Development of a primary hepatocyte-based preclinical spheroid model system for the analysis of Phase I and Phase II enzyme activity in vitro

Background: Lenvatinib is an oral tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1-4, RET, KIT and PDGFRβ. Anti-tumor activity has been observed in melanoma patients in Phase I studies. We applied an integrative supercomputer-driven analysis approach to identify biomarkers of lenvatinib treatment response in melanoma patients. Methods: Clinical data sets including tumor response data (RECIST), progression free survival (PFS), pharmacokinetic parameters (PK) and molecular data sets including baseline tumor gene expression (Affymetrix U133Plus2) and BRAF and NRAS mutational status, were collected from 18 patients with metastatic melanoma who received lenvatinib 10 mg orally twice daily in 28-day cycles. These clinical and molecular data sets were used to generate computational models developed using REFS (Reverse Engineering and Forward Simulation) modeling platform, which utilizes Bayesian network inference and simulations. Simulations were performed to identify biomarkers of lenvatinib treatment response. These potential predictive biomarkers were then tested for their ability to predict response to lenvatinib in preclinical models. Results: Using a model comprising gene expression, mutational status and PK data, REFS identified a panel of 18 potential predictive biomarkers of lenvatinib treatment response. Identified biomarkers were able to predict up to 89% of the observed variance in the tumor response data. A total of 32 identified genes including 6 candidate predictive biomarkers (TARBP2, CACNA1, C7ORF, RAP2A, SHMT1, IL22RA2) were further validated in a preclinical melanoma model system (n=12) and tissue bank samples with matched normal adjacent tissue (n=21). Expression of 14 genes correlated with relative tumor volume (r>0.35 or r<-0.35) in the preclinical model and expression levels of 19 genes were found to be higher in tumor compared to normal tissue (FC>2). Conclusions: Potential predictive biomarkers of lenvatinib treatment response in melanoma patients were identified by computational modeling and validated in a preclinical model system and tumor tissue bank samples. The identified biomarkers will be tested for their predictive value in an ongoing Phase 2 trial. Citation Format: Tadashi Kadowaki, Yasuhiro Funahashi, Junji Matsui, Kumar Pavan, Pallavi Sachdev, Jim O'Brien, Heming Xing, Paul D. McDonagh, Iya Khalil, Razelle Kurzrock, David S. Hong, John Nemunaitis. Data-driven computational modeling to identify biomarkers of response to lenvatinib (E7080) in melanoma. [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 4025. doi:10.1158/1538-7445.AM2013-4025

This paper seeks to shed light on an unwritten chapter in the history of Tartu semiotics, that is, to draw a parallel between Juri Lotman and Émile Benveniste on the status of natural language among other systems of signs. The tenet that language works as a ‘primary modelling system’ represents one of the trademarks of the Tartu-Moscow school. For Lotman, the primacy assigned to natural language in respect to other systems of signs lied in the fact that the former functions as a ‘model’ for the latter thus regarded as ‘secondary modelling systems’. Yet how does language carry out its function of being a model for other sign systems? Is language the only primary modelling system? This paper seeks to foster the abovementioned claim of the primacy of natural language and argues that this issue deserves a closer inspection. In order to follow this route, it suggests a parallel between Lotman and Benveniste arguing that there exist several points in common that lead to a convergence of positions between these two remarkable scholars. The paper explores such a possibility, arguing that Lotman’s and Benveniste’s positions open up an interesting debate with specific reference to the relations laid down between language and other system of signs.

Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

Non-Hodgkin’s lymphoma is the most commonly diagnosed hematologic cancer of adults in the United States, with the vast majority deriving from malignant B lymphocytes that express cell surface CD20. CD20 immunotherapy (Rituximab) is widely used to treat Non-Hodgkin’s lymphoma, even though the initial effectiveness of Rituximab varies widely amongst patients and typically wanes over time. The mechanisms through which lymphomas initially resist or gain resistance to immunotherapy are not well-established. To address this, a preclinical mouse model system was developed to comprehensively identify lymphoma transcriptomic changes that confer resistance to CD20 immunotherapy. The collection of spontaneous primary and familial lymphomas revealed that sensitivity to CD20 immunotherapy was not regulated by differences in CD20 expression, prior exposure to CD20 immunotherapy, nor serial in vivo passage. An unbiased forward exome screen of these primary lymphomas was used to validate the utility of this expansive lymphoma cohort, which revealed that increased lymphoma galectin-1 expression strongly correlated with resistance to immunotherapy. Genetically induced lymphoma galectin-1 expression ablated antibody-dependent lymphoma phagocytosis in vitro and lymphoma sensitivity to CD20 immunotherapy in vivo. Human NHLs also express elevated galectin-1 compared with non-malignant lymphocytes, demonstrating the ability of this preclinical model system to identify molecular targets that could be relevant to human therapy. This study therefore established a powerful preclinical model system that permits the comprehensive identification of the dynamic lymphoma molecular network that drives resistance to immunotherapy.

