01 ± 0.97 versus 2.20 ± 0.2, P = 0.024). On the other hand, treatment with M51R VSV resulted in fewer myeloid-derived suppressor cells relative to controls (10.66 ± 1.48 versus 14.47 ± 1.06, P = 0.035). M51R-treated peritoneal cavities also contained lower concentrations of immunosuppressive monocyte chemoattractant protein-1 and interleukin 6 cytokines relative to controls. ConclusionsOur findings suggest that M51R VSV alters the innate and adaptive immune responses in PSD from CRC. Future studies will delineate specific components of antitumor immunity that result in its therapeutic effect.

Abstract 3928: N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity

Abstract Background: Radiation is a mainstay of treatment in patients diagnosed with glioblastoma. Despite maximal surgical and chemo-radiotherapeutic treatment, the disease remains uniformly fatal, with local disease recurrence within the previously radiated field comprising the major pattern of progression. According to the molecular subtyping of GBM, there is a predominance of mesenchymal GBM cells within the recurrent tumor mass, with characteristic increased expression of CD44, N-cadherin, L1CAM, TWIST1 and phosphorylated STAT3. Moreover, these populations of cells are known to be resistant to additional salvage radiation. The current study aims to evaluate the role of N-cadherin in the relative radiation resistance of mesenchymal subtype GBM. Methods: Overall survival data from the TCGA was analyzed using Cox regression hazard ratios based on N-cadherin expression across multiple molecular subtypes of GBM along with age and MGMT covariates. Using the U3035 and U87MG cells, the analyses of N-cadherin expression or exogenous exposure in the setting of external beam radiation were performed via stable shRNA knockdowns or the use of recombinant human N-cadherin ectodomain-Fc fusion proteins in clonogenic survival assays. Orthotopic implantations were performed in NSG mice with the endpoints of overall survival and tumor size, as determined by luciferase imaging. Subcellular localization of RAD51 was accomplished by confocal microscopy. Statistical analyses were performed using SPSS and GraphPad Prism with significance at p<0.05. Results: N-cadherin expression above the median among the mesenchymal subtype of GBM in the TCGA dataset correlated with a significant decrease in overall survival (HR 1.924, p=0.029). The elimination of N-cadherin expression in normally high expressing U3035 GBM cells led to a significant increase in the ability of radiation to induce cell death in clonogenic assay (17.2% U3035 vs 2% U3035 Δ N-cadherin at 8Gy; 12.7% U3035 vs 1.75% U3035 Δ N-cadherin at 20Gy) and an increase in the presence of double stranded DNA breaks (DSBs) on TUNEL assay. N-cadherin ectodomain-Fc fusion protein exposure in U87 cells resulted in a decrease in NSG mouse mean OS (17.6±0.25 days) compared to control radiated mice (22.3±1.1 days). Conversely, N-cadherin knockdown in U3035 cells resulted in further decrease in tumor bioluminescence beyond that seen in 4Gy x 4 radiation treatment of native U3035 xenografts (21% vs 4% of baseline). Conclusions: The reduction of N-cadherin expression in mesenchymal U3035 GBM cells increases the efficacy of radiation-induced cell death. This finding is accompanied by a decrease in the nuclear localization of RAD51 following DSBs induced by ionizing radiation, indicating a potential crosstalk between DNA damage repair mechanisms and calcium dependent cell-cell adhesion. Citation Format: Christopher P. Cifarelli, J. Austin Vargo, Ian MacFawn, Steven M. Frisch. N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3928.

Abstract 3894: Determination of the efficacious Entrectinib exposures required for pathway inhibition and anti-tumor activity in a subcutaneous and intracranialTPM3-NTRK1mutant tumor model

