Human Lung Carcinoma Xenografts Research Articles

Metallic radionuclide-labeled tetrameric 2,6-diisopropylphenyl azides for cancer treatment.

This study proposes a new method for radionuclide therapy that involves the use of oligomeric 2,6-diisopropylphenyl azides and a chelator to form stable complexes with metallic radionuclides. The technique works by taking advantage of the endogenous acrolein produced by cancer cells. The azides react with the acrolein to give a diazo derivative that immediately attaches to the nearest organelle, effectively anchoring the radionuclide within the tumor. Preliminary in vivo experiments were conducted on a human lung carcinoma xenograft model, demonstrating the feasibility of this approach for cancer treatment.

Abstract 3439: An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts

Abstract Carcinoembyronic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in numerous solid tumors. CEA is constitutively released from tumor cells and reaches detectable levels in peripheral blood, making CEA targeting particularly challenging. Granzyme B is a key effector in immune-mediated killing of target cells, with cell death through both caspase-dependent and -independent mechanisms. We developed a fusion protein composed of granzyme B and a human scFv targeting the juxta-membrane epitope of CEA. The construct also incorporates a fragment of human IgG heavy chain to cause dimerization and provide increased in vivo circulation and antitumor efficacy. The GrB-Fc-CEA construct was cloned into a mammalian expression vector, transiently expressed by HEK-293E suspension cells, and secreted into the culture media. The final yield after purification and activation was 10-15 mg/L. The enzymatic activity of granzyme B in GrB-Fc-CEA was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against CEA-positive cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar range cytotoxicity against control (CEA-negative) cell lines. Importantly, GrB-Fc-CEA cytotoxicity against tumor cells in vitro was unaffected by exogenously-added CEA (200 ng/ml) extracellular domain, suggesting that the construct is not competitively absorbed by soluble CEA released by tumors and present in the circulation of patients. GrB-Fc-CEA internalized rapidly (within one hour) into the target cell lines A-431 and HT-1080/CEA whereas internalization was not observed with untargeted granzyme B, clearly demonstrating receptor-mediated internalization. GrB-Fc-CEA induced mitochondrial depolarization and caspase activation, consistent with the well-described granzyme B mechanism of action. Treatment of female nude mice bearing A-549 tumors with GrB-Fc-CEA (80 mg/kg over 5 doses) resulted in a significant growth inhibition of the established tumors compared to vehicle controls (1200 mm3 vs <40 mm3). Complete tumor regression was observed in seven of ten tumors in mice treated with GrB-Fc-CEA. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Thus, GrB-Fc-CEA appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3439.

Fish Oil, Plant Polyphenols, and Their Combinations Have No Tumor Growth Promoting Effects on Human Lung and Colon Carcinoma Xenograft Mice

The goal of this study was to evaluate if combinations of ingredients with known anti-cachexia benefits (Fish oil-FO with either curcumin or Green tea extract-GTE), have adverse effects on tumor growth, using human carcinoma xenograft mice models. FO (EPA/DHA 360 mg/kg bw), GTE (90 mg/kg bw), and curcumin (180 mg/kg bw) were administered orally, alone or in combination, to nude mice bearing either A549 human non-small cell lung carcinoma or SW620 human colon carcinoma tumors. Bodyweight, tumor growth, survival time, and other clinical endpoints were assessed. The ingredients either alone or in combinations were well tolerated in both lung and colon tumor-bearing mice. There were no significant group differences between individual or combination treatments for tumor growth (A549 or SW620) as measured by the median time in days to endpoint of tumor volume (TTE). TTE results indicate that these ingredients (alone or combinations) did not adversely impact tumor growth. No significant differences in body weights or survival were observed between controls and treatment groups indicating no adverse health effects of the ingredients. In conclusion, FO, GTE or curcumin administered as monotherapies and in combination were well tolerated and displayed no adverse effects on tumor growth in mouse xenograft models of lung and colon cancer.

