Anticoagulation targeting membrane-bound anionic phospholipids improves outcomes of traumatic brain injury in mice.

Severe traumatic brain injury (TBI) often causes an acute systemic hypercoagulable state that rapidly develops into consumptive coagulopathy. We have recently demonstrated that TBI-induced coagulopathy (TBI-IC) is initiated and disseminated by brain-derived extracellular vesicles (BDEVs) and propagated by extracellular vesicles (EVs) from endothelial cells and platelets. Here, we present results from a study designed to test the hypothesis that anticoagulation targeting anionic phospholipid-expressing EVs prevents TBI-IC and improves the outcomes of mice subjected to severe TBI. We evaluated the effects of a fusion protein (ANV-6L15) for improving the outcomes of TBI in mouse models combined with in vitro experiments. ANV-6L15 combines the phosphatidylserine (PS)-binding annexin V (ANV) with a peptide anticoagulant modified to preferentially target extrinsic coagulation. We found that ANV-6L15 reduced intracranial hematoma by 70.2%, improved neurological function, and reduced death by 56.8% in mice subjected to fluid percussion injury at 1.9 atm. It protected the TBI mice by preventing vascular leakage, tissue edema, and the TBI-induced hypercoagulable state. We further showed that the extrinsic tenase complex was formed on the surfaces of circulating EVs, with the highest level found on BDEVs. The phospholipidomic analysis detected the highest levels of PS on BDEVs, as compared with EVs from endothelial cells and platelets (79.1, 15.2, and 3.5 nM/mg of protein, respectively). These findings demonstrate that TBI-IC results from a trauma-induced hypercoagulable state and may be treated by anticoagulation targeting on the anionic phospholipid-expressing membrane of EVs from the brain and other cells.

Regulatory T cells restrict immunity and pathology in distal tissue sites following a localized infection.

Regulatory T cells (Tregs) are well-known to mediate peripheral tolerance at homeostasis, and there is a growing appreciation for their role in modulating infectious disease immunity. Following acute and chronic infections, Tregs can restrict pathogen-specific T cell responses to limit immunopathology. However, it is unclear if Tregs mediate control of pathology and immunity in distal tissue sites during localized infections. We investigated the role of Tregs in immunity and disease in various tissue compartments in the context of "mild" vaginal Zika virus infection. We found that Tregs are critical to generating robust virus-specific CD8 T cell responses in the initial infection site. Further, Tregs limit inflammatory cytokines and immunopathology during localized infection; a dysregulated immune response in Treg-depleted mice leads to increased T cell infiltrates and immunopathology in both the vagina and the central nervous system (CNS). Importantly, these CNS infiltrates are not present at the same magnitude during infection of Treg-sufficient mice, in which there is no CNS immunopathology. Our data suggest that Tregs are necessary to generate a robust virus-specific response at the mucosal site of infection, while Treg-mediated restriction of bystander inflammation limits immunopathology both at the site of infection as well as distal tissue sites.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity.

BACKGROUND: The paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor receptor 2 (HER2) is an attractive candidate for CAR T-cell therapy in humans but has the potential for eliciting on-target off-tumor toxicity. We developed an immunocompetent tumor model of CAR T-cell therapy targeting murine HER2 (mHER2) and examined the effect of CAR affinity, T-cell dose, and lymphodepletion on safety and efficacy. METHODS: Antibodies specific for mHER2 were generated, screened for affinity and specificity, tested for immunohistochemical staining of HER2 on normal tissues, and used for HER2-targeted CAR design. CAR candidates were evaluated for T-cell surface expression and the ability to induce T-cell proliferation, cytokine production, and cytotoxicity when transduced T cells were co-cultured with mHER2+ tumor cells in vitro. Safety and efficacy of various HER2 CARs was evaluated in two tumor models and normal non-tumor-bearing mice. RESULTS: Mice express HER2 in the same epithelial tissues as humans, rendering these tissues vulnerable to recognition by systemically administered HER2 CAR T cells. CAR T cells designed with single-chain variable fragment (scFvs) that have high-affinity for HER2 infiltrated and caused toxicity to normal HER2-positive tissues but exhibited poor infiltration into tumors and antitumor activity. In contrast, CAR T cells designed with an scFv with low-affinity for HER2 infiltrated HER2-positive tumors and controlled tumor growth without toxicity. Toxicity mediated by high-affinity CAR T cells was independent of tumor burden and correlated with proliferation of CAR T cells post infusion. CONCLUSIONS: Our findings illustrate the disadvantage of high-affinity CARs for targets such as HER2 that are expressed on normal tissues. The use of low-affinity HER2 CARs can safely regress tumors identifying a potential path for therapy of solid tumors that exhibit high levels of HER2.

Osteosarcoma PDX-Derived Cell Line Models for Preclinical Drug Evaluation Demonstrate Metastasis Inhibition by Dinaciclib through a Genome-Targeted Approach.