Galectin-1 drives lymphoma CD20 immunotherapy resistance: validation of a preclinical system to identify resistance mechanisms

Challenges in genomic analysis of model systems and primary tumors of pancreatic ductal adenocarcinoma

Drug development for cancer: Implications for chemical modifiers

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies

Sepsis and sepsis-associated multiorgan failure represent the major cause of mortality in intensive care units worldwide. Cardiovascular dysfunction, a key component of sepsis pathogenesis, has received much research interest, although research translatability remains severely limited. There is a critical need for more comprehensive preclinical sepsis models, with more clinically relevant end points, such as microvascular perfusion. The purpose of this study was to compare microcirculatory blood flow measurements, using a novel application of laser speckle contrast imaging technology, with more traditional hemodynamic end points, as part of a multiparameter monitoring system in preclinical models of sepsis. Our aim, in measuring mesenteric blood flow, was to increase the prognostic sensitivity of preclinical studies. In two commonly used sepsis models (cecal ligation and puncture, and lipopolysaccharide), we demonstrate that blood pressure and cardiac output are compromised postsepsis, but subsequently stabilize over the 24-h recording period. In contrast, mesenteric blood flow continuously declines in a time-dependent manner and in parallel with the development of metabolic acidosis and organ dysfunction. Importantly, these microcirculatory perturbations are reversed by fluid resuscitation, a mainstay intervention associated with improved outcome in patients. These data suggest that global hemodynamics are maintained at the expense of the microcirculation and are, therefore, not sufficiently predictive of outcome. We demonstrate that microcirculatory blood flow is a more sensitive biomarker of sepsis syndrome progression and believe that incorporation of this biomarker into preclinical models will facilitate sophisticated proof-of-concept studies for novel sepsis interventions, providing more robust data on which to base future clinical trials.

Recent advances in preclinical model systems for papillomaviruses

BackgroundPathogenic variations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) are associated with malignant hyperthermia (MH) susceptibility, a life-threatening hypermetabolic condition and RYR1-related myopathies (RYR1-RM), a spectrum of rare neuromuscular disorders. In RYR1-RM, intracellular calcium dysregulation, post-translational modifications, and decreased protein expression lead to a heterogenous clinical presentation including proximal muscle weakness, contractures, scoliosis, respiratory insufficiency, and ophthalmoplegia. Preclinical model systems of RYR1-RM and MH have been developed to better understand underlying pathomechanisms and test potential therapeutics.MethodsWe conducted a comprehensive scoping review of scientific literature pertaining to RYR1-RM and MH preclinical model systems in accordance with the PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O’Malley. Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019.ResultsOur search yielded 5049 publications from which 262 were included in this review. A majority of variants tested in RYR1 preclinical models were localized to established MH/central core disease (MH/CCD) hot spots. A total of 250 unique RYR1 variations were reported in human/rodent/porcine models with 95% being missense substitutions. The most frequently reported RYR1 variant was R614C/R615C (human/porcine total n = 39), followed by Y523S/Y524S (rabbit/mouse total n = 30), I4898T/I4897T/I4895T (human/rabbit/mouse total n = 20), and R163C/R165C (human/mouse total n = 18). The dyspedic mouse was utilized by 47% of publications in the rodent category and its RyR1-null (1B5) myotubes were transfected in 23% of publications in the cellular model category. In studies of transfected HEK-293 cells, 57% of RYR1 variations affected the RyR1 channel and activation core domain. A total of 15 RYR1 mutant mouse strains were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout. Porcine, avian, zebrafish, C. elegans, canine, equine, and drosophila model systems were also reported.ConclusionsOver the past 30 years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM.