Abstract 1485: E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway

Abstract Notch4 is selectively expressed on tumor endothelial cells and hypothesized to contribute to cancer malignancy through mechanisms including tumor angiogenesis regulation and promoting drug resistance. We developed E7011, a humanized IgG2κ antibody targeting Notch4 receptor signaling. To obtain anti-human Notch4 monoclonal antibody, human Notch4 3EGFR (epidermal growth factor repeat) domain - NRR (negative regulatory region) - SEAP (secreted alkaline phosphatase) - His protein was immunized to mice and 6698 hybridomas were obtained. By screening these clones with the Notch4 binding assay and Notch4-specific gal4-fusion luciferase reporter assay, mouse anti-human Notch4 mAb clone (6-3-A6) was selected. The CDRs (complementarity determining regions) of 6-3-A6 were grafted into the variable regions of human IgG2κ, and E7011, novel humanized anti-human Notch4 monoclonal antibody, was developed by screening in assays above. The biomolecular interaction analysis showed that E7011 is able to bind to human and mouse Notch4-NRR domain-SEAP-His, and inhibit human Notch4 receptor signaling in an antibody-dose dependent manner as well as 6-3-A6. IHC analysis showed that Notch4 was expressed on CD31 positive endothelial cells in Calu6 human non-small cell lung carcinoma xenograft tumor. Intravenous administration of E7011 at 1, 3, 10 mg/kg in twice a week significantly decreased tumor growth compared with control IgG treatment without severe body weight loss. We also investigated the functional tumor angiogenesis by hoechest dye-perfusion and staining with anti-CD31 antibody in the Calu6 xenograft model. E7011 treatment significantly decreased hoechst-positive perfusion area compared with vehicle treatment. In conclusion, E7011 is a novel antitumor agent that is a specific signal inhibitor targeting the Notch4 receptor and suppresses tumor growth through decreasing tumor vessel function in based on preclinical studies. Citation Format: Yusuke Adachi, Yoshimasa Sakamoto, Youya Nakazawa, Sho Tachino, Ken Ito, Takanori Abe, Yukinori Minoshima, Kana Hoshino-Negishi, Hideaki Ogasawara, Tomomi Kawakatsu, Miyuki Nishimura, Masashi Shimizu, Kazuhiro Tahara, Toshitaka Sato, Katsuhisa Suzuki, Kishan Agarwala, Masao Iwata, Yasuhiro Funahashi, Kenichi Nomoto, Yoichi Ozawa, Junji Matsui, Toshio Imai, Yu Kato. E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1485.

Graphdiyne-templated palladium-nanoparticle assembly as a robust oxygen generator to attenuate tumor hypoxia

Abstract Nanocatalysts have attracted many concerns in biomedical fields because their catalytic performance is robust and remains efficient even at a harsh condition compared to biological enzymes. However, the stability for both physical forms and the catalytic activity are the main challenges during biomedical applications. Here, we described a strategy to prepare a stable nanocatalyst that two-dimensional (2D) graphdiyne (GDY) serves as a template to immobilize catalytic palladium nanoparticles (PdNPs) on the GDY surface. In the presence of H2O2, the PdNPs/GDY composite functions as an oxygen generator to decompose H2O2 to produce molecular oxygen, which efficiently attenuates tumor hypoxia and delays tumor growth. According to X-ray absorption spectroscopy and computational simulation, we revealed that PdNPs mainly contribute to the decomposition of H2O2 into O2 companying with the variation in chemical forms of Pd in the presence of H2O2. Importantly, 2D GDY firmly anchors and disperses ultra-small PdNPs on the surface via Pd-C bonds to prevent the oxidation and aggregation of PdNPs that realizes sustainable and stable catalysis. This design endows the highly immobilized content of Pd on GDY, high catalytic activity and higher stability in catalytic reaction than commercial Pd/C and PdNPs/GO, accordingly. The PdNPs/GDY composite also exhibits as an effective and durable oxygen generator to decompose endogenous H2O2 and produces O2 inside solid tumors, attenuates tumor hypoxia, down-regulates HIF-1α expression and ultimately delays tumor growth in both human patient-derived squamous cell lung carcinoma xenograft model and 4T1 breast cancer xenografted mouse model. Moreover, in the combination with the chemotherapeutic agent, doxorubicin, PdNPs/GDY-based catalytic therapy achieved a significantly enhanced antitumor effect. Our findings demonstrate that the rational design of a nano-modulator for tumor hypoxia with long term stability is a promising way to enhance the outcome of tumor therapy.

Novel recombinant coxsackievirus B3 with genetically inserted basic peptide elicits robust antitumor activity against lung cancer.

Cancer therapy that utilizes oncolytic virus may offer an exciting alternative, and coxsackievirus B3 (CVB3) is a potent oncolytic virus. This study was to assess the oncolytic activities of novel recombinant CVB3 with genetically inserted basic peptides in lung cancer. Recombinant CVB3 was produced in Vero cells, with or without genetically inserted basic peptides. In vitro and in vivo experiments with nude mouse models bearing human lung carcinoma xenografts were performed to examine the antitumor activities. Cytokines and immune responses to the recombinant CVB3 were determined in cynomolgus monkeys. Recombinant CVB3 with genetically inserted basic peptides was associated with significantly higher pH values within tumors. Mice treated with recombinant CVB3 showed significantly less tumor progression, and recombinant CVB3 with genetically inserted basic peptides appeared to enhance tumor suppression. Recombinant CVB3 was associated with significantly less proliferation of various lung cancer cells without affecting proliferation of normal lung fibroblasts. The cytokine profiles of the cynomolgus monkeys were comparable among control group (normal saline solution) and those given recombinant CVB3 with or without fused basic peptides, with no induction of excessive cytokine or immune responses. In conclusions, recombinant CVB3, especially those with fused basic peptides, possess strong antitumor activities without eliciting excessive immune responses.

