Testing Candidate Therapies Research Articles

Murine models of advanced maternal age: a systematic review and meta-analysis.

Animal models have been developed to aid understanding of the increased incidence of adverse pregnancy complications observed in women of advanced maternal age (AMA). This systematic review of murine models of AMA demonstrates consistent effects of decreased litter size and fetal weight; this supports the future use of these models to determine pathophysiological mechanisms and test therapeutic strategies to improve poor pregnancy outcomes in AMA. Advanced maternal age (AMA; ≥35 years of age) is associated with an increased risk of adverse pregnancy outcomes. To explore causes of adverse pregnancy outcomes in AMA, and to test candidate therapies, an increasing number of murine AMA models have been developed. The aim of this study was to systematically review the literature to assess whether murine AMA models demonstrate a reproducible effect on pregnancy outcomes. PubMed, Ovid, Web of Science and Google Scholar were searched. Studies that reported on pregnancy outcomes in AMA mice and rats were included; the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool evaluated the risk of bias. Eleven mouse and six rat studies were included. AMA mice and rats had reduced litter size (standardised mean difference (SMD): -1.59, 95% confidence interval (CI): -1.84, -1.34 for mice; SMD: -1.66, 95% (CI): -2.09, -1.23 for rats) and reduced fetal weight (SMD: -0.87, 95% CI: -1.24, -0.49 for mice; SMD: -1.05, 95% CI: -1.40, -0.69 for rats). Placental weight was increased in AMA mice (SMD: 0.62, 95% CI: 0.16, 1.08). Subgroup analysis indicated that C57Bl/6 mice had less heterogeneity than other, mostly outbred, mouse strains with regards to litter size (C57 strain I2 = 68.2% vs other strain types I2 = 85.7%). The risk of bias was high, mostly due to the lack of methodological detail and unclear reporting of findings. Murine models of AMA demonstrate similar adverse pregnancy outcomes to those observed in large human epidemiological studies. The reproducible phenotypes in AMA murine models allow the exploration of mechanisms underpinning poor pregnancy outcomes and the pursuit of therapeutic interventions.

Abstract P3-08-03: Establishment of patient-derived xenograft tumor models from breast cancer patients for quantitative elemental profiles and testing candidate therapies

Abstract Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Triple negative breast cancer (TNBC) is among the most aggressive clinical manifestations of breast cancer (BC), and represents a significant clinical challenge to the effective treatment of early metastatic and treatment-refractory disease because of its poor outcomes. Therefore, there exists a need to develop pre-clinical models that retain the characteristics of the original TNBC tumor—e.g., PDX tumor models—to better understand the mechanisms of drug resistance in metastatic TNBC and to effectively evaluate the effects of anti-cancer drugs on patients with this disease. Furthermore, quantitative changes in cellular element signatures, which indicate levels of enzymatic activity, are emerging new biomarkers for cancer in the clinic, but are of yet systematically understudied in tumor samples from breast cancer patients or/and PDX models of these tumors. Due to limitations in current cellular element signature quantitation profiles, there also exists a need to investigate further. Seven samples of advanced BC patient pleural effusion were obtained from Northwestern Memorial Hospital to establish a PDX tumor model in immunodeficient NSG female mice using breast fat pad xenografting and to develop derived 3D spheroid cultures for anti-cancer drug evaluation. To authenticate, STR profiling against with original patient tumor DNA was conducted. Five developed BC PDX tumor models were shown by pathology to have highly heterogeneous characteristics and the metastatic features of the origin patient tumor. Liver and lung metastases were observed in breast fat pad xenografted PDX tumor mice. 3D tumor spheroid cultures were successfully established from original BC pleural effusion or/and PDX tumor cultures. Using the newly developed, highly sensitive, and reliable Wash-Free Inductively Coupled Plasma Mass Spectrometry (WF ICP-MS) method, we evaluated the inorganic phenotypes from samples of breast cancer patient tumor tissue and of the established PDX tumor to quantify the mobile elements (Na, K, and Ca) and less mobile elements (P, Mg, Mn, Fe, Cu, and Zn) simultaneously within the same sample. The data was collected for further analysis of breast cancer inorganic signatures from normal and tumor cells. Our results suggested that BC 3D spheroid cultures and PDX tumor models could serve as models to further study the mechanisms of MBC and serve as promising tools for in vivo and in vitro quick testing and mechanistic studies of novel antitumor drugs respectively. Identifying quantitative elemental profiles is fundamental to understanding the pathologies of various metal-related cancers and thus opens up new opportunities for disease management and therapeutic intervention Citation Format: Wenan Qiang, Haimei Chen, Yi Yang, Andrew Crawford, Demirkan B. Gursel, Jian-Jun Wei, Thomas O’Halloran, Massimo Cristofanilli. Establishment of patient-derived xenograft tumor models from breast cancer patients for quantitative elemental profiles and testing candidate therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-03.