Abstract Entrectinib is a selective and brain penetrant inhibitor with potency against TRKA, TRKB, TRKC, ROS1 and ALK currently in clinical development for the treatment of tumors harboring NTRK or ROS1 gene fusions. In multiple non-clinical models harboring NTRK or ROS1 gene fusions, entrectinib demonstrates potent tumor growth inhibition leading to tumor regressions. Entrectinib achieves exposure in the brain across multiple species with brain-to-plasma concentration ratios of 0.4 in mice, 0.6-1.5 in rats, and 1.4-2.2 in dogs after multiple oral administration. Previously Cook, et al. demonstrated that entrectinib demonstrates anti-tumor efficacy in a BCAN-NTRK1 glioma model, however only testing higher doses. Here we sought to better understand the effective exposures of entrectinib required for pathway inhibition and anti-tumor efficacy through dose-ranging efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) studies using the TPM3-NTRK1 KM12-Luciferase (KM12-Luc) tumor model inoculated subcutaneously or intracranially. In the subcutaneous setting, entrectinib treatment results in significant dose-dependent inhibition of TRKA pathway signaling for up to 12 hours and anti-tumor activity with doses of 5 mg/kg (dosed orally (PO), every day (QD)) and above resulting in strong pathway suppression and tumor regressions. PK analysis demonstrated that an exposure of AUC24h of ≥20 µM.hr was associated with potent TRKA pathway inhibition and tumor growth inhibition. In the intracranial setting, entrectinib similarly demonstrated a dose-dependent effect on inhibition of tumor bioluminescence and enhanced animal survival. Pathway inhibition was observed at all doses of entrectinib with significant activity observed at doses of 5 mg/kg, PO, QD and above. In contrast to tumors inoculated subcutaneously, entrectinib doses of 15 mg/kg, PO, twice daily (BID) or 30 mg/kg, PO, QD were required to strongly inhibit anti-tumor efficacy with the highest dose of 60 mg/kg, PO, BID achieving full suppression of intracranial tumor growth. These data help to establish a model connecting entrectinib drug exposure in circulation and in the brain to pathway suppression and anti-tumor efficacy in an NTRK gene fusion tumor model, which support clinical use of entrectinib in patients with brain NTRK or ROS1 gene fusion tumors. Citation Format: Cecile C. de la Cruz, Thomas Hunsaker, Faye Vazvaei, Dragomir Draganov, Li Yu, Mark Merchant. Determination of the efficacious Entrectinib exposures required for pathway inhibition and anti-tumor activity in a subcutaneous and intracranialTPM3-NTRK1mutant tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3894.

Development of a Bioluminescent BRCA1-Deficient Xenograft Model of Disseminated, High-Grade Serous Ovarian Cancer.

Successful translation of preclinical data relies on valid and comprehensive animal models. While high-grade serous ovarian cancer (HGSOC) is the most prevalent subtype, the most commonly used ovarian cancer cell lines are not representative of HGSOC. In addition, 50% of ovarian cancer patients present with dysfunctional BRCA1/2, however currently there is a shortage of BRCA-deficient models. By utilizing the OVCAR8 cell line, which contains a hypermethylated BRCA1 promoter, the aim of the current study was to establish and characterize an animal model for BRCA-deficient HGSOC. Transfection of the luciferase gene to OVCAR8 cells enabled bioluminescent imaging for real-time, non-invasive monitoring of tumor growth. The resulting model was characterized by peritoneal metastasis and ascites formation at late stages of disease. Immunohistochemical staining revealed high-grade serous histology in all resected tumor nodules. Immunoblotting and qPCR analysis demonstrated BRCA1 deficiency was maintained in vivo. Moderate to strong correlations were observed between bioluminescent signal and tumor weight. Lastly, intraperitoneal administration of carboplatin significantly reduced tumor growth as measured by bioluminescence. The current model demonstrated BRCA1 deficiency and a high resemblance of the clinical features of HGSOC. This model may be well-suited for evaluation of therapeutic efficacy in BRCA-deficient HGSOC.

Regional pressure-enabled drug delivery of anti-PD-1 agent for colorectal liver metastases improves anti-tumor activity without increased hepatic toxicity

Abstract Introduction Checkpoint inhibitors (CI) have greatly impacted the treatment of solid tumors. Success in the liver has been more limited. When intravenously infused, the CI is delivered predominantly to extrahepatic sites, which results in systemic side effects. Previously, we reported on regional delivery of cellular immunotherapy for liver tumors using pressure-enabled drug delivery (PEDD™, TriSalus™ Life Sciences, Inc.). We hypothesized that PEDD of CI would improve the therapeutic index (TI). Methods In a murine model of colorectal liver metastases (CRLM), we infused high (HP) or low pressure (LP) anti-PD-1 antibodies (RMP1-14) via the portal vein and compared to systemic tail vein delivery (SD) at 0, 0.3, 1.0 and 3.0 mg/kg. Tumor bioluminescence (TB), histopathology, and serologic analysis informed treatment effects and toxicity. Results At a dose of 3mg/kg, LP resulted in similar IVIS TB growth kinetics compared to SD 3 mg/kg at 7 days post-treatment (geometric mean, p = 0.94). In animals treated with a 10-fold lower dose (0.3 mg/kg), tumor growth was significantly lower with HP vs. LP (7.5-fold improvement, p = 0.04). Circulating serum anti-PD-1 levels were similar when we compared SD and LP at 1 and 3 mg/kg (Mean 2512.0 vs. 2250.2 ng/mL, p = 0.25). Comparatively, systemic exposure was significantly lower at 0.3 mg/kg LP (Mean 508.1 ng/mL, p < 0.0001). No increases in hepatotoxicity were seen with PEDD (AST p = 0.57, ALT p = 0.43). Conclusion PEDD of anti-PD-1 CI improved liver tumor control while also limiting systemic exposure. These findings support PEDD for anti-PD-1 therapy to improve TI. Phase 1 exploration of anti-PD-1 PEDD for patients with hepatic malignancy is thus warranted.

Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence.

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. In vivo optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice.

Poly(ethylene glycol)-block-poly(d,l-lactic acid) micelles containing oligo(lactic acid)8-paclitaxel prodrug: In Vivo conversion and antitumor efficacy

Poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles affect drug solubilization, and a paclitaxel (PTX) loaded-PEG-b-PLA micelle (PTX-PM) is approved for cancer treatment due to injection safety and dose escalation (Genexol-PM®) compared to Taxol®. However, PTX-PM is unstable in blood, has rapid clearance, and causes dose-limiting toxicity. We have synthesized a prodrug for PTX (7-OH), using oligo(lactic acid) as a novel pro-moiety (o(LA)8-PTX) specifically for PEG-b-PLA micelles, gaining higher loading and slower release of o(LA)8-PTX over PTX. Notably, o(LA)8-PTX prodrug converts into PTX by a backbiting reaction in vitro, without requiring esterases. We hypothesize that o(LA)8-PTX-loaded PEG-b-PLA micelles (o(LA)8-PTX-PM) has a lower Cmax and higher plasma AUC than PTX-PM for improved therapeutic effectiveness. In Sprague-Dawley rats at 10 mg/kg, compared to o(LA)8-PTX-PM (10% w/w loading) and PTX-PM (10%), o(LA)8-PTX-PM (50% w/w loading) produces a 2- and 3-fold higher plasma AUC0–24 of PTX, lactic acid-PTX, and o(LA)2-PTX (o(LA)0-2-PTX), respectively. For o(LA)8-PTX-PM at 10 and 50% w/w loading, PTX and lactic acid-PTX are major bioactive metabolites, respectively. Fast prodrug conversion of o(LA)8-PTX in vivo versus in vitro (by backbiting) suggests that o(LA)8 is a good substrate for esterases. At 60 mg/kg (qwx3), o(LA)8-PTX-PM (50%) has higher antitumor activity than o(LA)8-PTX-PM (10%) and PTX-PM (10%) in a syngeneic 4T1-luc breast tumor model based on measurements of tumor volume, 4T1-luc breast tumor bioluminescence, and survival. Importantly, intravenous administration of o(LA)8-PTX-PM is well tolerated by BALB/c mice. In summary, oligo(lactic acid)8-PTX is more compatible than PTX with PEG-b-PLA micelles, more stable, and may expand the role of PEG-b-PLA micelles from “solubilizer” into “nanocarrier” for PTX as a next-generation taxane for cancer.

Long Non-Coding RNA NONHSAT076754 Promotes Invasion and Metastasis in Epithelial Ovarian Cancer.

Although accumulating evidence suggests that long non-coding RNAs (lncRNAs) are critical determinants of ovarian cancer development and progression, reports of metastasis-associated lncRNAs are limited. Here, we focused on NONHSAT076754 and explored its expression level, clinical value, biological behavior and molecular basis in epithelial ovarian cancer (EOC) metastasis. The results showed that NONHSAT076754 expression was increased in EOC tissues and cell lines and that this expression was closely related with FIGO stage, high tumor grade and lymph node metastasis. Furthermore, NONHSAT076754 knockdown markedly inhibited EOC cell migration and invasion in vitro. Consistently, the in vivo data from both the bioluminescence imaging and tumor dissection revealed that depletion of NONHSAT076754 reduced EOC metastasis. Mechanically, the pro-metastatic activities of NONHSAT076754 were partially regulated by PTEN and HTATIP2. Further rescue assays validated that knockdown of HTATIP2 remarkably reversed NONHSAT076754 silencer-induced inhibition of EOC cell metastasis. These data indicate that NONHSAT076754 is a vital regulator of EOC metastasis, laying the foundation for lncRNA-based clinical management of EOC aggressiveness and metastasis.