Abstract 3905: Translational efficacy and safety modeling and simulation to support the clinical development of JNJ-64619178, a PRMT5 inhibitor

Abstract Protein arginine methyltransferase 5 (PRMT5) is an epigenetic enzyme with oncogenic properties. JNJ-64619178 (JNJ178) is a potent, selective, structurally unique PRMT5 inhibitor with good preclinical efficacy in inhibiting the growth of hematologic and solid tumor cell lines. Toxicology studies showed that JNJ178 decreased reticulocytes and neutrophils in rats and dogs. The objectives of translational modeling and simulation were to understand the exposure-response relationship of both safety and efficacy and provide guidance to the first-in-human clinical development of JNJ178. Experimental data for the PK/PD (Pharmacokinetics/Pharmacodynamics) modeling included: plasma concentration after single dose of JNJ178 in non-tumor bearing mice, plasma concentration and PD markers of dimethylation of arginine (%SDMA in plasma and %SMD1/3-Me2 in tumor, respectively) after multiple doses (1 to 10 mg/kg) QD (once daily) of JNJ178 in H1048 (human small cell lung carcinoma) xenografts, and tumor volume in four xenograft mouse models (A427, human lung carcinoma; H441, human lung adenocarcinoma; H520, human squamous cell lung carcinoma; and H1048). Plasma PK were first described by a standard two-compartment model and used as a driver of PD and tumor volume (efficacy). Plasma and tumor PD were modeled using an indirect response model. A hybrid tumor growth coupled with transit compartment mediated tumor killing model was used to fit the tumor volume data. To predict the safety profile of JNJ178, lifespan based indirect response model for erythropoiesis and Friberg myelosuppression model were used to simulate hemoglobin and neutrophil kinetics in human. The PK/PD model described the data well and validated the hypothesis that PD is driven by trough concentration. Based on the exposure-response relationship from the four xenograft models, the trough concentration needed to achieve tumor stasis for mouse was determined. In addition, the level of inhibition in tumor and plasma PD marker that was associated with tumor stasis was identified. Together with human PK parameters scaled using allometry, the dose range needed to achieve target therapeutic exposure for a typical human subject was predicted. Simulation results from erythropoiesis and Friberg myelosuppression models informed the optimal doing schedules for certain dose levels that would allow hematological toxicity to be manageable with <40% reduction in hemoglobin and >1.0 x 109/L neutrophil counts at all times. Overall, a translational modeling and simulation approach that considers safety and efficacy has been instrumental in the design of the first-in-human clinical development of PRMT5 inhibitor JNJ178 regarding selection of dose and schedule. Citation Format: Yue Guo, Nahor Haddish-Berhane, Hillary J. Millar, Tinne Verhulst, Tony Greway, Junguo Zhou, Loeckie DeZwart, Dana Gaffney, Joseph Portale, Dirk Brehmer, An Boeckx, Erika Van Heerde, Daniele Ouellet. Translational efficacy and safety modeling and simulation to support the clinical development of JNJ-64619178, a PRMT5 inhibitor [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 3905.

Targeted Delivery of Cisplatin-Derived Nanoprecursors via a Biomimetic Yeast Microcapsule for Tumor Therapy by the Oral Route.

Targeted therapy via the patient-friendly oral route remains the holy grail of chemotherapy for cancer. Herein we report a yeast-derived platform for targeted oral delivery of cisplatin (CDDP) that is one of the most effective drugs for chemotherapy of various types of cancers.Methods: The optimal conditions were first established to fabricate yeast microcapsules (YCs) with desirable loading capability. Then, CDDP-derived precursor nanoparticles (PreCDDP) were prepared and packaged into YC to produce orally deliverable PreCDDP/YC. The physiochemical properties, in vitro drug release profiles, in vitro antitumor activity, oral targeting capability, in vivo pharmacokinetics, and in vivo efficacy of the YC-based biomimetic delivery system were examined.Results: YCs obtained under the optimized condition showed desirable loading efficiency for quantum dots that were used as a model nanocargo. In vitro experiments demonstrated rapid endocytosis and prolonged retention of YC in macrophages. By electrostatic force-mediated self-deposition, PreCDDP was efficiently loaded into YC. PreCDDP/YC showed potent cytotoxicity in different tumor cells, indicating that PreCDDP loaded in YC maintained its antitumor activity after intracellular release. As compared to CDDP and PreCDDP, orally administered PreCDDP/YC displayed significantly higher bioavailability. Post oral delivery, YC could accumulate in A549 human lung carcinoma xenografts in mice, achieving by monocyte/macrophage-mediated translocation via the lymphatic system. Through this targeting effect, orally administered PreCDDP/YC showed desirable efficacy in A549 xenograft-bearing mice, which was comparable to that of free CDDP administered by intravenous injection. Orally administered free CDDP, however, did not afford antitumor effects. Furthermore, oral treatment with PreCDDP/YC displayed better safety than free CDDP administered via the oral or intravenous route.Conclusions: This biomimetic approach can serve as an effective strategy to develop targeted oral chemotherapies based on CDDP or its derivatives.