PURPOSE: Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, multiple models are needed to fully elucidate key aspects of disease biology and to recapitulate clinically relevant phenotypes. EXPERIMENTAL DESIGN: Matched patient samples, patient-derived xenografts (PDX), and PDX-derived cell lines were comprehensively evaluated using whole-genome sequencing and RNA sequencing. The in vivo metastatic phenotype of the PDX-derived cell lines was characterized in both an intravenous and an orthotopic murine model. As a proof-of-concept study, we tested the preclinical effectiveness of a cyclin-dependent kinase inhibitor on the growth of metastatic tumors in an orthotopic amputation model. RESULTS: PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication in a subset of cell lines. The cell lines were heterogeneous in their metastatic capacity, and heterogeneous tissue tropism was observed in both intravenous and orthotopic models. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden. CONCLUSIONS: The variation in metastasis predilection sites between osteosarcoma PDX-derived cell lines demonstrates their ability to recapitulate the spectrum of the disease observed in patients. We describe here a panel of new osteosarcoma PDX-derived cell lines that we believe will be of wide use to the osteosarcoma research community.

T cell-inflamed gene expression profile is associated with favorable disease-specific survival in non-hypermutated microsatellite-stable colorectal cancer patients.

BACKGROUND: The anti-tumor immune response plays a key role in colorectal cancer (CRC) progression and survival. The T cell-inflamed gene expression profile (GEP) is a biomarker predicting response to checkpoint inhibitor immunotherapy across immunogenic cancer types, but the prognostic value in CRC is unknown. We evaluated associations with disease-specific survival, somatic mutations, and examined its differentially expressed genes and pathways among 84 sporadic CRC patients from the Seattle Colon Cancer Family Registry. METHODS: Gene expression profiling was performed using Nanostring's nCounter PanCancer IO 360 panel. Somatic mutations were identified by a targeted DNA sequencing panel. RESULTS: The T cell-inflamed GEP was positively associated with tumor mutation burden and microsatellite instability high (MSI-H). Higher T cell-inflamed GEP had favorable CRC-specific survival (hazard ratio [HR] per standard deviation unit = 0.50, p = 0.004) regardless of hypermutation or MSI status. Analysis of recurrently mutated genes having at least 10 mutation carriers, suggested that the T cell-inflamed GEP is positively associated with RYR1, and negatively associated with APC. However, these associations were attenuated after adjusting for hypermutation or MSI status. We also found that expression of genes RPL23, EPCAM, AREG and ITGA6, and the Wnt signaling pathway was negatively associated with the T cell-inflamed GEP, which might indicate immune-inhibitory mechanisms. CONCLUSIONS: Our results show that the T cell-inflamed GEP is a prognostic biomarker in non-hypermutated microsatellite-stable CRC. This also suggests that patient stratification for immunotherapy within this CRC subgroup should be explored further. Moreover, reported immune-inhibitory gene expression signals may suggest targets for therapeutic combination with immunotherapy.

Development and characterization of new patient-derived xenograft (PDX) models of osteosarcoma with distinct metastatic capacities.

Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology, especially for highly aggressive cancers with a propensity for metastatic spread. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, a large panel of models is needed to fully elucidate key aspects of disease biology and to recapitulate clinically-relevant phenotypes. We describe the development and characterization of osteosarcoma patient-derived xenografts (PDXs) and a panel of PDX-derived cell lines. Matched patient samples, PDXs, and PDX-derived cell lines were comprehensively evaluated using whole genome sequencing and RNA sequencing. PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication (WGD) in a subset of cell lines. These cell line models were heterogeneous in their metastatic capacity and their tissue tropism as observed in both intravenous and orthotopic models. As proof-of-concept study, we used one of these models to test the preclinical effectiveness of a CDK inhibitor on the growth of metastatic tumors in an orthotopic amputation model. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden in this model.

Sustained Helicobacter pylori infection accelerates gastric dysplasia in a mouse model.

More than 80% of gastric cancer is attributable to stomach infection with Helicobacter pylori (Hp). Gastric preneoplastic progression involves sequential tissue changes, including loss of parietal cells, metaplasia and dysplasia. In transgenic mice, active KRAS expression recapitulates these tissue changes in the absence of Hp infection. This model provides an experimental system to investigate additional roles of Hp in preneoplastic progression, beyond its known role in initiating inflammation. Tissue histology, gene expression, the immune cell repertoire, and metaplasia and dysplasia marker expression were assessed in KRAS+ mice +/-Hp infection. Hp+/KRAS+ mice had severe T-cell infiltration and altered macrophage polarization; a different trajectory of metaplasia; more dysplastic glands; and greater proliferation of metaplastic and dysplastic glands. Eradication of Hp with antibiotics, even after onset of metaplasia, prevented or reversed these tissue phenotypes. These results suggest that gastric preneoplastic progression differs between Hp+ and Hp- cases, and that sustained Hp infection can promote the later stages of gastric preneoplastic progression.

