Humanized Mouse Model of Ovarian Cancer Recapitulates Patient Solid Tumor Progression, Ascites Formation, and Metastasis

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγnull (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

A guide to ancient protein studies

Palaeoproteomics is an emerging neologism used to describe the application of mass spectrometry-based approaches to the study of ancient proteomes. As with palaeogenomics (the study of ancient DNA), it intersects evolutionary biology, archaeology and anthropology, with applications ranging from the phylogenetic reconstruction of extinct species to the investigation of past human diets and ancient diseases. However, there is no explicit consensus at present regarding standards for data reporting, data validation measures or the use of suitable contamination controls in ancient protein studies. Additionally, in contrast to the ancient DNA community, no consolidated guidelines have been proposed by which researchers, reviewers and editors can evaluate palaeoproteomics data, in part due to the novelty of the field. Here we present a series of precautions and standards for ancient protein research that can be implemented at each stage of analysis, from sample selection to data interpretation. These guidelines are not intended to impose a narrow or rigid list of authentication criteria, but rather to support good practices in the field and to ensure the generation of robust, reproducible results. As the field grows and methodologies change, so too will best practices. It is therefore essential that researchers continue to provide necessary details on how data were generated and authenticated so that the results can be independently and effectively evaluated. We hope that these proposed standards of practice will help to provide a firm foundation for the establishment of palaeoproteomics as a viable and powerful tool for archaeologists, anthropologists and evolutionary biologists

Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2Rgamma(null) mice.

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rgamma(null) mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45(+) tumor-associated leukocytes within the xenograft are predominantly CD3(+) T cells with fewer CD138(+) plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-gamma in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-gamma and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45(+) cells. The majority of CD45(+) cells were CD3(+) and expressed a phenotype consistent with an effector memory T cell, consisting of CD4(+) or CD8(+) T cells that were CD45RO(+), CD44(+), CD62L(-), and CD25(-). Following adoptive transfer into non-tumor bearing NOD-scid IL2Rgamma(null) mice, these human T cells were found to expand in the spleen, produce IFN-gamma, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rgamma(null) tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods

C/EBPβ^-/- mice exhibit increased susceptibility to oral candidiasis in the context of cortisone-induced immunosuppression.

<p><b>(A)</b> C65BL/6 mice (“WT”) were treated with the indicated doses of cortisone acetate at days -1, +1 and +2 relative to infection. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. SHAM (n = 3), No cortisone control (n = 5), 60mg/kg (n = 9), 120/112mg/kg (n = 8), and 225mg/kg (n = 8). Data are pooled from 2 independent experiments. Bars indicate geometric mean with 95% CI. P<0.05 by t-test with Mann-Whitney correction: * vs NO CORT, ≠ vs 60mg/kg, # vs 120/112mg/kg and π vs 225mg/kg. (<b>B</b>) The indicated mice were infected orally as described in panel A. Cortisone acetate was administered subcutaneously on days -1, +1 and +2 relative to infection. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/+ 60 mg/kg (n = 16), C/EBPβ^+/- 60mg/kg (n = 16), C/EBPβ^-/- 60mg/kg (n = 10) and C/EBPβ^+/+ 225mg/kg (n = 4). <i>P</i><0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. Data are pooled from two independent experiments. <b>C.</b> Representative tongue sections from the indicated mice were stained with H&E or Periodic-acid Schiff (PAS). Scale bar indicates 200 μM. White arrows indicate hyphae.</p

Susceptibility of C/EBPβ^-/- mice to OPC correlates with expression of BD3.

<p><b>(A)</b> mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of <i>Gapdh</i>. Data are pooled from two independent experiments. <i>P</i><0.05 by student unpaired t-test. * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. (<b>B)</b> mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection and analyzed as in panel A. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 2), C/EBPβ^+/- (n = 2), and C/EBPβ^-/- (n = 2). Data are from one experiment. * <i>P</i><0.05 by student unpaired t-test. vs C/EBPβ^+/+, ≠ vs C/EBPβ^+/- and π vs C/EBPβ^-/-. (<b>C)</b> OKF6/TERT2 human oral keratinocytes were treated with 200 ng/ml IL-17 plus 2ng/ml TNFα or with 2×10⁶ HK <i>C</i>. <i>albicans</i> for 24 h. Complementary DNA was prepared and subjected to qPCR analysis to detect <i>DEFB4A</i>. Data are normalized to expression of <i>GAPDH</i> and represent absolute levels. Data are representative of 2 independent experiments. *<i>P</i><0.05 compared to unstimulated OKF6/TERT2 cells.</p

The susceptibility of C/EBPβ^-/- mice to OPC does not correlate with expression of prototypical IL-17-regulated genes.

<p>mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of <i>Gapdh</i>. Data are pooled from 2 independent experiments. <i>P</i><0.05 by student unpaired t-test: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg.</p

C/EBPβ^-/- mice exhibit increased susceptibility to systemic candidiasis but are resistant to oral candidiasis.

<p>(<b>A</b>) The indicated mice (n = 3 per group) were injected with <i>C</i>. <i>albicans</i> in the lateral tail vein. Time to sacrifice is indicated (days). *<i>P</i><0.05 versus (vs) C/EBPβ^+/- mice using Log-rank (Mantel-Cox). (<b>B</b>) The indicated mice were infected sublingually with <i>C</i>. <i>albicans</i> for 75 mins. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. Bars indicate geometric mean with 95% CI. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/- (n = 9), C/EBPβ^-/- (n = 3), C/EBPβ^+/+ plus 225mg/kg cortisone (n = 3); cortisone acetate was administered by subcutaneous injection on days -1, +1 and +2 relative to infection. <i>P</i><0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ WT, ≠ vs C/EBPβ^+/-, # vs C/EBPβ^-/-, π vs C/EBPβ^+/+ 225mg/kg. <b>C</b>. Weights of mice were assessed daily and graphed as percent of starting weight. * vs C/EBPβ^+/+ WT. <i>P</i><0.05 by t-test with Mann-Whitney correction. Experiment was performed once.</p