Single Mouse Research Articles

Single Mouse Testing Demonstrates Value.

Single mouse testing may be an efficient approach to assess cancer therapies in children. In a prospective study, researchers established that single mouse models provided results similar to conventional testing, saving resources to study more tumor types.

Irs1S57X Heterozygous Mutant Mice Display Normal Hematopoiesis and Phenotypic Features, While Homozygous Knockout Exhibit High Fetal or Postnatal Lethality

Introduction: Pharmacological inhibition of insulin receptor substrate 1 (IRS1) protein has been demonstrated to promote antineoplastic effects in hematological disorders, including myeloproliferative neoplasms, chronic myeloid leukemia and acute lymphoblastic leukemia. However, the role of IRS1 in normal hematopoiesis has not yet been elucidated. In this scenario, using a murine Irs1 knockout model represents an interesting tool to evaluate the role of this gene in normal hematopoiesis. B6.129S2-Irs1smla mouse carries a spontaneous nonsense mutation in serine 57 (Irs1S57X), which in homozygosis produces a knockout animal for Irs1, characterized by a small size. Throughout the development of the study, we noticed that the successive mating between Irs1smla heterozygous animals did not result in homozygous mice among the offspring. In view of these findings, the present study aimed to compare the hematological parameters of wild type and heterozygous mice for the Irs1S57X mutation and to describe the fetal lethality of homozygous mice. Methods: Protocols for mouse experiments were approved by the Animal Ethics Committee of the Institution. B6.129S2-Irs1smla/J mice (Jackson Laboratory, stock number: 007240) from a pure C57BL/6 background were mated to produce homozygous animals. For peripheral blood analysis, mice were evaluated monthly at different age ranges (8-10; 12-14; 16-18; 20-22 weeks of age). Samples were collected in heparin and submitted to automated blood cell count. The hematological parameters evaluated included white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT) and platelets. For fetal lethality evaluation, mating was performed between heterozygous mice. Females were followed up daily to check for the presence of a vaginal plug. The time that the plug was identified was considered as 0.5 days post coitus (dpc) or D+0.5. After the plug was found, the females were accommodated separately until the date of euthanasia. Euthanasia of the females was performed on gestational days D+9.5 (n=1), D+12.5 (n=1), D+15.5 (n=2) and D+18.5 (n=2). The fetuses were carefully separated from the maternal material to avoid contamination. Genotyping was performed by Sanger sequencing. Results: After 28 intercrossing events, a total of 101 heterozygotes, 46 wild type mice and 1 homozygous mouse were obtained, with an average of 4.9 (± 1.6) born alive per female. The absence of homozygous knockout adult mice precluded the inclusion of this genotype in the statistical analysis. The single Irs1 knockout mouse that survived to weaning was euthanized at 9 weeks of age for analysis. The Irs1-deficient mouse was male and characterized by low body weight (15.64 grams); 6.8x109/LWBC; 9.79x1012/L RBC, 16.4 g/dl HGB; 48.5% HCT and 311x109/Lplatelets. Wild type animals and heterozygotes were followed up for four months from 8 weeks of age, being weighed and submitted to peripheral blood analysis monthly. Among wild type animals (n= 7 females and 4 males) and heterozygotes (n= 7 females and 12 males) there were no significant differences in body weight and hematological parameters of peripheral blood, at all age ranges evaluated (all p>0.05). Irs1S57X mutation, in homozygosis, promotes fetal or postnatal lethality. Progeny genotyping demonstrated that homozygous fetuses developed until gestational day 18.5. Full-term deliveries (n=2 females) resulted in 17 pups, including 11 born alive and 6 miscarried/dead immediately after birth; the stillborn were exclusively homozygous. Conclusions: Irs1S57X mutation, in heterozygous state, does not result in phenotypic alterations in weight and hematological parameters. In this study, spontaneous truncated homozygosis mutation Irs1S57X, from a pure C57BL/6 genetic background, resulted in high fetal or postnatal lethality. High mortality was also described in a knockout model for SerpinB2, which spontaneously acquired the Irs1 S57X mutation, resulting in the total loss of Irs1 protein expression (WESTRICK et al., 2010). Irs1-deficient models generated by targeted disruption do not exhibit significative reduction in survival (ARAKI et al., 1994; TAMEMOTO et al., 1994). These data suggest that the elevated mortality observed in this study may not directly be related to Irs1 deficiency. Based on these findings, further hematologic studies of B6.129S2-Irs1smla/J mice should be focused on fetal hematopoiesis. Disclosures Traina: AbbVie: Other: Principal Investigator for Protocol Number M15-656 ; University of São Paulo at Ribeirão Preto Medical School: Current Employment.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

Assessment of the Genotoxic Potential of Cizolirtine a Substance-P and Calcitonin Gene-Related Peptide Release Modulator.