A Novel Rabbit Dry Eye Model Induced by a Controlled Drying System.

PurposeTo establish an environment-induced dry eye model in rabbits using a controlled drying system (CDS).MethodsRabbits were randomly divided into two groups. The rabbits in the dry group were housed in the CDS, in which the relative humidity, airflow, and temperature were controlled at 22% ± 4%, 3 to 4 m/s, and 23°C to 25°C for 14 days. The rabbits in the control group were housed in a normal environment at the same time. A Schirmer test, fluorescein staining, and lissamine green staining were performed. On day 14, the eyeballs and lacrimal glands were processed for evaluating the corneal epithelial thickness, inflammatory cell infiltration index, goblet cell density, and expression of the MUC5AC protein and caspase-3 protein. The mRNA expression of the involved inflammatory genes was analyzed.ResultsThe CDS was able to maintain a dry environment, in which the tear production decreased, and the ocular surface staining increased over time in the rabbits. In the dry group, the corneal epithelium became thinner, inflammatory cells were noted, goblet cells and MUC5AC proteins decreased, and the increased levels of caspase-3 proteins and inflammatory cytokines were observed in the ocular surface tissues and lacrimal glands.ConclusionsThis CDS could create a dry environment, in which the rabbits exhibited a pathological change in dry eye similar to that in humans.Translational RelevanceThis model would be helpful in offering a platform to identify and test candidate therapies for environment-induced dry eye and to explore its underlying mechanisms.

Abstract 1046: Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients

Abstract Inflammatory breast cancer (IBC) is the most aggressive clinical manifestation of breast cancer (BC), with striking erythema of the overlying skin, and exhibiting features of early metastatic and treatment-refractory disease. Malignant cells and infiltrating leukocytes, e.g. tumor-associated microphages (TAMs) commonly designated "M2" macrophages, play a major part in metastasis and treatment resistance. TAMs help promote tumor growth and progression. There is a significant need for preclinical models that retain the characteristics of the original patient tumor for investigating mechanisms of IBC metastasis and for testing candidate therapies. Six samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from five IBC or metastatic breast cancer (MBC) patients were obtained from Northwestern Memorial Hospital. IBC cells were used for 3D tumor spheroid cultures, and for developing PDX tumor models in immunodeficient mice. Short Tandem Repeat profiling against with original patient tumor DNA was conducted to establish patient DNA identify, and to serve as a reference for authenticating derivative tumor models. The tumor genomic mutation analysis indicated the most common alterations of five IBC or MBC patients were TP53 (75%), CCND1 (75%), BRCA2 (50%), NF1 (50%), FGFR1 (50%), CDK6 (50%), EGFR (50%), MYC (50%), BRAF (50%). Examination IBC pleural effusion cellular composition indicated ~60% were tumor cells and ~40% were M2 TAMs (CD163+). In early 3D spheroid IBC culture, a symbiotic phenomenon was observed between tumor cells and attached TAMs; Anti-cancer drugs GW2580 and Pexidartinib inhibited 32% and 68% of cell proliferation, respectively, when added to spheroid cultures at 100 nanomolar. Histopathologic analysis of derivative IBC and MBC PDX revealed highly heterogeneous characteristics and metastatic features characteristic of corresponding patient tumors. Subcutaneous tumors also demonstrated metastasis to liver and lung tissue. NSG mice engrafted with original IBC pleural effusion cells or derived spheroid cultures manifested a variety of inflammatory clinical symptoms. Mice engrafted with original pleural effusion cells developed palpable tumor and manifested most IBC-characteristic including reddish and ulcerated skin lesions necessitating euthanasia within 2 months of cell engraftment. Mice engrafted with > 6 passage or <4 passages of spheroid cultures (lower % of TAMs present) manifested lesser skin inflammation and lesion, and developed tumors of ~ 1 cm within 2-3 months. This may indicate the role of TAMs in the development of IBC skin lesion. Our results indicate IBC 3D spheroid cultures and PDX tumor models recapitulate clinical characteristics observed in IBC patients, and will prove useful for studying mechanisms of IBC metastasis as well as responsiveness to investigational therapies. Citation Format: Wenan Qiang, Youbin Zhang, Demirkan Gursel Gursel, Jian-Jun Wei, Charles David James, Thomas V. O'Halloran, Massimo Cristofanilli. Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients [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 1046.