A novel scavenging tool for cancer biomarker discovery based on the blood-circulating nanoparticle protein corona

The prominent discrepancy between the significant investment towards plasma biomarker discovery and the very low number of biomarkers currently in clinical use stresses the need for discovery technologies. The discovery of protein biomarkers present in human blood by proteomics is tremendously challenging, owing to the large dynamic concentration range of blood proteins. Here, we describe the use of blood-circulating lipid-based nanoparticles (NPs) as a scavenging tool to comprehensively analyse the blood proteome. We aimed to exploit the spontaneous interaction of NPs with plasma proteins once injected in the bloodstream, known as ‘protein corona’, in order to facilitate the capture of tumor-specific molecules. We employed two different tumor models, a subcutaneous melanoma model (B16-F10) and human lung carcinoma xenograft model (A549) and comprehensively compared by mass spectrometry the in vivo protein coronas formed onto clinically used liposomes, intravenously administered in healthy and tumor-bearing mice. The results obtained demonstrated that blood-circulating liposomes surface-capture and amplify a wide range of different proteins including low molecular weight (MW) and low abundant tumor specific proteins (intracellular products of tissue leakage) that could not be detected by plasma analysis, performed in comparison. Most strikingly, the NP (liposomal) corona formed in the xenograft model was found to consist of murine host response proteins, as well as human proteins released from the inoculated and growing human cancer cells. This study offers direct evidence that the in vivo NP protein corona could be deemed as a valuable tool to enrich the blood proteomic analysis and to allow the discovery of potential biomarkers in experimental disease models.

Enhanced Cancer Immunotherapy by Chimeric Antigen Receptor-Modified T Cells Engineered to Secrete Checkpoint Inhibitors.

Purpose: Despite favorable responses of chimeric antigen receptor (CAR)-engineered T-cell therapy in patients with hematologic malignancies, the outcome has been far from satisfactory in the treatment of solid tumors, partially owing to the development of an immunosuppressive tumor microenvironment. To overcome this limitation, we engineered CAR T cells secreting checkpoint inhibitors (CPI) targeting PD-1 (CAR.αPD1-T) and evaluated their efficacy in a human lung carcinoma xenograft mouse model.Experimental Design: To evaluate the effector function and expansion capacity of CAR.αPD1-T cells in vitro, we measured the production of IFNγ and T-cell proliferation following antigen-specific stimulation. Furthermore, the antitumor efficacy of CAR.αPD1-T cells, CAR T cells, and CAR T cells combined with anti-PD-1 antibody was determined using a xenograft mouse model. Finally, the underlying mechanism was investigated by analyzing the expansion and functional capacity of TILs.Results: Human anti-PD-1 CPIs secreted by CAR.αPD1-T cells efficiently bound to PD-1 and reversed the inhibitory effect of PD-1/PD-L1 interaction on T-cell function. PD-1 blockade by continuously secreted anti-PD-1 attenuated the inhibitory T-cell signaling and enhanced T-cell expansion and effector function both in vitro and in vivo In the xenograft mouse model, we demonstrated that the secretion of anti-PD-1 enhanced the antitumor activity of CAR T cells and prolonged overall survival.Conclusions: With constitutive anti-PD-1 secretion, CAR.αPD1-T cells are more functional and expandable, and more efficient at tumor eradication than parental CAR T cells. Collectively, our study presents an important and novel strategy that enables CAR T cells to achieve better antitumor immunity, especially in the treatment of solid tumors. Clin Cancer Res; 23(22); 6982-92. ©2017 AACR.

Glycybridins A-K, Bioactive Phenolic Compounds from Glycyrrhiza glabra.

In an attempt to discover bioactive agents from the herbal medicine Glycyrrhiza glabra (widely known as licorice), 11 new phenolic compounds, glycybridins A-K (1-11), along with 47 known phenolics (12-58) were isolated. Their structures were elucidated on the basis of extensive NMR and MS analyses as well as experimental and computed ECD data. According to the clinical therapeutic effects of licorice, enzyme or cell-based bioactivity screenings of 1-58 were conducted. A number of compounds significantly activate Nrf2, inhibit tyrosinase or PTP1B, inhibit LPS-induced NO production and NF-κB transcription, and inhibit the proliferation of human cancer cells (HepG2, SW480, A549, MCF7). Glycybridin D (4) showed moderate cytotoxic activities against the four cancer cell lines, with IC50 values ranging from 4.6 to 6.6 μM. Further studies indicated that 4 (10 mg/kg, ip) decreased tumor mass by 39.7% on an A549 human lung carcinoma xenograft mice model, but showed little toxicity.