The analysis of the genotoxic potential of cizolirtine, a compound being developed as a drug for analgesia and for urinary incontinence, was carried out using a battery of in vitro and in vivo assays as recommended in the guidelines for medicinal products. Negative results were obtained in an Ames test (up to 5000 µg/plate), in a Mouse Lymphoma assay (up to 2000 µg/ml) and in a single dose mouse bone marrow micronucleus assay (up to 300 mg/kg). In a human lymphocyte chromosome aberration assay, a slight statistical increase in the frequency of cells with chromosome aberrations including gaps was reported for the concentrations of 200 and 1600 μg/ml at the 24-h sampling time. This minor increase in chromosome aberrations was considered of questionable biological relevance since it was moderate, was within the laboratory historical control values, did no show a dose-dependent effect and was not observed at similar concentrations in a repeat assay. Taking into considerations the results obtained in the different in vitro and in vivo assays and a weight-of-evidence analysis, it suggests that cizolirtine would not pose a genotoxic risk when administered to humans.

Diet-induced obesity in genetically diverse collaborative cross mouse founder strains reveals diverse phenotype response and amelioration by quercetin treatment in 129S1/SvImJ, PWK/EiJ, CAST/PhJ, and WSB/EiJ mice

Significant evidence suggests protective effects of flavonoids against obesity in animal models, but these often do not translate to humans. One explanation for this disconnect is use of a few mouse strains (notably C57BL/6 J) in obesity studies. Obesity is a multifactorial disease. The underlying causes are not fully replicated by the high-fat C57BL/6 J model, despite phenotypic similarities. Furthermore, the impact of genetic factors on the activities of flavonoids is unknown. This study was designed to explore how diverse mouse strains respond to diet-induced obesity when fed a representative flavonoid. A subset of Collaborative Cross founder strains (males and females) were placed on dietary treatments (low-fat, high-fat, high-fat with quercetin, high-fat with quercetin and antibiotics) longitudinally. Diverse responses were observed across strains and sexes. Quercetin appeared to moderately blunt weight gain in male C57 and both sexes of 129S1/SvImJ mice, and slightly increased weight gain in female C57 mice. Surprisingly, quercetin dramatically blunted weight gain in male, but not female, PWK/PhJ mice. For female mice, quercetin blunted weight gain (relative to the high-fat phase) in CAST/PhJ, PWK/EiJ and WSB/EiJ mice compared to C57. Antibiotics did not generally result in loss of protective effects of quercetin. This highlights complex interactions between genetic factors, sex, obesity stimuli, and flavonoid intake, and the need to move away from single inbred mouse models to enhance translatability to diverse humans. These data justify use of genetically diverse Collaborative Cross and Diversity Outbred models which are emerging as invaluable tools in the field of personalized nutrition.

Absence of R-Ras1 and R-Ras2 causes mitochondrial alterations that trigger axonal degeneration in a hypomyelinating disease model.

Fast synaptic transmission in vertebrates is critically dependent on myelin for insulation and metabolic support. Myelin is produced by oligodendrocytes (OLs) that maintain multilayered membrane compartments that wrap around axonal fibers. Alterations in myelination can therefore lead to severe pathologies such as multiple sclerosis. Given that hypomyelination disorders have complex etiologies, reproducing clinical symptoms of myelin diseases from a neurological perspective in animal models has been difficult. We recently reported that R-Ras1-/- and/or R-Ras2-/- mice, which lack GTPases essential for OL survival and differentiation processes, present different degrees of hypomyelination in the central nervous system with a compounded hypomyelination in double knockout (DKO) mice. Here, we discovered that the loss of R-Ras1 and/or R-Ras2 function is associated with aberrant myelinated axons with increased numbers of mitochondria, and a disrupted mitochondrial respiration that leads to increased reactive oxygen species levels. Consequently, aberrant myelinated axons are thinner with cytoskeletal phosphorylation patterns typical of axonal degeneration processes, characteristic of myelin diseases. Although we observed different levels of hypomyelination in a single mutant mouse, the combined loss of function in DKO mice lead to a compromised axonal integrity, triggering the loss of visual function. Our findings demonstrate that the loss of R-Ras function reproduces several characteristics of hypomyelinating diseases, and we therefore propose that R-Ras1-/- and R-Ras2-/- neurological models are valuable approaches for the study of these myelin pathologies.

Effects of Acrylic Tunnel Enrichment on Anxiety-Like Behavior, Neurogenesis, and Physiology of C57BL/6J Mice.