The Complexity of Biomechanics Causing Primary Blast-Induced Traumatic Brain Injury: A Review of Potential Mechanisms.

Primary blast-induced traumatic brain injury (bTBI) is a prevalent battlefield injury in recent conflicts, yet biomechanical mechanisms of bTBI remain unclear. Elucidating specific biomechanical mechanisms is essential to developing animal models for testing candidate therapies and for improving protective equipment. Three hypothetical mechanisms of primary bTBI have received the most attention. Because translational and rotational head accelerations are primary contributors to TBI from non-penetrating blunt force head trauma, the acceleration hypothesis suggests that blast-induced head accelerations may cause bTBI. The hypothesis of direct cranial transmission suggests that a pressure transient traverses the skull into the brain and directly injures brain tissue. The thoracic hypothesis of bTBI suggests that some combination of a pressure transient reaching the brain via the thorax and a vagally mediated reflex result in bTBI. These three mechanisms may not be mutually exclusive, and quantifying exposure thresholds (for blasts of a given duration) is essential for determining which mechanisms may be contributing for a level of blast exposure. Progress has been hindered by experimental designs, which do not effectively expose animal models to a single mechanism and by over-reliance on poorly validated computational models. The path forward should be predictive validation of computational models by quantitative confirmation with blast experiments in animal models, human cadavers, and biofidelic human surrogates over a range of relevant blast magnitudes and durations coupled with experimental designs, which isolate a single injury mechanism.

The promise of futility trials in neurological diseases.

Double-blinded randomized controlled trials (RCTs) have contributed much important evidence to guide treatment decisions in neurology. RCTs are relatively straightforward to conduct, provided that they investigate common diseases, have clearly defined outcome measures, and are of short duration. In neurology, however, many diseases are uncommon, have no consensus outcome measures, and develop over decades. Basic research into neurological diseases continues to identify candidate therapies faster than they can be tested for their clinical utility, leading to a 'translational gap'. Futility trials were initially developed in oncology to efficiently test candidate therapies in phase II trials. As single-arm unblinded studies, futility trials are relatively easy to conduct, and they generally require smaller sample sizes than RCTs. In this article, we discuss futility models, highlighting their advantages as well as challenges to their application in several neurological diseases, including Parkinson disease, stroke and multiple sclerosis.

Degraded speech sound processing in a rat model of fragile X syndrome

Fragile X syndrome is the most common inherited form of intellectual disability and the leading genetic cause of autism. Impaired phonological processing in fragile X syndrome interferes with the development of language skills. Although auditory cortex responses are known to be abnormal in fragile X syndrome, it is not clear how these differences impact speech sound processing. This study provides the first evidence that the cortical representation of speech sounds is impaired in Fmr1 knockout rats, despite normal speech discrimination behavior. Evoked potentials and spiking activity in response to speech sounds, noise burst trains, and tones were significantly degraded in primary auditory cortex, anterior auditory field and the ventral auditory field. Neurometric analysis of speech evoked activity using a pattern classifier confirmed that activity in these fields contains significantly less information about speech sound identity in Fmr1 knockout rats compared to control rats. Responses were normal in the posterior auditory field, which is associated with sound localization. The greatest impairment was observed in the ventral auditory field, which is related to emotional regulation. Dysfunction in the ventral auditory field may contribute to poor emotional regulation in fragile X syndrome and may help explain the observation that later auditory evoked responses are more disturbed in fragile X syndrome compared to earlier responses. Rodent models of fragile X syndrome are likely to prove useful for understanding the biological basis of fragile X syndrome and for testing candidate therapies.