Co-delivery of VEGF siRNA and Etoposide for Enhanced Anti-angiogenesis and Anti-proliferation Effect via Multi-functional Nanoparticles for Orthotopic Non-Small Cell Lung Cancer Treatment

Targeting tumor angiogenesis pathway via VEGF siRNA (siVEGF) has shown great potential in treating highly malignant and metastatic non-small cell lung cancer (NSCLC). However, anti-angiogenic monotherapy lacked sufficient antitumor efficacy which suffered from malignant tumor proliferation. Therefore, the combined application of siVEGF and chemotherapeutic agents for simultaneous targeting of tumor proliferation and angiogenesis has been a research hotspot to explore a promising NSCLC therapy regimen.Methods: We designed, for the first time, a rational therapy strategy via intelligently co-delivering siVEGF and chemotherapeutics etoposide (ETO) by multi-functional nanoparticles (NPs) directed against the orthotopic NSCLC. These NPs consisted of cationic liposomes loaded with siVEGF and ETO and then coated with versatile polymer PEGylated histidine-grafted chitosan-lipoic acid (PHCL). We then comprehensively evaluated the anti-angiogenic and anti-proliferation efficiency in the in vitro tumor cell model and in bioluminescent orthotopic lung tumor bearing mice model.Results: The NPs co-delivering siVEGF and ETO exhibited tailor-made surface charge reversal features in mimicking tumor extracellular environment with improved internal tumor penetration capacity and higher cellular internalization. Furthermore, these NPs with flexible particles size triggered by intracellular acidic environment and redox environment showed pinpointed and sharp intracellular cargo release guaranteeing adequate active drug concentration in tumor cells. Enhanced VEGF gene expression silencing efficacy and improved tumor cell anti-proliferation effect were demonstrated in vitro. In addition, the PHCL layer improved the stability of these NPs in neutral environment allowing enhanced orthotopic lung tumor targeting efficiency in vivo. The combined therapy by siVEGF and ETO co-delivered NPs for orthotopic NSCLC simultaneously inhibited tumor proliferation and tumor angiogenesis resulting in more significant suppression of tumor growth and metastasis than monotherapy.Conclusion: Combined application of siVEGF and ETO by the multi-functional NPs with excellent and on-demand properties exhibited the desired antitumor effect on the orthotopic lung tumor. Our work has significant potential in promoting combined anti-angiogenesis therapy and chemotherapy regimen for clinical NSCLC treatment.

The Novel Combination of Nitroxoline and PD-1 Blockade, Exerts a Potent Antitumor Effect in a Mouse Model of Prostate Cancer.

Programmed cell death protein 1 (PD-1) blockade is a promising therapeutic strategy against prostate cancer. Nitroxoline has been found to have effective anticancer properties in several cancer types. We investigated the efficacy of a combination therapy involving nitroxoline and PD-1 blockade in a prostate cancer mouse model. In our in vitro analysis, we found that nitroxoline inhibited the viability and proliferation of the mouse prostate cancer cell line RM9-Luc-PSA. Additionally, nitroxoline downregulated the expressions of phospho-PI3 kinase, phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-GSK-3β, Bcl-2, and Bcl-xL. Nitroxoline also downregulated programmed death-ligand 1 (PD-L1) expression levels in prostate cancer cell line and tumor tissue. In our murine prostate cancer orthotopic model, nitroxoline plus PD-1 blockade synergistically suppressed tumor growth when compared with nitroxoline or PD-1 blockade alone, leading to reductions in tumor weight, bioluminescence tumor signals, and serum prostate-specific antigen levels. Furthermore, fluorescence-activated cell sorting analysis showed that the combination strategy significantly enhanced antitumor immunity by increasing CD44+CD62L+CD8+ memory T cell numbers and reducing myeloid-derived suppressor cell numbers in peripheral blood. In conclusion, our findings suggest that nitroxoline plus PD-1 blockade may be a promising treatment strategy in patients with prostate cancer.

PNCK depletion inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells in vitro and in vivo.

Purpose: Recent studies indicate that pregnancy upregulated non-ubiquitous calmodulin kinase (PNCK) is significantly up-regulated in breast and renal carcinomas. However, the expression profile and its biological relevance of PNCK in nasopharyngeal carcinoma (NPC) have not been elucidated.Methods: The expression level of PNCK was detected in specimens of NPC (n=10) and normal tissues (n=10) by real-time PCR and immunohistochemistry. Celigo Cell Counting and MTT assay were used to measure cell viability. Apoptosis was detected by flow cytometric analysis and caspases 3/7 activity assay. Real-time PCR and Western blotting were performed to evaluate the expression of PNCK. The bioluminescence imaging was used to evaluate the effects of PNCK knockdown on tumor growth using a xenograft animal model. The global gene expression profile was determined in wild type and PNCK-depleted CNE-2 cells via transcriptomics analysis. For mechanical investigation, the changes of PI3K/AKT/mTOR signaling pathway were detected by Western blotting.Results: The mRNA and protein levels of PNCK were increased in human NPC samples. In vitro experiments showed that shRNA or CRISPR-Cas9 mediated silencing of PNCK inhibited proliferation and induced apoptosis in NPC cells. In addition, in vivo assay revealed that knockdown of PNCK suppressed tumor growth. Consistently, a significant reduction of tumor bioluminescence in mice inoculated with PNCK-knockdown cells compared to that of control cells. In gene expression, the transcriptomics analysis revealed that there were 589 upregulated genes and 589 downregulated genes in PNCK-knockdown cells. Ingenuity Pathway Analysis (IPA) identified significant changes of PI3K/AKT/mTOR signaling pathway in PNCK-knockdown cells. Furthermore, western blot analysis revealed that interference with PNCK reduced the phosphorylation levels of PI3K, AKT and mTOR in CNE-2 cells.Conclusion: This study for the first time demonstrates that knockdown of PNCK could suppress growth and induce apoptosis of NPC cells both in vitro and in vivo by regulating PI3K/AKT/mTOR signaling pathway. These findings suggest that PNCK might be a novel therapeutic target for NPC treatment.