Environmental enrichment for mice lags behind the standard enrichment offered to other laboratory rodents due to concerns about environmental variability and, in specific contexts, aggression. Our objective in this study was to evaluate concerns that the introduction of structural enrichment in the form of a single red acrylic mouse tunnel into murine housing may confound study findings. We measured effects on anxiety-like behaviors (elevated zero maze and open field activity), hippocampal neurogenesis, body weight gain, and physiologic markers of stress (adrenal gland weight, plasma corticosterone concentration, and neutrophil:lymphocyte ratio). Male and female C57BL/6J mice were randomly assigned to one of 2 groups: a standard-housed control group with enrichment consisting of paper nesting material, or an enriched group that received a single acrylic tunnel in addition to nesting material. All results fell within biologically normal ranges regardless of treatment, and variability (standard deviation) was not significantly different between groups for any measure. Mice in the enriched group showed modest differences during open field testing suggestive of decreased anxiety, traveling farther and depositing fewer fecal boli than standard-housed mice. Male mice in the tunnel-enriched group gained more body weight than standard-housed male mice. No significant effects by treatment were found in neurogenic or physiologic parameters. These results indicate that provision of simple structural enrichment is unlikely to have confounding effects on murine anxiety-like behaviors, neurogenesis, body weight gain, or physiologic parameters. We therefore recommend the inclusion of simple structural enrichment, such as an acrylic tunnel, to the standard environmental enrichment of social housing and nesting material for mice.

37 Poster Discussion - Prospective validation of single mouse testing (SMT) by the pediatric preclinical testing consortium (PPTC)

37 Poster Discussion - Prospective validation of single mouse testing (SMT) by the pediatric preclinical testing consortium (PPTC)

Photooxidation mediated by 11-cis and all-trans retinal in single isolated mouse rod photoreceptors.

Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-cis retinal continually supplied to photoreceptors. The released all-trans retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-cis and all-trans retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-cis or all-trans retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-cis retinal. The results indicate that both 11-cis and all-trans retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans retinal generated by light through its reduction to retinol minimizes all-trans retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-cis retinal, 11-cis-retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.

Monitoring AuNP Dynamics in the Blood of a Single Mouse Using Single Particle Inductively Coupled Plasma Mass Spectrometry with an Ultralow-Volume High-Efficiency Introduction System.

Gold nanoparticles (AuNPs) are increasingly being used as diagnostic and therapeutic agents owing to their excellent properties; however, there is not much data available on their dynamics in vivo on a single particle basis in a single mouse. Here, we developed a method for the direct analysis of nanoparticles in trace blood samples based on single particle inductively coupled plasma-mass spectrometry (spICP-MS). A flexible, highly configurable, and precisely controlled sample introduction system was designed by assembling an ultralow-volume autosampler (flow rate in the range of 5-5000 μL/min) and a customized cyclonic spray chamber (transfer efficiency up to 99%). Upon systematic optimization, the detection limit of the nanoparticle size (LODsize) of AuNPs in ultrapure water was 19 nm, and the detection limit of the nanoparticle number concentration (LODNP) was 8 × 104 particle/L. Using a retro-orbital blood sampling method and subsequent dilution, the system was successfully applied to track the dynamic changes in size and concentration for AuNPs in the blood of a single mouse, and the recovery for the blood sample was 111.74%. Furthermore, the concentration of AuNPs in mouse blood reached a peak in a short period of time and, then, gradually decreased. This study provides a promising technique for analyzing and monitoring the size and concentration of nanoparticles in ultralow-volume blood samples with low concentrations, making it a powerful tool for analyzing and understanding the fate of nanoparticles in vivo.

Handheld endomicroscope using a fiber-optic harmonograph enables real-time and in vivo confocal imaging of living cell morphology and capillary perfusion

Confocal laser endomicroscopy provides high potential for noninvasive and in vivo optical biopsy at the cellular level. Here, we report a fully packaged handheld confocal endomicroscopic system for real-time, high-resolution, and in vivo cellular imaging using a Lissajous scanning fiber-optic harmonograph. The endomicroscopic system features an endomicroscopic probe with a fiber-optic harmonograph, a confocal microscope unit, and an image signal processor. The fiber-optic harmonograph contains a single mode fiber coupled with a quadrupole piezoelectric tube, which resonantly scans both axes at ~ 1 kHz to obtain a Lissajous pattern. The fiber-optic harmonograph was fully packaged into an endomicroscopic probe with an objective lens. The endomicroscopic probe was hygienically packaged for waterproofing and disinfection of medical instruments within a 2.6-mm outer diameter stainless tube capable of being inserted through the working channel of a clinical endoscope. The probe was further combined with the confocal microscope unit for indocyanine green imaging and the image signal processor for high frame rate and high density Lissajous scanning. The signal processing unit delivers driving signals for probe actuation and reconstructs confocal images using the auto phase matching process of Lissajous fiber scanners. The confocal endomicroscopic system was used to successfully obtain human in vitro fluorescent images and real-time ex vivo and in vivo fluorescent images of the living cell morphology and capillary perfusion inside a single mouse.

DamID to Map Genome-Protein Interactions in Preimplantation Mouse Embryos.