Ephrin receptor A2 (EphA2) is a member of the Ephrin receptor family of cell-cell junction proteins and is both highly overexpressed in several solid tumors and associated with poor prognosis in patients. EphA2 is considered a high-value target for cancer therapeutics, but development of an antibody drug conjugate targeting EphA2 (MEDI-547) was stopped in early clinical development after severe adverse events, including bleeding toxicity were seen (Annunziata et al, 2013). We have developed a series of Bicycle Toxin Conjugates comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to EphA2, which is covalently linked to a toxin payload via a cleavable linker. The small size of BTCs offers a significant advantage over other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation, renal clearance and improved tumor penetration. BT5528 was selected from a panel of >75 Bicycle Toxin Conjugates (BTCs) with variations in Bicycle binder, molecular spacer, cleavable linker and toxin payload, based on in vivo efficacy and tolerability. BT5528 is effective in EphA2-expressing xenograft models, with efficacy seen from 1mg/kg qw and complete tumor regression at 3mg/kg qw. Rapid, complete regression of tumors is seen across a range of EphA2-expressing cell lines, including HT1080 fibrosarcoma line, MDA-MB-231 triple negative breast cancer line and NCI-H1975 lung cancer line and NSCLC PDX models. In order to test the benefit of the enhanced tumor penetration of BTCs, NSCLC PDX tumors were grown to >1000mm3 before commencing treatment. Complete regression of tumor was seen following 3 weeks of dosing. The bleeding events reported in early clinical trials of MEDI-547 (EphA2 ADC) were consistent with effects on the coagulation system in preclinical models. Dose-limiting toxicology in non-human primate (NHP) was reportedly Disseminated Intravascular Coagulation (DIC) and changes were seen in APTT and D-Dimer, with associated elevations in liver enzymes. Toxicology studies of BT5528 in rat and NHP showed no laboratory or clinical evidence of DIC, with normal levels of platelets, D-Dimer, APTT, and other coagulation-related parameters. Bicycle Toxin Conjugates (BTC) targeting EphA2 show potent antitumor activity in a range of solid tumor xenograft models. The BTCs show profound efficacy, without the limiting toxicity observed with previous Antibody Drug Conjugate approaches. IND-enabling studies for BT5528 are currently underway. Citation Format: Gavin Bennett, Philip Huxley, Amy Brown, Gemma Mudd, Peter U. Park, Nicholas Keen. BT5528, a Bicycle Toxin Conjugate targeting EphA2 has potent anti-tumor activity without bleeding or coagulation abnormalities in preclinical 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 5854.

BackgroundBladder cancer is the fourth most common cancer in American men with chances of 1 in 27 developing this form of cancer. Despite the progress in treating these patients with...

Poetry is an important challenge for semiotics, and a special area of study for the Tartu-Moscow semiotic school, since the first volume of Sign Systems Studies was Juri Lotman’s monograph Lectures on Structural Poetics (1964). From then on the concept of poetry as one of the secondary modelling systems has evolved, since in relation to poetry, the primary modelling system is natural language. In this paper, the concept of semiotic system has been re-examined and the treatment of primary and secondary semiotic systems has been significantly revised. A semiotic system can be characterized not only by its internal structure and other systems to which it is related, but also by the field upon what it is realized. The latter aspect has gained almost no attention in any treatment of semiotics; the execution of a sign is understood in the spirit of Saussure and Hjelmslev as a material realization of an abstract element (for instance, a chess piece knight can be realized with wood or plastic, but it can also remain purely virtual). At first, distinction is made between language and sign system. Every sign system consists of language and field. There are three different kinds of fields: 1) just a background – footprints on sand are a sign on the background of sand; 2) a material structured field (a football ground or a chess board in the game called Chapayev) and 3) an abstract structured field, which in its turn consists of other fields (for instance, the chess board which consists of 64 fields). Differently from a football ground, a chess board can be a purely virtual one on which virtual pieces are moved (for instance, in case of blindfold or correspondence chess). The field in its turn can be language and one language can use another language as its field. In this case we speak of primary and secondary sign systems. For instance, the prosodic system of language is a field for a verse metre, while the semantic system of language is a field for a narrative.

Trastuzumab (Herceptin®) is a humanized monoclonal antibody designed to bind and inhibit the function of the human epidermal growth factor receptor2 (HER2)/erbB2 receptor. Trastuzumab has demonstrated clinical activity in several types of HER2-overexpressing epithelial tumors, such as breast and metastatic gastric or gastroesophageal junction cancer. Relapse and therapeutic resistance, however, still occur in a subset of patients treated with regimens including trastuzumab, despite significant improvements in response rates, survival and quality of life. To investigate the potential mechanisms of acquired therapeutic resistance to trastuzumab, we developed a preclinical model of human ovarian cancer cells, SKOV-3 Herceptin-resistant (HR), and examined the corresponding changes in gene expression profiles. SKOV-3HR cells were developed by invivo serial passaging of parental trastuzumab-sensitive SKOV-3cells. Following four rounds of serial transplantation of 'break-through' xenograft tumors under trastuzumab treatment, significant and reproducible differences in the effects of trastuzumab treatment between SKOV-3HR and SKOV-3 cells invivo and invitro were revealed. SKOV-3HR cells retained HER2protein expression but were unaffected by the antiproliferative effects of trastuzumab. The trastuzumab binding affinity for SKOV-3HR cells was diminished, despite these cells having more binding sites for trastuzumab. Microarray expression profiling (MEP) was performed to determine the genes involved in the resistance mechanism. Functional analysis revealed the differential expression of genes potentially involved in angiogenesis, metastasis, differentiation and proliferation, such as mucin1(MUC1). Immunohistochemical staining of SKOV-3HR cells demonstrated a marked overexpression of MUC1. Based on these data, we hypothesize that the overexpression of MUC1 may hinder trastuzumab binding to HER2receptors, abrogating the antitumor effects of trastuzumab and thus could contribute to resistance to therapy. Moreover, the resultant MEP preclinical gene signature in this preclinical model system may provide the basis for further investigation of potential clinical mechanisms of resistance to trastuzumab.