The Pex1-G844D mouse: A model for mild human Zellweger spectrum disorder

Zellweger spectrum disorder (ZSD) is a disease continuum that results from inherited defects in PEX genes essential for normal peroxisome assembly. These autosomal recessive disorders impact brain development and also cause postnatal liver, adrenal, and kidney dysfunction, as well as loss of vision and hearing. The hypomorphic PEX1-G843D missense allele, observed in approximately 30% of ZSD patients, is associated with milder clinical and biochemical phenotypes, with some homozygous individuals surviving into early adulthood. Nonetheless, affected children with the PEX1-G843D allele have intellectual disability, failure to thrive, and significant sensory deficits. To enhance our ability to test candidate therapies that improve human PEX1-G843D function, we created the novel Pex1-G844D knock-in mouse model that represents the murine equivalent of the common human mutation. We show that Pex1-G844D homozygous mice recapitulate many classic features of mild ZSD cases, including growth retardation and fatty livers with cholestasis. In addition, electrophysiology, histology, and gene expression studies provide evidence that these animals develop a retinopathy similar to that observed in human patients, with evidence of cone photoreceptor cell death. Similar to skin fibroblasts obtained from ZSD patients with a PEX1-G843D allele, we demonstrate that murine cells homozygous for the Pex1-G844D allele respond to chaperone-like compounds, which normalizes peroxisomal β-oxidation. Thus, the Pex1-G844D mouse provides a powerful model system for testing candidate therapies that address the most common genetic cause of ZSD. In addition, this murine model will enhance studies focused on mechanisms of pathogenesis.

Experimental Induction of Type 2 Diabetes in Aging-Accelerated Mice Triggered Alzheimer-Like Pathology and Memory Deficits

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease constituting ~95% of late-onset non-familial/sporadic AD, and only ~5% accounting for early-onset familial AD. Availability of a pertinent model representing sporadic AD is essential for testing candidate therapies. Emerging evidence indicates a causal link between diabetes and AD. People with diabetes are >1.5-fold more likely to develop AD. Senescence-accelerated mouse model (SAMP8) of accelerated aging displays many features occurring early in AD. Given the role played by diabetes in the pre-disposition of AD, and the utility of SAMP8 non-transgenic mouse model of accelerated aging, we examined if high fat diet-induced experimental type 2 diabetes in SAMP8 mice will trigger pathological aging of the brain. Results showed that compared to non-diabetic SAMP8 mice, diabetic SAMP8 mice exhibited increased cerebral amyloid-β, dysregulated tau-phosphorylating glycogen synthase kinase 3β, reduced synaptophysin immunoreactivity, and displayed memory deficits, indicating Alzheimer-like changes. High fat diet-induced type 2 diabetic SAMP8 mice may represent the metabolic model of AD.

Animal models of neurologic disorders: a nonhuman primate model of spinal cord injury.

Primates are an important and unique animal resource. We have developed a nonhuman primate model of spinal cord injury (SCI) to expand our knowledge of normal primate motor function, to assess the impact of disease and injury on sensory and motor function, and to test candidate therapies before they are applied to human patients. The lesion model consists of a lateral spinal cord hemisection at the C7 spinal level with subsequent examination of behavioral, electrophysiological, and anatomical outcomes. Results to date have revealed significant neuroanatomical and functional differences between rodents and primates that impact the development of candidate therapies. Moreover, these findings suggest the importance of testing some therapeutic approaches in nonhuman primates prior to the use of invasive approaches in human clinical trials. Our primate model is intended to: 1) lend greater positive predictive value to human translatable therapies, 2) develop appropriate methods for human translation, 3) lead to basic discoveries that might not be identified in rodent models and are relevant to human translation, and 4) identify new avenues of basic research to "reverse-translate" important questions back to rodent models.