Investigating the chromatin landscape of the early mammalian embryo is essential to understand how epigenetic mechanisms may direct reprogramming and cell fate allocation. Genome-wide analyses of the epigenome in preimplantation mouse embryos have recently become available, thanks to the development of low-input protocols. DNA adenine methyltransferase identification (DamID) enables the investigation of genome-wide protein-DNA interactions without the requirement of specific antibodies. Most importantly, DamID can be robustly applied to single cells. Here we describe the protocol for performing DamID in single oocytes and mouse preimplantation embryos, as well as single blastomeres, using a Dam-LaminB1 fusion to generate high-resolution lamina-associated domain (LAD) maps. This low-input method can be adapted for other proteins of interest to faithfully profile their genomic interaction, allowing us to interrogate the chromatin dynamics and nuclear organization during the early mammalian development.

Mannose-binding lectin (MBL) and the lectin complement pathway play a role in cutaneous ischemia and reperfusion injury

ObjectivesCutaneous ischemia/reperfusion (CI/R) injury has shown to play a significant role in chronic wounds such as decubitus ulcers, diabetic foot ulcers, atherosclerotic lesions, and venous stasis wounds. CI/R also plays a role in free tissue transfer in reconstructive microsurgery and has been linked to clinical burn-depth progression after thermal injury. While the role of the complement system has been elucidated in multiple organ systems, evidence is lacking with respect to its role in the skin. Therefore, we evaluated the role of the complement system in CI/R injury.MethodsUsing a single pedicle skin flap mouse model of acute CI/R, we performed CI/R in wild-type (WT) mice and complement knock out (KO) mice, deficient in either C1q (C1q KO; classical pathway inhibition), mannose-binding lectin (MBL null; lectin pathway inhibition) or factor B (H2Bf KO; alternative pathway inhibition). Following 10 h ischemia and 7 days reperfusion, mice were sacrificed, flaps harvested and flap viability assessed via Image J software. The flap necrotic area was expressed as % total flap area. In another group, mice were sacrificed following CI/R with 10 h ischemia and 48 h reperfusion. Two cranial skin flap samples were taken for gene expression analysis of IL1b, IL6, TNFα, ICAM1, VCAM1, IL10, IL13 using real-time polymerase chain reaction (RT-PCR).ResultsFollowing CI/R, MBL null mice had a statistically significant smaller %necrotic flap area compared to WT mice (10.6 vs. 43.1%; p<0.05) suggesting protection from CI/R. A significantly reduced mean %necrotic flap area was not seen in either C1q KO or H2Bf KO mice relative to WT (22.9 and 31.3 vs. 43.1%; p=0.08 and p=0.244, respectively). There were no statistically significant differences between groups for markers of inflammation (TNFα, ICAM1, VCAM1, IL1b, IL6). In contrast, mRNA levels of IL10, a regulator of inflammation, were significantly increased in the MBL null group (p=0.047).ConclusionsWe demonstrated for the first time a significant role of MBL and the lectin complement pathway in ischemia/reperfusion injury of the skin and a potential role for IL10 in attenuating CI/R injury, as IL10 levels were significantly increased in the tissue from the CI/R-protected MBL null group.

REGULATION OF OVIDUCT HOMEOSTASIS AND FERTILITY BY Pax2 AND Pax8 GENES

In mouse and humans, the development regulatory genes Pax2 and Pax8 are expressed in ovarian duct surface epithelia, yet the function of these genes and proteins in adult females remains unclear. By Generating double & single KO mouse modules of Pax2 and Pax8 we investigate their roles in maintaining the integrity of the oviductal cells. Our studies directly address aspects of oviduct epithelial homeostasis and the effects on fertility. To create Pax2, Pax8, and Pax2/8 double mutants specifically in oviduct epithelia and characterize changes in gene expression, epithelial cell integrity, and fertility. We will implement detailed analyses of oviducts isolated at various times post tamoxifen administration from mice with conditional Pax2, Pax8, or both alleles, using the Ovigp1-CreER driver. Mice will also carry reporter allele (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo) to mark all active Cre expressing cells with cell surface EGFP. In addition to defining novel functions for Pax proteins in the oviduct, we will utilize state-of-the-art methods for single cell analyses, transcriptomics, and chromatin remodeling. Mutation of Pax2 and Pax8 in adult female oviducts results in significant changes in gene expression and morphology. Loss of both Pax2 and Pax8 resulted in infertility (Table 1). To date, most of the preliminary data was obtained from Pax2/8 double mutants since we were concerned with redundancy and expected the most significant phenotypes in the double mutants. Analyses of histology, immunohistochemistry and changes in gene expression of total RNA from whole oviducts isolated after tamoxifen administration. For a rapid first pass screen, we compared controls (Pax2f/f;Pax8f/f) to single Pax mutants and Pax2/8 double mutants (Pax2f/f;Pax8f/f; Ovigp1-CreER) by Affymetrix microarrays. These data show hundreds of changes in gene expression levels upon Pax2 or Pax8 deletion, with significant overlap between the Pax2 and Pax8 mutants but also expression changes unique to each single mutant.Tabled 1TABLE 1. Fertility of Oviduct Specific Pax MutantsGenotype # females # plugs Litters Litter sizePax2f/f, Pax8f/f, Ovi-Cre 5 15 0 0Pax2f/f, Pax8f/f, 4 12 12 6-9Pax8f/f, Ovi-Cre 3 6 2 6-7 Open table in a new tab Little is known regarding the mechanisms underlying epithelial homoeostasis, the proteins that determine cell fates, and epithelial integrity in the adult oviduct. Pax8 expressing secretory cells are thought to give rise to ciliated cells, which help move the oocyte down the duct and Pax2 is associated with cilia motility and physiology. Using a conditional KO model of Pax2 & Pax8 will allow us to understand those mechanisms and identify future novel therapeutic targets for diagnostics and treatments

