Inhibition of Hedgehog Signaling Antagonizes Serous Ovarian Cancer Growth in a Primary Xenograft Model

Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, basal cell, small cell lung, pancreatic, prostate and ovarian. This investigation was designed to determine if inhibition of this pathway could inhibit serous ovarian cancer growth.We utilized an in vivo pre-clinical model of serous ovarian cancer to characterize the anti-tumor activity of Hh pathway inhibitors cyclopamine and a clinically applicable derivative, IPI-926. Primary human serous ovarian tumor tissue was used to generate tumor xenografts in mice that were subsequently treated with cyclopamine or IPI-926.Both compounds demonstrated significant anti-tumor activity as single agents. When IPI-926 was used in combination with paclitaxel and carboplatinum (T/C), no synergistic effect was observed, though sustained treatment with IPI-926 after cessation of T/C continued to suppress tumor growth. Hh pathway activity was analyzed by RT-PCR to assess changes in Gli1 transcript levels. A single dose of IPI-926 inhibited mouse stromal Gli1 transcript levels at 24 hours with unchanged human intra-tumor Gli1 levels. Chronic IPI-926 therapy for 21 days, however, inhibited Hh signaling in both mouse stromal and human tumor cells. Expression data from the micro-dissected stroma in human serous ovarian tumors confirmed the presence of Gli1 transcript and a significant association between elevated Gli1 transcript levels and worsened survival.IPI-926 treatment inhibits serous tumor growth suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may hold promise as a novel therapeutic target, especially in the maintenance setting

ALDH Activity Selectively Defines an Enhanced Tumor-Initiating Cell Population Relative to CD133 Expression in Human Pancreatic Adenocarcinoma

Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends. cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice. phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy

Synchronizing Allelic Effects of Opposing Quantitative Trait Loci Confirmed a Major Epistatic Interaction Affecting Acute Lung Injury Survival in Mice

Increased oxygen (O2) levels help manage severely injured patients, but too much for too long can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and even death. In fact, continuous hyperoxia has become a prototype in rodents to mimic salient clinical and pathological characteristics of ALI/ARDS. To identify genes affecting hyperoxia-induced ALI (HALI), we previously established a mouse model of differential susceptibility. Genetic analysis of backcross and F2 populations derived from sensitive (C57BL/6J; B) and resistant (129X1/SvJ; X1) inbred strains identified five quantitative trait loci (QTLs; Shali1-5) linked to HALI survival time. Interestingly, analysis of these recombinant populations supported opposite within-strain effects on survival for the two major-effect QTLs. Whereas Shali1 alleles imparted the expected survival time effects (i.e., X1 alleles increased HALI resistance and B alleles increased sensitivity), the allelic effects of Shali2 were reversed (i.e., X1 alleles increased HALI sensitivity and B alleles increased resistance). For in vivo validation of these inverse allelic effects, we constructed reciprocal congenic lines to synchronize the sensitivity or resistance alleles of Shali1 and Shali2 within the same strain. Specifically, B-derived Shali1 or Shali2 QTL regions were transferred to X1 mice and X1-derived QTL segments were transferred to B mice. Our previous QTL results predicted that substituting Shali1 B alleles onto the resistant X1 background would add sensitivity. Surprisingly, not only were these mice more sensitive than the resistant X1 strain, they were more sensitive than the sensitive B strain. In stark contrast, substituting the Shali2 interval from the sensitive B strain onto the X1 background markedly increased the survival time. Reciprocal congenic lines confirmed the opposing allelic effects of Shali1 and Shali2 on HALI survival time and provide unique models to identify their respective quantitative trait genes and to critically assess the apparent bidirectional epistatic interactions between these major-effect loci

Mixed-strain housing for female C57BL/6, DBA/2, and BALB/c mice: validating a split-plot design that promotes refinement and reduction