Neuroanatomical correlation of behavioral deficits in the CCI model of TBI

Traumatic brain injury (TBI) is the leading cause of death and disability both in combat and civilian situations with limited treatment options including surgical removal of hematoma, ventricular drainage and use of hyperosmotic agents that restrict secondary injury following TBI. Availability of appropriate model system with full-range characterization of anatomical and behavioral components correlative with brain injury provides a pre-clinical platform to test candidate therapies for clinical translation. Modeling of TBI using controlled cortical impact injury (CCI) is largely considered to be close to clinical TBI and hence CCI models have been widely used in pre-clinical TBI research. Most studies reported so far using CCI models were presented with a limited behavioral characterization and lacked its correlation with the signature histopathology of TBI. Current investigation validated a detailed sensomotor and cognitive behavioral characterization correlative with diffuse axonal injury—the signature histopathology of TBI, in the CCI mouse model of TBI. Present study offers a comprehensively characterized model of TBI that can be used to investigate cellular and molecular mechanisms underlying TBI and to test candidate therapies in developing novel and effective treatments for TBI.

Molecular Genetics and Biomarkers of Polyglutamine Diseases

Polyglutamine diseases are hereditary neurodegenerative disorders caused by an abnormal expansion of a trinucleotide CAG repeat, which encodes a polyglutamine tract. To date, nine polyglutamine diseases are known: Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA) and six forms of spinocerebellar ataxia (SCA). The diseases are inherited in an autosomal dominant fashion except for SBMA, which shows an X-linked pattern of inheritance. Although the causative gene varies with each disorder, polyglutamine diseases share salient genetic features as well as molecular pathogenesis. CAG repeat size correlates well with the age of onset in each disease, shows both somatic and germline instability, and has a strong tendency to further expand in successive generations. Aggregation of the mutant protein followed by the disruption of cellular functions, such as transcription and axonal transport, has been implicated in the etiology of neurodegeneration in polyglutamine diseases. Although animal studies have provided promising therapeutic strategies for polyglutamine diseases, it remains difficult to translate these disease-modifying therapies to the clinic. To optimize "proof of concept", the process for testing candidate therapies in humans, it is of importance to identify biomarkers which can be used as surrogate endpoints in clinical trials for polyglutamine diseases.

Autologous Lacrimal–Lymphoid Mixed-cell Reactions Induce Dacryoadenitis in Rabbits

Autoimmune dacryoadenitis, such as occurs in Sjögren's syndrome, is a frequent cause of lacrimal insufficiency, which in turn can cause dry eye. Rabbits are used frequently to test ocular therapies. Our goal is to develop a rabbit model of autoimmune dacryoadenitis to identify and test candidate therapies. Our approach arises from the observations that lacrimal gland epithelial cells stimulate proliferation in cultured autologous lymphocyte preparations and that an anti-MHC II antibody blocks this proliferation. The purpose of this study was to determine if injecting this proliferating autologous mixed cell reaction could induce dacryoadenitis in rabbits. After establishing that irradiated lacrimal gland epithelial cells stimulate proliferation in autologous peripheral blood lymphocytes, irradiated cells from a single lacrimal gland were co-cultured with autologous lymphocytes and after 5 days the mixed cell reaction, or components of the reaction, were injected into the contralateral lacrimal gland of the donor rabbit. After 2 weeks, the injected glands were removed and lymphocytic infiltration quantitated using digital image analysis of immunostained histological sections. Injecting an autologous mixed cell reaction of co-cultured irradiated lacrimal gland epithelial cells and lymphocytes reliably induced abundant periductal foci of >200 lymphocytes expressing CD18 and/or a rabbit thymic lymphocyte antigen (RTLA). Injection of medium or autologous lymphocytes alone elicited little response; injections of lymphocytes cultured with lysates of lacrimal gland epithelial cells elicited variable, modest responses. These lysates did not stimulate proliferation in the mixed cell reaction and proliferation was not observed if a porous membrane separated co-cultured lacrimal gland cells and lymphocytes. The results demonstrate that injecting an autologous mixed cell reaction of lacrimal gland epithelial cells and lymphocytes reliably creates a model of autoimmune dacryoadenitis. The relative ineffectiveness of components of the reaction to do the same supports the hypothesis that lacrimal gland epithelial cells trigger or exacerbate lacrimal autoimmune disease by presentation of autoantigens via MHC II. This experimental system can aid efforts to further understand mechanisms of diseases, and to identify and test candidate therapies.