Abstract 561: Preclinical efficacy data for the anti-CEACAM5-DM4 ADC SAR408701 supports further development in lung and gastro-intestinal cancers

Abstract Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a cell-surface glycoprotein highly expressed in several tumor types including non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and gastric cancer (GC). SAR408701 is an antibody-drug conjugate (ADC) which consists of anti-CEACAM5 antibody conjugated to the maytansinoid DM4 cytotoxic agent, a highly potent tubulin inhibitor. SAR408701 was investigated in a First in Human trial (FIH) in patients with advanced solid tumors. During the early dose escalation phase, preliminary anti-tumor activity was observed with objective response reported in 3 patients with CRC and 1 patient with GC. In an interim analysis of an ongoing expansion cohort of patients with non-squamous NSCLC and with CEACAM5 expression in ≥ 50% of tumor cells, 25% overall response rate (ORR) (8/32 evaluable patients) and 37.5% (12 patients) stable disease were observed. In parallel to FIH trial, several preclinical studies were performed to support the clinical development of SAR408701 in NSCLC, CRC and GC. A single mouse trial (SMT) was carried out on a panel of 28 patent-derived xenografts (PDX) covering the top 3 indications. The SMT format employs a single mouse per PDX model and treatment arm and uses RECIST criteria as readout. Results showed an efficacy profile in the preclinical trial close to that obtained in clinic with an ORR of 10% in CRC, of 50% in lung cancers and of 37.5% in GC. The efficacy of SAR408701 is correlated with CEACAM5 expression in lung and gastric indications. To further assess the potential of SAR408701 in NSCLC, preclinical investigations have been done to determine if an ADC-DM4 could be beneficially combined with immune checkpoint inhibitors (ICIs). The capacity of DM4-treated tumor cells to immunize immune competent mice to the tumor was evaluated, indicating that DM4 can induce immunogenic cell death. This result supports the development of SAR408701 in combination with ICIs in NSCLC. The combination of SAR408701 with 4 different standard of care (SoC) therapies were tested in 2 CRC PDX models, one sensitive and one resistant to SAR408701 alone. Combinations of SAR408701 were evaluated with either FOLFOX regimen, FOLFIRI regimen, TAS-102 or cetuximab. All combinations resulted in significantly better antitumor activity than single agent arms in both resistant and sensitive PDXs without an increase in toxicity and showed a therapeutic synergy. These results suggest that SAR408701 can be advantageously combined with SoC in gastro-intestinal (GI) cancers. In summary, preclinical data show that SAR408701 displays a strong antitumor activity as a single agent in NSCLC and in combination with SOC in GI cancers, supporting further development in these indications. Furthermore, SAR408701 could be a potential good candidate to be combined with ICIs in NSCLC. Citation Format: Stephanie Decary, Olivier Lemaitre, Anne-Marie Lefebvre, Alexandra Ferrier, Marielle Chiron, Dmitri Wiederschain, Anne-Sophie Lacoste-Bourgeacq, Nathalie Le Bail, Sukhvinder Sidhu, Cecile Combeau, Mustapha Chadjaa, Celine Nicolazzi. Preclinical efficacy data for the anti-CEACAM5-DM4 ADC SAR408701 supports further development in lung and gastro-intestinal cancers [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 561.

Abstract 5634: The single mouse trial format predicts the sensitivity towards checkpoint inhibitor treatment in NSCLC PDX models in HuCD34NCG/CRL mice, an innovative triple immune deficient mouse strain