Combined Local Pulmonary and Systemic Delivery of AT2R Gene by Modified TAT Peptide Nanoparticles Attenuates Both Murine and Human Lung Carcinoma Xenografts in Mice

To evaluate the potential of cell-penetrating peptide-based delivery of apoptosis-inducer gene in cancer therapy, a modified HIV-1 TAT peptide (dimerized TAT peptide, dTAT) was studied. The dTAT and plasmid DNA (pDNA) complexes (dTAT-pDNA) were condensed using calcium chloride (dTAT-pDNA-Ca2+). This simple nonviral formulation approach showed high levels of gene expression in vitro without any cytotoxicity. In mouse studies, a single intratracheal (IT) aerosol spray or 2 intravenous (IV) injections of the dTAT, apoptosis-inducer gene, angiotensin II type 2 receptor (AT2R), and Ca2+ complexes (dTAT-pAT2R-Ca2+) significantly attenuated the acutely growing mouse Lewis lung carcinoma allografts in mouse lungs. Furthermore, single IT (p = 0.054) and the combination of IT and IV (p < 0.05) administrations of dTAT-pAT2R-Ca2+ markedly attenuated slowly growing and relatively large-sized H358 human bronchioloalveolar carcinoma xenografts in mouse lungs. These results indicate that the dTAT-pDNA-Ca2+ effectively delivered the gene to cancer cells by either IT or IV administration although the local pulmonary delivery of the dTAT-pAT2R-Ca2+ showed more effective growth inhibition of orthotopic lung cancer grafts. Thus, the present study offers preclinical proof of concept that a dTAT-based nonviral gene delivery method via IT administration may be an effective lung cancer gene therapy.

Abstract 5516: Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts

Abstract The anti-mitotic taxanes belong to one of the most important families of anticancer drugs that have broad anti-tumor activity and are widely used for a variety of human cancer types. However, the taxanes are limited by a number of serious pharmacological and toxicological effects. We are developing a unique delivery system for docetaxel, comprised of a PEGylated liposomal nanoparticle containing a prodrug of docetaxel to improve solubility, tolerability and increase efficacy through improved pharmacokinetics and biodistribution. In this study we evaluated the biodistribution of MNK-010, a liposomal formulation of a docetaxel prodrug in immunodeficient mice bearing A549 Human Non-Small Cell Lung Carcinoma (NSCLC) xenografts. This study tested the hypothesis that MNK-010 can produce greater plasma and tumor accumulation of docetaxel than with delivery of standard of care free docetaxel in tumor-bearing mice. Female nude mice bearing A549 human NSCLC xenografts were given a single intravenous (IV) dose of MNK-010 (40 mg/kg docetaxel molar equivalent) or free docetaxel (50 mg/kg). For each treatment group, three animals were sacrificed at various time points between 1 hr and 504 hr post injection. After euthanasia, blood, tumor, liver, kidney, spleen, lung and skeletal muscle specimens were collected. Tissue and blood samples were assayed to determine docetaxel concentrations. Administration of MNK-010 IV provided a reservoir for the continual slow release of docetaxel in the circulation and in tumors. MNK-010 increased systemic docetaxel exposure (AUC) ten-fold greater than free docetaxel injected at similar doses. MNK-010 produced sustained docetaxel levels in tumor through the 21 day observation period, resulting in four-fold greater docetaxel tumor exposure (AUC) compared to free docetaxel. The increase in MNK-010 plasma exposure as well as the enhanced permeability effect observed with liposomes likely contributed to the increased tumor exposure. In addition to improved accumulating in tumor tissue, docetaxel derived from MNK-010 also accumulated in liver, spleen and kidney. These tissues contain phagocytic cells (macrophages or mesangial) and serve as organs of elimination for chemotherapeutics. Docetaxel derived from MNK-010 was not detectable in skeletal muscle and was only found in lung over the first 24 hours post administration. In contrast, IV injection of free docetaxel produced high initial concentrations of docetaxel in all tissues evaluated, before falling quickly over time. Tumor levels decreased below levels of quantitation after nine days. This study demonstrated that Mallinckrodt Pharmaceuticals’ PEGylated liposomal docetaxel prodrug MNK-010 provided a reservoir for the continual release of docetaxel into the systemic circulation and produced an enhanced accumulation of docetaxel in A549 human NSCLC xenografts in mice compared to the standard of care docetaxel. Citation Format: Richard M. Fitch, James H. Wible, James A. Blackledge, William D. McGhee. Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts. [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 5516. doi:10.1158/1538-7445.AM2015-5516

Evaluation of efficacy of a new MEK inhibitor, RO4987655, in human tumor xenografts by [(18)F] FDG-PET imaging combined with proteomic approaches.