Targeting CD74 in multiple myeloma with the novel, site-specific antibody-drug conjugate STRO-001.

STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.

Continuous low-dose infusion of patupilone increases the therapeutic index in mouse and rat tumour models.

Patupilone is a microtubule-targeted cytotoxic agent with clinical efficacy, but causes diarrhoea in more than 80% of patients. The efficacy and tolerability of patupilone delivered continuously by subcutaneous (s.c.) mini-pumps [(mini-pump dose (MPD)] or by intravenous bolus administration [intravenous bolus dose (IVBD)] were compared preclinically to determine whether the therapeutic index could be improved. The antiproliferative potency in vitro of patupilone was determined by measuring total cell protein. Tumours were grown s.c. in rats (A15) or nude mice (KB31, KB8511) or intracranially in nude mice (NCI-H460-Luc). Efficacy was monitored by measuring tumour volumes, bioluminescence or survival. Toxicity was monitored by body weight and/or diarrhoea. Total drug levels in blood, plasma, tissues or dialysates were quantified ex-vivo by liquid chromatography-mass spectroscopy/mass spectroscopy. Patupilone was potent in vitro with GI50s of 0.24-0.28 nmol/l and GI90s of 0.46-1.64 nmol/l. In rats, a single IVBD of patupilone dose dependently inhibited the growth of A15 tumours, but also caused dose-dependent body weight loss and diarrhoea, whereas MPD achieved similar efficacy, but no toxicity. In mice, MPD showed efficacy similar to that of IVBD against KB31 and KB8511 tumours, but with reduced toxicity. In a mouse intracranial tumour model, IVBD was more efficacious than MPD, consistent with patupilone concentrations in the brain. MPD provided constant plasma levels, whereas IVBD had very high C0/Cmin ratios of 70-280 (rat) or 8000 (mouse) over the dosing cycle. Overall, the correlation of plasma and tumour levels with response indicated that a Cave of at least GI90 led to tumour stasis. Continuous low concentrations of patupilone by MPD increased the therapeutic index in s.c. rodent tumour models compared with IVBD by maintaining efficacy, but reducing toxicity.

Anti-prostate cancer activity of 8-hydroxyquinoline-2-carboxaldehyde-thiosemicarbazide copper complexes in vivo by bioluminescence imaging.

Copper 8-hydroxyquinoline-2-carboxaldehyde-thiosemicarbazide complex (CuHQTS) is a copper complex with strong anticancer activity against cisplatin-resistant neuroblastoma and prostate cancer cells in vitro by cell proliferation assay or fluorescent microscopic imaging. This study aimed to evaluate anti-prostate cancer activity of CuHQTS in vivo by bioluminescence imaging (BLI) and tumor size measurement, using athymic nu/nu mice implanted with prostate cancer cells carrying luciferase reporter gene (Luc-PC3). Growth of Luc-PC3 cells (1 × 105 cells) implanted in athymic nu/nu mice treated with CuHQTS for 2weeks was suppressed by measurement of luciferase signals (6.18 × 107 to 5.36 × 107p/s/cm2/sr) with BLI, compared with luciferase signals of Luc-PC3 cells (4.66 × 107 to 1.51 × 108 p/s/cm2/sr, p < 0.05) in the mice treated with normal saline of placebo control. Moreover, the size of PC-3 xenograft tumor (126.5 ± 34.2mm3) in athymic nu/nu mice treated with CuHQTS was significantly smaller than the size of PC-3 xenograft tumor (218.6 ± 48.0mm3, p < 0.05) in athymic nu/nu mice treated with normal saline of placebo control, suggesting in vivo tumor growth inhibition activity of CuHQTS on prostate cancer. The findings of this study support further investigation of CuHQTS as a promising new anticancer agent for the treatment of metastatic prostate cancer refractory to anticancer drugs currently available.