Abstract Immunotherapy is revolutionizing cancer treatment. As a consequence, there is an urgent need for predictive preclinical models to support compound development with the aim of bringing optimal drug candidates into clinical trials. Patient-derived xenografts (PDX) are the gold-standard in cancer drug development. One major drawback of PDX is the lack of an immunological competent host. To overcome this hurdle triple immunodeficient mice are humanized with hematopoietic stem cells (HSC) to enable the interaction between human immune cells and human tumor cells in a rodent host. The current study uses HSC humanized NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) mice, HuCD34NCG, to evaluate the sensitivity of five NSCLC PDX models towards anti CTLA-4 (Ipilimumab), anti-PD-1 (Nivolumab) and the combination thereof. We identified one predicted responder and one non-responder and in a single mouse trial approach (SMT) and confirmed the results in a conventional experimental set-up with n=5 per treatment arm. Infiltration of human immune cells was detected by flow cytometry in tumor tissue (TIL) and hematopoietic organs. Serum of tumor bearing animals was analyzed for human and mouse cytokine secretion pre- and post-treatment. The NCG mouse strain was genetically characterized by whole exome sequencing (WES) and the mutational profile compared to other common mouse strains. The mutational profile showed the highest overlap with the genome data from other triple immune compromised mice and with NOD/SCID indicating their close hereditary relationship. Non-tumor bearing HuCD34NCG mice depicted engraftment of human CD45+ cells in peripheral blood of &amp;gt;25% and &amp;gt;60% in bone marrow and spleen after 8-12 wks post injection. Thus, they fulfilled enrollment criteria for conducting a drug screen for immune modulating compounds. The five NSCLC PDX models showed a distinct sensitivity profile ranging from partial remission (tumor growth inhibition, TGI, of 87.5% compared to untreated control) to progression (no TGI). In all models the combination treatment was the most active therapy (TGI: 60%), followed by Ipilimumab (TGI: 55%) and Nivolumab (TGI: 48%). Nevertheless, in selected models anti-CTLA-4 treatment was the most efficacious therapy. The two models examined in more detail LXFE 397 (=responder, TGI 55% in both formats) and LXFA 400 (non-responder, TGI 2%SMTvs 5%, n=5/group) displayed the same sensitivity pattern in the conventional set-up as compared to the SMT screening approach. The TIL analysis revealed a pronounced rise in the CD3+ subset and increased CD4+/CD8+ ratio in the most effective treatment arms. Subsequent serum analyses will help identify possible biomarkers predicting drug response towards checkpoint inhibition. Taken together our study proves that the HuCD34NCG mouse is a fully functional drug development tool. In combination with highly predictive PDX tumor models this in vivo platform provides a further step to support the development of new drugs targeting the host immune response. Citation Format: Julia Schüler, Jenny Rowe, Eva Oswald, Stephen Festin. The single mouse trial format predicts the sensitivity towards checkpoint inhibitor treatment in NSCLC PDX models in HuCD34NCG/CRL mice, an innovative triple immune deficient mouse strain [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 5634.

Abstract LB-217: Preclinical evaluation of trastuzumab deruxtecan (T-DXd; DS-8201a), a HER2 antibody-drug conjugate, in pediatric solid tumors by the Pediatric Preclinical Testing Consortium (PPTC)

Abstract Introduction: HER2 is expressed in a subset of pediatric solid tumors and is a target of interest for innovative immune therapies including CAR-T cells and antibody-drug conjugates (ADCs). We evaluated preclinical efficacy of T-DXd, a humanized monoclonal HER2 antibody conjugated to a topoisomerase 1 inhibitor payload, DXd, in solid tumor patient derived xenograft (PDX) models. Methods: 7 osteosarcoma (OS), 2 rhabdoid tumor (RT) and 3 Wilms tumor (WT) PDX models with varying HER2 expression were tested using 10 mice per group. ERBB2 mRNA expression was determined using RNA seq. T-DXd or vehicle control was administered IV to mice harboring flank tumors at a dose of 5mg/kg on Day 1. Standard PPTC statistical methods were employed for EFS for treatment (T) and control (C) groups, minimum relative tumor volume (minRTV) and objective response measure. Results: Among PPTC solid tumor models ERBB2 mRNA expression was observed across multiple histologies, with highest expression observed for WT (median = 22 FPKM), followed by RT, OS, and Ewing sarcoma. The relationship between HER2 expression by IHC and by RNAseq was inconsistent. Mice tolerated T-DXd with minimal toxicity. T-DXd significantly prolonged EFS in 5/7 OS, 2/2 RT and 3/3 WT PDX models (see table). Complete response (CR) or maintained CR were observed for 4 of 5 WT and RT models, while stable disease was the best response among OS models. Conclusions: T-DXd exhibits single agent anti-tumor activity in models of pediatric solid tumors. Single mouse testing experiments will be used to extend knowledge of the range of activity of T-DXd for pediatric solid tumors. Correlations between ERBB2 gene expression, HER2 expression by IHC, and genetic changes associated with camptothecin sensitivity are ongoing and will be presented. Clinical trials assessing efficacy of a HER2-directed ADC in pediatric patients with HER2-expressing tumors should be considered. EFS T - C(days)EFS T/C Ratiop-value Gehan-WilcoxonminRTVmean±SDminRTVp-valueObjective Response MeasureOS-120.31.85p &amp;lt; 0.0011.152±0.090p = 0.023PD1OS-230.12.76p &amp;lt; 0.0010.815±0.183p &amp;lt; 0.001PD2OS-94.11.14p = 0.1031.334±0.203p = 1.000PD1OS-1734.01.97p = 0.0091.172±0.179p = 0.017PD1OS-3111.91.84p &amp;lt; 0.0011.676±0.395p = 0.015PD1OS-3361.34.87p &amp;lt; 0.0010.486±0.220p &amp;lt; 0.001SDOS-603.81.11p = 0.0161.084±0.103p = 0.007PD1BT29 (RT)&amp;gt; 58.4&amp;gt; 3.28p &amp;lt; 0.0010.019±0.041p &amp;lt; 0.001MCRRBD2 (RT)&amp;gt; 97.3&amp;gt; 13.57p &amp;lt; 0.0010.021±0.066p &amp;lt; 0.001CRKT-10 (WT)&amp;gt; 85.1&amp;gt; 7.57p &amp;lt; 0.0010.000±0.000p &amp;lt; 0.001MCRKT-11 (WT)18.32.62p &amp;lt; 0.0010.954±0.486p &amp;lt; 0.001PD2KT-13 (WT)&amp;gt; 71.5&amp;gt; 3.69p &amp;lt; 0.0010.019±0.060p &amp;lt; 0.001MCR Citation Format: Pooja Hingorani, Wendong Zhang, Raushan Kurmasheva, Zhongting Zhang, Yifei Wang, Zhaohui Xu, Michael Roth, Jonathan Gill, Douglas Harrison, Stephen Erickson, Edward A. Kolb, Malcolm Smith, Peter Houghton, Richard Gorlick. Preclinical evaluation of trastuzumab deruxtecan (T-DXd; DS-8201a), a HER2 antibody-drug conjugate, in pediatric solid tumors by the Pediatric Preclinical Testing Consortium (PPTC) [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 LB-217.