Abstract Background Inefficient experimental designs are common in animal-based biomedical research, wasting resources and potentially leading to unreplicable results. Here we illustrate the intrinsic statistical power of split-plot designs, wherein three or more sub-units (e.g. individual subjects) differing in a variable of interest (e.g. genotype) share an experimental unit (e.g. a cage or litter) to which a treatment is applied (e.g. a drug, diet, or cage manipulation). We also empirically validate one example of such a design, mixing different mouse strains -- C57BL/6, DBA/2, and BALB/c -- within cages varying in degree of enrichment. As well as boosting statistical power, no other manipulations are needed for individual identification if co-housed strains are differentially pigmented, so also sparing mice from stressful marking procedures. Methods The validation involved housing 240 females from weaning to 5 months of age in single- or mixed- strain trios, in cages allocated to enriched or standard treatments. Mice were screened for a range of 26 commonly-measured behavioural, physiological and haematological variables. Results Living in mixed-strain trios did not compromise mouse welfare (assessed via corticosterone metabolite output, stereotypic behaviour, signs of aggression, and other variables). It also did not alter the direction or magnitude of any strain- or enrichment-typical difference across the 26 measured variables, or increase variance in the data: indeed variance was significantly decreased by mixed- strain housing. Furthermore, using Monte Carlo simulations to quantify the statistical power benefits of this approach over a conventional design demonstrated that for our effect sizes, the split- plot design would require significantly fewer mice (under half in most cases) to achieve a power of 80 %. Conclusions Mixed-strain housing allows several strains to be tested at once, and potentially refines traditional marking practices for research mice. Furthermore, it dramatically illustrates the enhanced statistical power of split-plot designs, allowing many fewer animals to be used. More powerful designs can also increase the chances of replicable findings, and increase the ability of small-scale studies to yield significant results. Using mixed-strain housing for female C57BL/6, DBA/2 and BALB/c mice is therefore an effective, efficient way to promote both refinement and the reduction of animal-use in research

The Effects of Breeding Protocol in C57BL/6J Mice on Adult Offspring Behaviour

Animal experiments have demonstrated that a wide range of prenatal exposures can impact on the behaviour of the offspring. However, there is a lack of evidence as to whether the duration of sire exposure could affect such outcomes. We compared two widely used methods for breeding offspring for behavioural studies. The first involved housing male and female C57Bl/6J mice together for a period of time (usually 10–12 days) and checking for pregnancy by the presence of a distended abdomen (Pair-housed; PH). The second involved daily introduction of female breeders to the male homecage followed by daily checks for pregnancy by the presence of vaginal plugs (Time-mated; TM). Male and female offspring were tested at 10 weeks of age on a behavioural test battery including the elevated plus-maze, hole board, light/dark emergence, forced swim test, novelty-suppressed feeding, active avoidance and extinction, tests for nociception and for prepulse inhibition (PPI) of the acoustic startle response. We found that length of sire exposure (LSE) had no significant effects on offspring behaviour, suggesting that the two breeding protocols do not differentially affect the behavioural outcomes of interest. The absence of LSE effects on the selected variables examined does not detract from the relevance of this study. Information regarding the potential influences of breeding protocol is not only absent from the literature, but also likely to be of particular interest to researchers studying the influence of prenatal manipulations on adult behaviour

Co-Housing Rodents with Different Coat Colours as a Simple, Non-Invasive Means of Individual Identification:Validating Mixed-Strain Housing for C57BL/6 and DBA/2 Mice

Standard practice typically requires the marking of laboratory mice so that they can be individually identified. However, many of the common methods compromise the welfare of the individuals being marked (as well as requiring time, effort, and/or resources on the part of researchers and technicians). Mixing strains of different colour within a cage would allow them to be readily visually identifiable, negating the need for more invasive marking techniques. Here we assess the impact that mixed strain housing has on the phenotypes of female C57BL/6 (black) and DBA/2 (brown) mice, and on the variability in the data obtained from them. Mice were housed in either mixed strain or single strain pairs for 19 weeks, and their phenotypes then assessed using 23 different behavioural, morphological, haematological and physiological measures widely used in research and/or important for assessing mouse welfare. No negative effects of mixed strain housing could be found on the phenotypes of either strain, including variables relevant to welfare. Differences and similarities between the two strains were almost all as expected from previously published studies, and none were affected by whether mice were housed in mixed- or single-strain pairs. Only one significant main effect of housing type was detected: mixed strain pairs had smaller red blood cell distribution widths, a measure suggesting better health (findings that now need replicating in case they were Type 1 errors resulting from our multiplicity of tests). Furthermore, mixed strain housing did not increase the variation in data obtained from the mice: the standard errors for all variables were essentially identical between the two housing conditions. Mixed strain housing also made animals very easy to distinguish while in the home cage. Female DBA/2 and C57BL/6 mice can thus be housed in mixed strain pairs for identification purposes, with no apparent negative effects on their welfare or the data they generate. This suggests that there is much value in exploring other combinations of strains