Abstract LB-122: Significant cytotoxic and immumomodulatory effects of continuous low dose intravesical gemcitabine in rodent bladder tumor models

Abstract Traditional gemcitabine bladder instillations (40 mg/mL) exhibit limited efficacy possibly due to micturition and saturable nucleotide uptake limiting tumor exposure. The present study investigates gemcitabine's anti-tumor and immunomodulatory effects after continuous low dose intravesical administration to enhance bladder tumor exposure. The direct effects of low dose gemcitabine at nominal bladder urine concentrations of 0, 20, 40, or 80 µg/ml were studied in athymic nude rats with human MIBC cells, T24-TurboFP635, injected into the bladder. After 3 days tumor growth, gemcitabine or vehicle was perfused into the bladder over 5 days, N=6 per group. Tumor bioluminescence was measured on day 5. A companion study in immunocompetent Wistar rats inoculated with syngeneic NBTII bladder tumors provided tumors for immunocyte analysis. A significant reduction in bladder tumor bioluminescence of 47.6%, 70.5% and 91.2% vs control was observed in the 20, 40, and 80 µg/ml groups, respectively. Flow cytometric analysis of residual tumors in immunocompetent rats demonstrated a significant decrease of 85.2% in the proportion of tumor T regulatory cells to T effector cells. Gemcitabine immunogenicity was studied in immunocompetent rats with NBTII cells injected into the bladder and subcutaneous flank tissue; flank tumors were implanted either simultaneously with bladder tumors or 14 days after bladder tumor implantation. Control groups received only flank tumors, N=15, or bladder and flank tumors with vehicle perfusion, N=10. Treatment groups, N=10, received two 5-day bladder perfusions, yielding gemcitabine urine concentrations of 10, 20, and 40 µg/ml which were separated by 7 days. Flank tumor growth was similar with tumor doubling times of 4.5 days after subcutaneous injections of 1.0, 2.5, or 5.0 x 106 cells and was unchanged with concomitant bladder tumor implantation and vehicle perfusion. During the first gemcitabine perfusion cycle, flank tumor growth was fully arrested, with tumor sizes averaging only 50% of controls in rats with concomitant bladder tumors; this effect was maintained during the 7-day drug free period in the 60 µg/ml group. Initiation of the second perfusion resulted in rapid flank tumor ablation in all but one animal completing the study. A similar priming effect was observed in the 15 and 30 ug/ml groups. Delaying flank tumor implantation to 3 days before the second perfusion not only prevented tumor growth but completely ablated all tumors. The results suggest continuous low dose intravesical gemcitabine inhibits bladder tumor growth, shifts the tumor microenvironment toward effector T cell dominance and induces a systemic immune response sufficient to suppress distant disease. Perhaps for the first time, the results also demonstrate a cytotoxic agent can induce a significant abscopal response when administered under proper conditions. Citation Format: Dennis H. Giesing, Don Reynolds, Vikas Agarwal, Chris Cutie, Balaji Ramachandran, Aravindakshan Jayaprakash, Satheesh Rajendiran, Purnand Sarma. Significant cytotoxic and immumomodulatory effects of continuous low dose intravesical gemcitabine in rodent bladder tumor models [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 LB-122.