BackgroundInhibition of mitogen-activated protein kinase (MEK, also known as MAPK2, MAPKK), a key molecule of the Ras/MAPK (mitogen-activated protein kinase) pathway, has shown promising effects on B-raf-mutated and some RAS (rat sarcoma)-activated tumors in clinical trials. The objective of this study is to examine the efficacy of a novel allosteric MEK inhibitor RO4987655 in K-ras-mutated human tumor xenograft models using [18F] FDG-PET imaging and proteomics technology.Methods[18F] FDG uptake was studied in human lung carcinoma xenografts from day 0 to day 9 of RO4987655 therapy using microPET Focus 120 (CTI Concorde Microsystems, Knoxville, TN, USA). The expression levels of GLUT1 and hexokinase 1 were examined using semi-quantitative fluorescent immunohistochemistry (fIHC). The in vivo effects of RO4987655 on MAPK/PI3K pathway components were assessed by reverse phase protein arrays (RPPA).ResultsWe have observed modest metabolic decreases in tumor [18F] FDG uptake after MEK inhibition by RO4987655 as early as 2 h post-treatment. The greatest [18F] FDG decreases were found on day 1, followed by a rebound in [18F] FDG uptake on day 3 in parallel with decreasing tumor volumes. Molecular analysis of the tumors by fIHC did not reveal statistically significant correlations of GLUT1 and hexokinase 1 expressions with the [18F] FDG changes. RPPA signaling response profiling revealed not only down-regulation of pERK1/2, pMKK4, and pmTOR on day 1 after RO4987655 treatment but also significant up-regulation of pMEK1/2, pMEK2, pC-RAF, and pAKT on day 3. The up-regulation of these markers is interpreted to be indicative of a reactivation of the MAPK and activation of the compensatory PI3K pathway, which can also explain the rebound in [18F] FDG uptake following MEK inhibition with RO4987655 in the K-ras-mutated human tumor xenografts.ConclusionsWe have performed the first preclinical evaluation of a new MEK inhibitor, RO4987655, using a combination of [18F] FDG-PET imaging and molecular proteomics. These results provide support for using preclinical [18F] FDG-PET imaging in early, non-invasive monitoring of the effects of MEK and perhaps other Ras/MAPK signaling pathway inhibitors, which should facilitate a wider implementation of clinical [18F] FDG-PET to optimize their clinical use.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-014-0034-6) contains supplementary material, which is available to authorized users.

Abstract LB-358: Soy improves radiotherapy for lung tumors and mitigates radiation-induced injury to lung tissue.

Abstract Radiotherapy of locally-advanced non-small cell lung cancer (NSCLC) is limited by pneumonitis and fibrosis caused by radiation-induced injury to normal lung tissue. We have investigated the effect of a high thoracic dose of radiation combined with soy isoflavones in a pre-clinical model of orthotopic A549 human lung carcinoma xenografts in nude mice. Mice bearing established lung tumors (&amp;gt;300µm) were pretreated with soy for 3 days starting day 15 after A549 cell injection, and then received 10 Gy irradiation administered to the whole lung. Soy treatment, given orally at 1mg/day (50mg/kg) was continued for 5 days per week for 4 more weeks, then lungs were perfused with formalin and processed for histology. The effect of the therapy on the tumor cells, the surrounding tumor microenvironment and lung tissue structures was further analyzed using novel immunostaining and quantitative techniques. Compared to large invasive lung tumor nodules (up to 1-2 x 106 µm2) in untreated mice, the combined therapy caused a significant inhibition of tumor progression in lungs resulting in few residual small tumor nodules (0.02-0.07 x 106 µm2, p&amp;lt;0.05) with a low index of proliferation (mean of 16 positive nuclei for Ki-67 compared to 147 in control tumors, p&amp;lt;0.001). Pneumonitis was evaluated by morphometric measurements of the thickness of alveolar septa on H&amp;E stained lung tissue sections. Soy isoflavones reduced the extent of pneumonitis induced by radiation resulting in 30% of the lung tissue with thickened septa compared to 45% observed in lungs treated with radiation only. Fibrosis staining using Masson's Trichrome revealed that radiation caused a dramatic increase in collagen fibers supporting the vessel and bronchiole walls and this effect was mitigated by soy isoflavones. Triple immunofluorescent staining of lung vessel walls was performed on lung tissue sections using antibodies specific to endothelial cells (anti-CD31), pericytes (anti-SMA) and collagen (anti-collagen IV). This technique revealed that soy isoflavones combined with radiation decreased the fraction of vessels with damaged basement membrane to 25% down from 42% in radiation-alone treated lungs. Thus, these studies demonstrate a differential effect of soy isoflavones on augmenting tumor destruction induced by radiation while acting as radioprotectors for the surrounding lung tissue. Soy isoflavones reduced pneumonitis and fibrosis, and preserved the integrity of lung structures including alveolar septa, bronchioles and vessels, probably by mitigation of the inflammatory response induced by radiation. Complementing cancer radiotherapy with soy isoflavones has the potential to improve its effectiveness on the tumor target and reduce dose-limiting radiotoxicity to the normal lung and could be applied in the treatment of advanced stage NSCLC. Citation Format: Gilda Gali Hillman, Vinita Singh-Gupta, David J. Hoogstra, Lisa Abernathy, Joseph Rakowski, Christopher Yunker, Fazlul H. Sarkar, Andre A. Konski, Fulvio Lonardo, Michael C. Joiner. Soy improves radiotherapy for lung tumors and mitigates radiation-induced injury to lung tissue. [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 LB-358. doi:10.1158/1538-7445.AM2013-LB-358