Abstract 4181: The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes

Abstract Introduction: Exportin 1 (XPO1/CRM1) plays a central role in the export of proteins from the nucleus including those with tumor suppressor and growth regulatory activity. Eltanexor (KPT-8602) is a second-generation Selective Inhibitor of Nuclear Export (SINE) that specifically blocks XPO1 cargo interactions and is under clinical evaluation for adults with cancer (NCT02649790). In previous Pediatric Preclinical Testing Consortium (PPTC) studies, eltanexor showed potent in vivo activity against acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs). In an effort to understand whether eltanexor exhibited subtype-specific activity in pediatric ALL we carried out a single-mouse trial (SMT) against an intended 90 pediatric ALL PDXs. Methods: Pediatric ALL PDXs, an orthotopic model of the disease, were inoculated into NSG mice in an SMT format (a single mouse inoculated with a single PDX was treated with a single drug). Engraftment and drug responses were assessed by weekly enumeration of the % human leukemic blasts in the peripheral blood (%huCD45+/HLA-ABC+). Treatment commenced when the median %huCD45+/HLA-ABC+ exceeded 1%, and mice received eltanexor at 12.5 mg/kg per oral daily x 5 for 4 weeks. The baseline level of the %huCD45+/HLA-ABC+ in each mouse served as its own control. PDX responses to treatment were assessed by time to event, the maximum decrease in %huCD45+/ABC+ at any point after treatment initiation, and by stringent Objective Response Measures (ORMs) modeled after the clinical setting (Houghton et al, Pediatr Blood Cancer, 2007, 49:928-40). Results: Eltanexor was well tolerated with a mean maximum weight loss of 5.5% across all mice. SMT testing was successful in all 90 pediatric ALL PDXs representative of B-ALL (typical B-ALL, Philadelphia chromosome-positive ALL [Ph+-ALL] and Ph-like ALL), T-ALL (typical T-ALL and early T-cell precursor ALL [ETP-ALL]), and MLL-rearranged ALL (MLLr-ALL). Event-free survival (EFS) of mice ranged from 2.6-137 days, and 61 (68%) of mice experienced EFS that extended beyond the treatment window. Regressions of ≥50% from baseline were observed in 56 (62%) of mice, and these were most pronounced in T-ALL (14/18, 78%) and Ph-like ALL (11/19, 58%). ORMs classified as Complete Response (CR) or Maintained CR (MCR) were elicited in 43 (48%) of PDXs, and again these were more common in T-ALL (12/18, 67%). The ALL subtypes that exhibited the poorest responses to eltanexor were Ph+-ALL and ETP-ALL, although with relatively small sample size (n=3 and 6, respectively). ORMs in the SMT correlated highly with historical conventional drug testing (R=0.94, P&amp;lt;0.001, n=12). Conclusions: Eltanexor exhibits potent single-agent in vivo activity against PDXs derived from a broad range of ALL subtypes including T-ALL and Ph-like ALL and warrants further investigation in pediatric ALL. (Supported by NCI Grants CA199222 &amp; CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Connor D. Jones, Stephen W. Erickson, Beverly A. Teicher, TJ Unger, Yosef Landesman, Malcolm A. Smith. The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes [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 4181.