Abstract 5917: A novel lipid nanoparticle (NBF-006) encapsulating glutathione S-transferase P (GSTP) siRNA for the treatment of KRAS-driven non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers and KRAS mutation occurs in 25-30% of NSCLC. Therapeutics directly targeting KRAS mutant proteins are currently not available. We developed a novel siRNA therapeutic against Glutathione S-Transferase P (GSTP), a gene target that is highly expressed in KRAS mutant NSCLC and is also involved in modulating RAS signaling pathway proteins. GSTP has been known as a Phase II detoxification enzyme and a modulator of MAP kinase-related cell-signaling pathways. Therefore, developing a GSTP inhibiting siRNA may be an effective therapeutic approach to treat KRAS mutant NSCLC. The lead drug product candidate (NBF-006) is a proprietary siRNA-based lipid nanoparticle (LNP) with a size of less than 70 nm, comprising GSTP siRNA (NDT-05-1040) and a novel ionizable, non-immunogenic, biodegradable lipid developed to target tumors in the lung. Our studies evaluated biodistribution and in vivo antitumor activities of NBF-006 in KRAS mutant NSCLC xenograft models. NBF-006 delivered siRNA molecules favorably to the lungs and tumor as compared to other tissues, with 70-80% of tumor cells showing siRNA uptake. Significant tumor growth inhibition (P &amp;lt;0.05) was observed in xenograft models derived from A549 (KRASMUT/TP53WT) and H23 (KRASMUT/TP53MUT) NSCLC cells, with notable tumor regression in A549 tumors and highly significant tumor regression in the H23 tumors following once-a-week administration. In the bioluminescent orthotopic lung tumor model, the average luminescent signal response in the NBF-006 treatment group was markedly lower than the vehicle control group (14.3 vs. 69.6 million photon/second, P &amp;lt;0.05). Additionally, in a surgically implanted orthotopic lung tumor model, the survival rate of the NBF-006 treatment group was significantly prolonged (P &amp;lt;0.005) at a 4 mg/kg dose compared to the vehicle control group. Finally, NBF-006 treatment was well tolerated in the preclinical efficacy models. In summary, we conclude that NBF-006 is selectively delivered to lung tumor tissue and its considerable uptake by tumor cells results in significant tumor growth inhibition and overall survival benefit as demonstrated in our preclinical models of KRAS mutant NSCLC. *These authors contributed equally. Citation Format: Zhihong O'Brien*, Li Wang*, Bharat Majeti*, Jeanpierre Clamme, Robiel Baclig, James Chu, Steven Fong, Jens Harborth, Jose Ibarra, Haiqing Yin, Jing Yu, Clayton Zhang, Roger Adami, Sonya Zabludoff, Wenbin Ying. A novel lipid nanoparticle (NBF-006) encapsulating glutathione S-transferase P (GSTP) siRNA for the treatment of KRAS-driven non-small cell lung cancer [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 5917.

Abstract 2549: Development of CAR T-cell therapy targeting glypican-3 in liver cancer

Abstract Background: Glypican-3 (GPC3) is a candidate therapeutic target in hepatocellular carcinoma (HCC). We have generated the HN3 and hYP7 antibodies that recognize the N-terminus and C-terminus of GPC3, respectively. Here, we engineered human T cells that express GPC3-specific chimeric antigen receptors (CARs) and evaluated their potential for the treatment of HCC. Methods: The GPC3-specific CARs (CAR.HN3 and CAR.hYP7) contain a CD3ζ chain and the 4-1BB co-stimulatory endodomain along with a truncated human EGFR polypeptide (huEGFRt) that retains the cetuximab epitope. A set of GPC3 positive-liver cancer cell lines including Hep3B, HepG2 and Huh7 were engineered to express the firefly luciferase gene. These cells were co-cultured with GPC3 CAR T cells at various ratios for 24 hours. A bioluminescent luciferase reporter assay was then used to evaluate the cytolytic activity of effector CAR T cells. We also evaluated the anti-tumor activity of CAR T cells in xenograft models with intraperitoneal injection of luciferase-expressing HCC cells into NOD/SCID/IL2gnull (NSG) mice. The tumor growth was measured via bioluminescence tumor imaging. Results: We found that the CAR.hYP7 cells targeting the C-terminus of GPC3 showed the highest cytolytic activity against GPC3-positive HCC cells. In two HCC intraperitoneal xenograft models (Hep3B and HepG2), luciferase-expressing HCC cells preferentially grew on the mouse liver. More importantly, we found that a single intraperitoneal treatment with a high dose of CAR.hYP7 cells (40 million per mouse) exhibited sustainable antitumor efficacy in the Hep3B model and all of the mice survived after 9 weeks post CAR T-cell treatment without recurrence. The serum levels of alpha fetoprotein (AFP) in mice treated with a high dose of CAR.hYP7 cells were less than 50 ng/ml, the threshold value for HCC diagnosis. Additionally, a single intraperitoneal treatment with a low dose of CAR.hYP7 cells (10 million per mouse) caused regression of liver tumor xenografts. In the low dose CAR.hYP7 group, Hep3B tumors in the mice grew locally and restricted to the fat tissues far from the mouse liver, indicating that CAR T cells prevented tumors from seeding and growing in the liver. Although CAR.HN3 cells targeting the N- terminus of GPC3 also exhibited high cytotoxicity to HCC cells in vitro, no significant Hep3B tumor growth inhibition was seen in the mice treated with CAR.HN3 cells. Conclusion: Our work suggests that GPC3-targeting CAR T cells, in particular CAR.hYP7, is a promising therapeutic intervention for liver cancer that can be translated to human use. Citation Format: Dan Li, Nan Li, Yifan Zhang, Haiying Fu, Madeline B. Torres, Qun Wang, Tim F. Greten, Mitchell Ho. Development of CAR T-cell therapy targeting glypican-3 in liver cancer [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 2549.