Improving the Antitumor Activity of Squalenoyl‐Paclitaxel Conjugate Nanoassemblies by Manipulating the Linker between Paclitaxel and Squalene

A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to overcome the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2'-OH of paclitaxel through a 1,4-cis,cis-dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an efficient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl-paclitaxel nanoparticles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis-squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2'-squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor efficacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis-deca-5,8-dienoyl linker shows comparable antitumor efficacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

Abstract LB-264: Axitinib improves radiotherapy for murine xenograft lung tumors

Abstract Biological agents are currently being tested to improve the outcome of chemo-radiotherapy for locally advanced non-small cell lung cancer (NSCLC). The anti-angiogenic drug axitinib (AG-013736, Pfizer), is a small molecule receptor tyrosine kinase (RTKs) inhibitor. Axitinib selectively targets VEGF receptors 1, 2 and 3 (VEGFR), has a high potency for VEGFR-2 and inhibits the signal transduction induced by VEGF binding to VEGFR. Axitinib used in a phase II trial for advanced NSCLC demonstrated a promising one-year survival rate with manageable toxicities. We have tested the combination of axitinib with radiotherapy in a pre-clinical model of orthotopic A549 model of human lung carcinoma xenografts in nude mice. Mice-bearing established A549 lung tumor nodules were treated orally and daily with axitinib at 25mg/kg/day concomitant with daily fractionated radiation for 5 consecutive days. Radiation was administered to the left lung at a dose 2 Gy per day and then, axitinib treatment was continued for 5 days per week for 4 more weeks. A mild decrease in overall weight was observed in mice treated with axitinib or axitinib and radiation indicating that axitinib was safe and well tolerated, probably as a result of its short-half life (2-5h). No systemic effect on the vasculature of other organs including the heart, kidney and liver was observed by histology. Axitinib caused decreased tumor cellularity in lung nodules associated with a heavy infiltration of inflammatory cells. Mice treated with axitinib and radiation had a significant decrease in the size and number of lung tumor nodules which contained degenerating giant tumor cells and inflammatory infiltrates. Morphometric measurements of tumor nodules confirmed that the size of tumor nodules was significantly smaller in the irradiated left lung and was further reduced by combination with axitinib compared to that of mice treated with single modality (p&amp;lt;0.05). Ki-67 staining showed a marked decrease in the number of proliferating tumor cells. In the surrounding lung tissue, scattered intra-alveolar hemorrhage was estimated to cover 10 to 20% of the lung surface in treated mice but it was comparable to that observed in untreated mice. Axitinib caused mild chronic interstitial pneumonia and combined axitinib and radiation treatment was associated with moderate interstitial pneumonia. In independent experiments, treatment of lung tumors with a single dose of radiation (10 Gy) administered to the lungs and 10 weeks of axitinib showed a complete inhibition of tumor growth in the lungs. Histology showed the presence of distinctive lymphohistiocytic nodules with no detectable viable tumor cells. This treatment was well tolerated with minimal weight loss. These findings suggest a potential novel approach for the treatment of locally advanced NSCLC by combining radiotherapy with the anti-angiogenic drug axitinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-264. doi:1538-7445.AM2012-LB-264

The heat shock protein 90 inhibitor, AT13387, displays a long duration of action in vitro and in vivo in non‐small cell lung cancer

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, l-lactic acid salt) a novel, high-affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho-signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho-signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387's long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer-acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues.

A Novel p53-Based Strategy for Protection of Bone Marrow and Gastrointestinal (GI) Track From Total Body Irradiation (TBI)-Induced Damage.

