A multi-modal data resource for investigating topographic heterogeneity in patient-derived xenograft tumors.

Patient-derived xenografts (PDXs) are an essential pre-clinical resource for investigating tumor biology. However, cellular heterogeneity within and across PDX tumors can strongly impact the interpretation of PDX studies. Here, we generated a multi-modal, large-scale dataset to investigate PDX heterogeneity in metastatic colorectal cancer (CRC) across tumor models, spatial scales and genomic, transcriptomic, proteomic and imaging assay modalities. To showcase this dataset, we present analysis to assess sources of PDX variation, including anatomical orientation within the implanted tumor, mouse contribution, and differences between replicate PDX tumors. A unique aspect of our dataset is deep characterization of intra-tumor heterogeneity via immunofluorescence imaging, which enables investigation of variation across multiple spatial scales, from subcellular to whole tumor levels. Our study provides a benchmark data resource to investigate PDX models of metastatic CRC and serves as a template for future, quantitative investigations of spatial heterogeneity within and across PDX tumor models

A Wisp3 Cre-knockin Allele Produces Efficient Recombination in Spermatocytes during Early Prophase of Meiosis I

Individuals with the autosomal recessive skeletal disorder Progressive Pseudorheumatoid Dysplasia have loss-of-function mutations in WISP3, and aberrant WISP3 expression has been detected in tumors from patients with colon and breast cancer. In mice however, neither absence nor over-expression of WISP3 was found to cause a phenotype, and endogenous Wisp3 expression has been difficult to detect. To confirm that Wisp3 knockout mice have no phenotype and to identify potential sites of endogenous Wisp3 expression, we generated mice with a knockin allele (Wisp3GFP-Cre) designed to express Green Fluorescent Protein (GFP) and Cre-recombinase instead of WISP3. Heterozygous and homozygous knockin mice were fertile and indistinguishable from their wild-type littermates, confirming that mice lacking Wisp3 have no phenotype. We could not detect GFP-expression from the knockin allele, but we could detect Cre-expression after crossing mice with the knockin allele to Cre-reporter mice; the double heterozygous offspring had evidence of Cre-mediated recombination in several tissues. The only tissue that had high levels of Cre-mediated recombination was the testis, where recombination in spermatocytes occurred by early prophase of meiosis I. As a consequence, males that were double heterozygous for a Wisp3GFP-Cre and a floxed allele only contributed a recombined allele to their offspring. We detected no evidence of Cre-mediated recombination in the female ovary, although when double heterozygous females contributed the reporter allele to their offspring it had recombined ~7% of the time. Wisp3GFP-Cre expression therefore occurs less frequently and most likely at a later stage of oocyte development in female mice compared to male mice. We conclude that although WISP3 is dispensable in mice, male mice with a Wisp3GFP-Cre allele (Jackson Laboratory stock # 017685) will be useful for studying early prophase of meiosis I and for efficiently recombining floxed alleles that are passed to offspring

Selective pattern of motor system damage in gamma-synuclein transgenic mice mirrors the respective pathology in amyotrophic lateral sclerosis

AbstractAmyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system

Induction of Lrp5 HBM-causing mutations in Cathepsin-K expressing cells alters bone metabolism

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, μCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8–17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population

Psychosocial effects of an Ebola outbreak at individual, community and international levels.

The 2013-2016 Ebola outbreak in Guinea, Liberia and Sierra Leone was the worst in history with over 28,000 cases and 11,000 deaths. Here we examine the psychosocial consequences of the epidemic. Ebola is a traumatic illness both in terms of symptom severity and mortality rates. Those affected are likely to experience psychological effects due to the traumatic course of the infection, fear of death and experience of witnessing others dying. Survivors can also experience psychosocial consequences due to feelings of shame or guilt (e.g. from transmitting infection to others) and stigmatization or blame from their communities. At the community level, a cyclical pattern of fear occurs, with a loss of trust in health services and stigma, resulting in disruptions of community interactions and community break down. Health systems in affected countries were severely disrupted and overstretched by the outbreak and their capacities were significantly reduced as almost 900 health-care workers were infected with Ebola and more than 500 died. The outbreak resulted in an increased need for health services, reduced quality of life and economic productivity and social system break down. It is essential that the global response to the outbreak considers both acute and long-term psychosocial needs of individuals and communities. Response efforts should involve communities to address psychosocial need, to rebuild health systems and trust and to limit stigma. The severity of this epidemic and its long-lasting repercussions should spur investment in and development of health systems