Abstract 2111: The tumor microenvironment of preclinical tumor models may have an impact on the therapeutic index of nanotherapeutics

Abstract Rapid development in nanotechnology allows the incorporation of multiple therapeutic agents (e.g. liposomes) into nanoparticle with a size range from 1 to 1000 nm. These nanocarrier systems provide new approaches to treat aggressive malignancy. Nano-based delivery systems hold an advantage over traditional small molecule therapy in that they can deliver drugs preferentially to tumors due to the enhanced permeability and retention (EPR) effect. On one hand, high permeability of the tumor vessels and a lack of functional lymphatic vessels results in the EPR effect, driving nanoparticle extravasation. On the other hand, these phenomena lead to high interstitial fluid pressure (IFP), limiting nanoparticle extravasation. In this study, we have used dynamic-contrast enhanced (DCE) MRI with small (Dotarem, 5 nm) and large (Vistarem, 25 nm) contrast agents (CAs) to examine tumor vessel permeability in SJSA-1 osteosarcoma tumor-bearing rats. An inversion-recovery true fast imaging with steady state precession (TrueFISP) was employed to determine the transfer constant Ktrans in subcutaneous and intra-tibia tumor models. When the tumor cells were injected in both niches within the same animal, Ktrans was significantly lower in intra-tibia tumors with both CAs, and blood vessels significantly less and larger compared to subcutaneous tumors. Moreover, Ktrans negatively correlated with tumor bioluminescence for the intra-tibia model, suggesting that EPR decreased inversely with tumor size, while the opposite was noticed for the subcutaneous tumors. These data demonstrate for the first time that the EPR of tumors with a similar size within the same animal can be influenced by the microenvironment. Moreover, they show that the therapeutic index of a nanotherapeutics may be different if it's determined with either an orthotopic or ectopic model. Citation Format: Stephane Ferretti, Nicolau Beckmann, laura Holzer, Michael Obrecht, Marjorie Berger, Michael Rugaard Jensen. The tumor microenvironment of preclinical tumor models may have an impact on the therapeutic index of nanotherapeutics [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 2111.

PD-1 blockade in combination with zoledronic acid to enhance the antitumor efficacy in the breast cancer mouse model

BackgroundBlockade of PD-1 receptor may provide proof of concepts for the activity of an immune-modulation approach for the treatment of breast cancer (BC). Zoledronic acid (ZA) has been proven to inhibit angiogenesis, invasion, and adhesion of tumor cells. The aim of this study was to investigate the potential of monoclonal antibody against T cell checkpoint PD-1 in combination with chemotherapeutic drug ZA in BC mouse model.MethodsThe 4 T1-fLuc mouse BC model was used in this study. The anti-tumor efficacy of anti-PD-1 antibody alone or in combination with ZA was monitored by measuring bioluminescence imaging (BLI) and tumor volume. At the end of study, the flow cytometry was used to determine the immune cell population in tumors after different treatment.ResultsThe results showed that mice treated with the combination therapy of anti-PD-1 antibody plus ZA exhibited better antitumor response compared to untreated controls or single therapy with no obvious toxicity.ConclusionOur study provides preclinical evidence for the enhanced BC treatment benefit through targeting co-signal molecules by combining anti-PD-1 antibody plus ZA treatment.

A Xenograft Model of Vestibular Schwannoma and Hearing Loss.

Microsurgical implantation of mouse merlin-deficient Schwann cells (MD-SC) into the cerebellopontine angle of immunodeficient rats will initiate tumor formation, hearing loss, and vestibular dysfunction. The progress in identifying effective drug therapies for treatment of Neurofibromatosis type II (NF2) is limited by the availability of animal models of VS that develop hearing loss and imbalance. A microsurgical technique for implanting MD-SCs onto the cochleovestibular nerve of rats was developed. Ten Rowett Nude rats were implanted with either ∼10 MD-SCs expressing luciferase (N = 5) or vehicle (N = 5). Rats received bioluminescence imaging, auditory brainstem response testing, and were observed for head tilt every 2 weeks after surgery, for a total of 6 weeks. Tumors were harvested and processed with hematoxylin & eosin staining and immunohistochemistry was performed for S100. Rats implanted with MD-SCs developed significantly higher tumor bioluminescence measurements and hearing threshold shifts at multiple frequencies by the 4th and 6th weeks post-implantation, compared with control rats. Rats implanted with MD-SCs also developed gross tumor. The tumor volume was significantly greater than nerve volumes obtained from rats in the control group. All rats with tumors developed a head tilt, while control rats had no signs of vestibular dysfunction. Tumors demonstrated histological features of schwannoma and express S100. Using this microsurgical technique, this xenograft rat model of VS develops tumors involving the cochleovestibular nerve, shifts in hearing thresholds, and vestibular dysfunction. This animal model can be used to investigate tumor-mediated hearing loss and perform preclinical drug studies for NF2.