Antitumor activity of an engineered decoy receptor targeting CLCF1-CNTFR signaling in lung adenocarcinoma.

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.

Genome-Informed Targeted Therapy for Osteosarcoma.

Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for more than 30 years. Osteosarcoma is characterized by widespread and recurrent somatic copy-number alterations (SCNA) and structural rearrangements. In contrast, few recurrent point mutations in protein-coding genes have been identified, suggesting that genes within SCNAs are key oncogenic drivers in this disease. SCNAs and structural rearrangements are highly heterogeneous across osteosarcoma cases, suggesting the need for a genome-informed approach to targeted therapy. To identify patient-specific candidate drivers, we used a simple heuristic based on degree and rank order of copy-number amplification (identified by whole-genome sequencing) and changes in gene expression as identified by RNA sequencing. Using patient-derived tumor xenografts, we demonstrate that targeting of patient-specific SCNAs leads to significant decrease in tumor burden, providing a road map for genome-informed treatment of osteosarcoma. SIGNIFICANCE: Osteosarcoma is treated with a chemotherapy regimen established 30 years ago. Although osteosarcoma is genomically complex, we hypothesized that tumor-specific dependencies could be identified within SCNAs. Using patient-derived tumor xenografts, we found a high degree of response for "genome-matched" therapies, demonstrating the utility of a targeted genome-informed approach.This article is highlighted in the In This Issue feature, p. 1.

Influence of Genetic Background on Hematologic and Histopathologic Alterations during Acute Granulocytic Anaplasmosis in 129/SvEv and C57BL/6J Mice Lacking Type I and Type II Interferon Signaling.

The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR-/-/IFNGR-/-) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.

Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity.

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2',7'-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Japanese Bobtail: vertebral morphology and genetic characterization of an established cat breed.

Several cat breeds are defined by morphological variation of the tail. The Japanese Bobtail is a breed that has been accepted for registration only within the past 50 years; however, the congenital kinked tail variants defining this breed were documented in the Far East centuries ago and the cats are considered 'good luck' in several Asian cultures. The recent discovery of the mutation for the tailless Manx phenotype has demonstrated that the Japanese Bobtail does not have a causative mutation in the same gene (T-Box). Here, a simple segregation analysis of cats bred from a pedigreed Japanese Bobtail demonstrated a simple autosomal dominant mode of inheritance with variable expression of the tail length and kink placement. Unexpectedly, radiological examinations of the entire vertebral column of kink-tailed cats indicated variation from the normal vertebral feline formula (C7, T13, L7, S3, Cd20-24), including cats with mostly one reduction of thoracic vertebrae (C7, T12, L7, S3), and an average of 15.8 caudal vertebrae. A few cats had variation in the number of cervical vertebrae. Several transitional vertebrae and anomalous ribs were noted. One cat had a bifid vertebra in the tail. Most cats had hemivertebrae that were usually included in the tail kink, one of which was demonstrated by gross pathology and histopathology. The abnormal vertebral formula or the placement of the kink in the tail did not coincide with morbidity or mortality.

To the Root of the Curl: A Signature of a Recent Selective Sweep Identifies a Mutation That Defines the Cornish Rex Cat Breed.

The cat (Felis silvestris catus) shows significant variation in pelage, morphological, and behavioral phenotypes amongst its over 40 domesticated breeds. The majority of the breed specific phenotypic presentations originated through artificial selection, especially on desired novel phenotypic characteristics that arose only a few hundred years ago. Variations in coat texture and color of hair often delineate breeds amongst domestic animals. Although the genetic basis of several feline coat colors and hair lengths are characterized, less is known about the genes influencing variation in coat growth and texture, especially rexoid - curly coated types. Cornish Rex is a cat breed defined by a fixed recessive curly coat trait. Genome-wide analyses for selection (di, Tajima's D and nucleotide diversity) were performed in the Cornish Rex breed and in 11 phenotypically diverse breeds and two random bred populations. Approximately 63K SNPs were used in the analysis that aimed to localize the locus controlling the rexoid hair texture. A region with a strong signature of recent selective sweep was identified in the Cornish Rex breed on chromosome A1, as well as a consensus block of homozygosity that spans approximately 3 Mb. Inspection of the region for candidate genes led to the identification of the lysophosphatidic acid receptor 6 (LPAR6). A 4 bp deletion in exon 5, c.250_253_delTTTG, which induces a premature stop codon in the receptor, was identified via Sanger sequencing. The mutation is fixed in Cornish Rex, absent in all straight haired cats analyzed, and is also segregating in the German Rex breed. LPAR6 encodes a G protein-coupled receptor essential for maintaining the structural integrity of the hair shaft; and has mutations resulting in a wooly hair phenotype in humans.