Expression of a Degradation‐Resistant β‐Catenin Mutant in Osteocytes Protects the Skeleton From Mechanodeprivation‐Induced Bone Wasting

Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β‐catenin—an intracellular signaling node in the canonical Wnt pathway—in disuse mechanotransduction is not defined. Using the β‐catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen‐inducible, osteocyte‐selective Cre driver, we evaluated the effects of degradation‐resistant β‐catenin on bone properties during disuse. We hypothesized that if β‐catenin plays an important role in Wnt‐mediated osteoprotection, then artificial stabilization of β‐catenin in osteocytes would protect the limbs from disuse‐induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum‐toxin (botox)‐induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (µCT), and histomorphometry. Activation of the βcatCA allele in tail‐suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox‐treated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tail‐suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail‐suspended β‐catenin stabilized mice and tail‐suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β‐catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease

Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre) are active in multiple non-skeletal tissues

In humans, somatic activating mutations in PIK3CA are associated with skeletal overgrowth. In order to determine if activated PI3K signaling in bone cells causes overgrowth, we used Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to somatically activate a disease-causing conditional Pik3ca allele (Pik3caH1047R) in osteoblasts and osteocytes. We observed Tg(BGLAP-Cre);Pik3caH1047R/+ offspring were born at the expected Mendelian frequency. However, these mice developed cutaneous lymphatic malformations and died before 7 weeks of age. In contrast, Tg(DMP1-Cre);Pik3caH1047R/+ offspring survived and had no cutaneous lymphatic malformations. Assuming that Cre-activity outside of the skeletal system accounted for the difference in phenotype between Tg(BGLAP-Cre);Pik3caH1047R/+ and Tg(DMP1-Cre);Pik3caH1047R/+ mice, we developed sensitive and specific droplet digital PCR (ddPCR) assays to search for and quantify rates of Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated recombination in non-skeletal tissues. We observed Tg(BGLAP-Cre)-mediated recombination in several tissues including skin, muscle, artery, and brain; two CNS locations, hippocampus and cerebellum, exhibited Cre-mediated recombination in >5% of cells. Tg(DMP1-Cre)-mediated recombination was also observed in muscle, artery, and brain. Although we cannot preclude that differences in phenotype between mice with Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated PIK3CA activation are due to Cre-recombination being induced at different stages of osteoblast differentiation, differences in recombination at non-skeletal sites are the more likely explanation. Since unanticipated sites of recombination can affect the interpretation of data from experiments involving conditional alleles, we recommend ddPCR as a good first step for assessing efficiency, leakiness, and off-targeting in experiments that employ Cre-mediated or Flp-mediated recombination

Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

Etoposide and other topoisomerase II-targeted drugs are important anticancer therapeutics. Unfortunately, the safe usage of these agents is limited by their indiscriminate induction of topoisomerase II-mediated DNA cleavage throughout the genome and by a lack of specificity toward cancer cells. Therefore, as a first step toward constraining the distribution of etoposide-induced DNA cleavage sites and developing sequence-specific topoisomerase II-targeted anticancer agents, we covalently coupled the core of etoposide to oligonucleotides centered on a topoisomerase II cleavage site in the PML gene. The initial sequence used for this ‘oligonucleotide-linked topoisomerase inhibitor’ (OTI) was identified as part of the translocation breakpoint of a patient with acute promyelocytic leukemia (APL). Subsequent OTI sequences were derived from the observed APL breakpoint between PML and RARA. Results indicate that OTIs can be used to direct the sites of etoposide-induced DNA cleavage mediated by topoisomerase IIα and topoisomerase IIβ. OTIs increased levels of enzyme-mediated cleavage by inhibiting DNA ligation, and cleavage complexes induced by OTIs were as stable as those induced by free etoposide. Finally, OTIs directed against the PML-RARA breakpoint displayed cleavage specificity for oligonucleotides with the translocation sequence over those with sequences matching either parental gene. These studies demonstrate the feasibility of using oligonucleotides to direct topoisomerase II-mediated DNA cleavage to specific sites in the genome