AbstractAbstract 1454 Purpose:Recognizing p53 activation as the primary mechanism that underlies radiation-induced damage to bone marrow and GI track, we aim to explore the use of low dose arsenic-induced transient p53 inhibition as a novel strategy for radiotherapy protection. Methods:Expanding from our recent finding that low-dose of arsenic can suppress radiation-induced p53 activation and subsequent apoptosis, we used a mouse model to test the potential of arsenic in protection of bone marrow and GI track against TBI induced damage. Arsenic pretreatment was carried out by feeding sex-matched mice (4-6 weeks of age) with water with or without 1.0 mg/L sodium arsenic for three days. Mice were randomized into following groups; control; arsenic only; irradiation only; arsenic and irradiation. Mice were then irradiated with 2Gy TBI daily for 7 days. Radiation-induced bone marrow and GI track damages were analyzed histologically with H&E staining 4 weeks after last treatment. Animal body weight was monitored as a measure of toxicity. To test the prediction that arsenic would not provide protection for malignant cells because of their defect in p53, a lung carcinoma cell line NCI-H358 was used to generate a mouse xenograft model. Treatments were initiated three weeks after implantation when the lung carcinoma cells developed into readily visible tumor with an average volume of approximately 100 mm3. Tumor volumes were measured periodically. Tumor volume was calculated using the equation: (volume = length × width × depth × 0.5236 mm3). Two independent experiments were done and the tumor volumes are means ± SE from total of 10 mice per group. [Display omitted] Results:Consistent with published results, mice treated with irradiation significantly lost their body weight. TBI-induced body weight loss was effectively prevented by arsenic pretreatment (Fig. 1A). In line with the data of body weight change, radiation treatments were associated with severe damages to small intestine and bone marrow cells, and remarkably, such damages were significantly reduced by low-dose arsenic pretreatment (Fig. 1B). The results together demonstrate the potential of low-dose arsenic to effectively protect bone marrow and GI track to radiation-induced damage. [Display omitted] In tumor xenograft models, the tumor volume of the control group continued to increase with time. Arsenic treatment did not have any detectable effect on growth of the implanted tumors. As expected, irradiation with 2Gy of TBI daily for 7 days resulted in marked tumor regression. Arsenic pretreatment showed little effect on irradiation-induced tumor suppression (Fig. 2), indicating that low-dose arsenic pretreatment does not detectably affect the efficacy of radiation, at least in the human lung carcinoma xenograft mouse model. Collectively, the results demonstrate that a brief treatment with low-dose arsenic is associated with a marked protection of bone marrow and GI track without compromising the ability of irradiation to kill carcinoma cells. Conclusion:The animal studies have provided proof-of-principle for the use of low-dose arsenic in radiotherapy protection of bone marrow and GI track, serving as strong rationales to initiate clinical trials. Disclosures:No relevant conflicts of interest to declare.

Abstract 5217: Evaluation of the therapeutic efficacy of MEK inhibitor (TAK-733) using [18F]FDG-PET in the human lung xenograft model A549

Abstract Introduction: TAK-733 is a novel, potent, selective, non-ATP competitive, allosteric inhibitor of MEK kinase, which has a potential to inhibit cancer proliferation and survival. [18F]Fluorodeoxyglucose ([18F]FDG) is an imaging biomarker for glucose metabolism detectable using positron emission tomography (PET) and widely used in clinical cancer diagnosis and is increasingly used to assess response to anti-cancer therapy. The aim of this study was to evaluate the therapeutic efficacy of TAK-733 using [18F]FDG-PET in nude rats bearing A549 (human lung carcinoma) xenografts. Methods: A549 tumor fragments were subcutaneously implanted in female RNU nude rats. Treatments began when the mean estimated tumor mass for all groups was 250mg (Day 1). TAK-733 was orally administered at 0 (vehicle), 1, 3 and 10 mg/kg daily for two weeks (n = 8 / group). PET scans were performed before treatment (Day 0) and on Days 2, 4, 7, 10 and 14. PET image acquisition was performed for 13 min at 1.5 hr after [18F]FDG injection (400 μCi). Tracer accumulations in tumor tissue were quantified as mean standard uptake value (SUVmean) and percentage of injected dose (%ID). Results: TAK-733 showed dose-dependent inhibition of tumor growth and [18F]FDG uptake in tumor tissue. TAK-733, at 10mg/kg, produced a statistically significant difference in tumor mass (Day 14) compared to the vehicle group (31% T/C) and in SUVmean (Day 2) compared to the vehicle group. The statistical significance for the 10mg/kg did not persist however, and was only observed at a single time point for tumor mass and SUVmean. However, a significant response in SUVmean (Day 2) occurred before significant tumor growth inhibition (Day 14). In this treatment group, the SUVmean value on Day 2 (1.38 ± 0.26) was lower than that observed at pre-treatment, Day 0 (1.60 ± 0.30), and statistically lower compared to the SUVmean values in the vehicle (1.54 ± 0.46) and 1mg/kg groups (1.53 ± 0.29) on Day 2. The SUVmean value in the vehicle and 1 and 3 mg/kg groups showed an increase over time. On Day 14, the SUVmean value at 10 mg/kg (1.45 ± 0.33) was statistically significantly lower compared to the vehicle group (2.16 ± 0.33). TAK-733 produced a dose-dependent %ID response. 10mg/kg TAK-733 produced the lowest %ID for every imaging time point, while the vehicle group had the highest %ID at every imaging time point. There were no statistically significant differences in %ID response. However, %ID did not show statistically significant decreased values compared to the vehicle group, suggesting that SUVmean may be a more sensitive parameter for [18F]FDG uptake than %ID. Conclusion: [18F]FDG-PET enabled an early indication of later tumor growth in response to TAK-733 treatment in this model. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5217.