Abstract 2229: A panel of NSCLC patient derived xenografts displays a distinct sensitivity profile towards checkpoint inhibitor treatment in vitro and in vivo

Abstract The field of cancer immunology is rapidly moving towards innovative therapeutic strategies. As a consequence there is a need for robust and predictive preclinical platforms for assessing therapies. The current project aims to establish a drug screening workflow bridging between innovative 3D in vitro assays and humanized mouse models based on the same PDX model panel. A total of six different lung cancer patient derived xenograft models (NSCLC PDX) were transduced to express nuclear RFP, cultivated as 3D spheroids and co-cultured with human peripheral blood monocytes (PBMC). Spheroid fluorescent intensity was monitored every 4 hours over a total of 120h. Efficacy of different checkpoint inhibitors was determined using a live cell imaging technology (IncuCyte S3, Sartorius). In vivo a total of 14 different NSCLC PDX were screened for their sensitivity towards α-CTLA-4, α-PD-1 or the combination thereof. With n=1 per treatment arm and model and the study design followed the screening approach of the single mouse trial (SMT). Human immune cell infiltrates of tumor (=TILs), peripheral blood, spleen and bone marrow (determined by flow cytometry, FC, and IHC) and secretion of human and mouse cytokines in murine serum (determined by cytokine array &amp; Luminex based technology) were determined in all models.In the spheroid assay as well as in vivo all three treatment arms displayed a discrete activity pattern throughout the PDX panel. The activity pattern of the 14 PDX mirrored the clinical diversity of tumor responses to checkpoint inhibitor treatment ranging from complete remission to resistance. The sensitivity towards checkpoint inhibitors was stable in a distinct PDX model across the two drug development platforms. Tumor models with high tumor infiltrating lymphocyte (TIL) rates (&amp;gt;5%) in the untreated control group were more susceptible towards checkpoint inhibitor treatment than models with low TIL rates. In either case, numbers of TILs were markedly increased in the treatment groups as compared to control vehicle. The analysis of human and mouse cytokines in the serum of tumor bearing mice led to the identification of a cytokine pattern specific for PDX models sensitive towards checkpoint inhibitor treatment. 11 human cytokines were upregulated in the sensitive tumor models (e.g. GM-CSF, IL-4 &amp; CX3CL1). The use of PDX based innovative 3D in vitro models in combination with humanized mouse models enables screening campaigns in the immune-oncology field using clinically relevant tumor models. The predictivity of the 3D spheroid platform towards the in vivo humanized mouse assay is a critical feature as it allows the optimal selection of promising drug candidates to be profiled in more detail. Citation Format: Robert Nunan, Eva Oswald, Ilona Aylott, Friedrich Feuerhake, Rhiannon Jenkinson, Julia Schüler. A panel of NSCLC patient derived xenografts displays a distinct sensitivity profile towards checkpoint inhibitor treatment in vitro and in vivo [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 2229.

Increased talin–vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections

AbstractTalin and vinculin, both actin-cytoskeleton-related proteins, have been documented to participate in establishing bacterial infections, respectively, as the adapter protein to mediate cytoskeleton-driven dynamics of the plasma membrane. However, little is known regarding the potential role of the talin–vinculin complex during spotted fever group rickettsial and Ebola virus infections, two dreadful infectious diseases in humans. Many functional properties of proteins are determined by their participation in protein–protein complexes, in a temporal and/or spatial manner. To resolve the limitation of application in using mouse primary antibodies on archival, multiple formalin-fixed mouse tissue samples, which were collected from experiments requiring high biocontainment, we developed a practical strategic proximity ligation assay (PLA) capable of employing one primary antibody raised in mouse to probe talin–vinculin spatial proximal complex in mouse tissue. We observed an increase of talin–vinculin spatial proximities in the livers of spotted fever Rickettsiaaustralis or Ebola virus-infected mice when compared with mock mice. Furthermore, using EPAC1-knockout mice, we found that deletion of EPAC1 could suppress the formation of spatial proximal complex of talin–vinculin in rickettsial infections. In addition, we observed increased colocalization between spatial proximity of talin–vinculin and filamentous actin-specific phalloidin staining in single survival mouse from an ordinarily lethal dose of rickettsial or Ebola virus infection. These findings may help to delineate a fresh insight into the mechanisms underlying liver specific pathogenesis during infection with spotted fever rickettsia or Ebola virus in the mouse model.The authors observed an increase of talin-vinculin spatial proximities in the livers of spotted fever Rickettsiaaustralis or Ebola virus-infected mice when compared to mock mice. Furthermore, using EPAC1 knockout mice, they found that deletion of EPAC1 suppresses the formation of spatial proximal complex of talin-vinculin in rickettsial infections.