Inducible generalized activation of hSTING-N154S expression in mice leads to lethal hypercytokinemia: a model for "cytokine storm".

Excessive levels of circulating proinflammatory mediators, known as "hypercytokinemia," that are generated by overwhelming immune system activation can lead to death due to critical organ failure and thrombotic events. Hypercytokinemia has been frequently associated with a variety of infectious and autoimmune diseases, with severe acute respiratory syndrome coronavirus 2 infection currently being the commonest cause, of what has been termed the cytokine storm. Among its various functions within the host, STING (stimulator of interferon genes) is critical in the defense against certain viruses and other pathogens. STING activation, particularly within cells of the innate immune system, triggers potent type I interferon and proinflammatory cytokine production. We thus hypothesized that generalized expression of a constitutively active STING mutant in mice would lead to hypercytokinemia. To test this, a Cre-loxP-based system was used to cause the inducible expression of a constitutively active hSTING mutant (hSTING-N154S) in any tissue or cell type. Herein, we employed a tamoxifen-inducible ubiquitin C-CreERT2 transgenic to obtain generalized expression of the hSTING-N154S protein, thereby triggering the production of IFN-ß and multiple proinflammatory cytokines. This required euthanizing the mice within 3 to 4 d after tamoxifen administration. This preclinical model will allow for the rapid identification of compounds aimed at either preventing or ameliorating the lethal effects of hypercytokinemia.

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).

Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis.

The disruption of polynucleotide kinase/phosphatase (PNKP) in colorectal cancer (CRC) cells deficient in phosphatase and tensin homolog (PTEN) is expected to lead to the loss of cell viability by a process known as synthetic lethality. In previous studies, we have reported on the encapsulation of a novel inhibitor of PNKP, namely, A83B4C63, in polymeric micelles and its activity in slowing the growth of PTEN-deficient CRC cells as well as subcutaneous xenografts. In this study, to enhance drug delivery and specificity to CRC tumors, the surface of polymeric micelles carrying A83B4C63 was modified with GE11, a peptide targeting epidermal growth factor receptor (EGFR) overexpressed in about 70% of CRC tumors. Using molecular dynamics (MD) simulations, we assessed the binding site and affinity of GE11 for EGFR. The GE11-modified micelles, tagged with a near-infrared fluorophore, showed enhanced internalization by EGFR-overexpressing CRC cells in vitro and a trend toward increased primary tumor homing in an orthotopic CRC xenograft in vivo. In line with these observations, the GE11 modification of polymeric micelles was shown to positively contribute to the improved therapeutic activity of encapsulated A83B4C63 against HCT116-PTEN-/- cells in vitro and that of orthotopic CRC xenograft in vivo. In conclusion, our results provided proof of principle evidence for the potential benefit of EGFR targeted polymeric micellar formulations of A83B4C63 as monotherapeutics for aggressive and metastatic CRC tumors but at the same time highlighted the need for the development of EGFR ligands with improved physiological stability and EGFR binding.

Intratumoral STING activation causes durable immunogenic tumor eradication in the KP soft tissue sarcoma model.

Introduction: Soft tissue sarcomas (STS) are highly metastatic, connective-tissue lineage solid cancers. Immunologically, sarcomas are frequently characterized by a paucity of tumor infiltrating lymphocytes and an immune suppressive microenvironment. Activation of the STING pathway can induce potent immune-driven anti-tumor responses within immunogenic solid tumors; however, this strategy has not been evaluated in immunologically cold sarcomas. Herein, we assessed the therapeutic response of intratumoral STING activation in an immunologically cold murine model of undifferentiated pleomorphic sarcoma (UPS). Materials and Results: A single intratumoral injection of the murine STING agonist, DMXAA resulted in durable cure in up to 60% of UPS-bearing mice. In mice with synchronous lung metastases, STING activation within hindlimb tumors resulted in 50% cure in both anatomic sites. Surviving mice all rejected UPS re-challenge in the hindlimb and lung. Therapeutic efficacy of STING was inhibited by lymphocyte deficiency but unaffected by macrophage deficiency. Immune phenotyping demonstrated enrichment of lymphocytic responses in tumors at multiple timepoints following treatment. Immune checkpoint blockade enhanced survival following STING activation. Discussion: These data suggest intratumoral activation of the STING pathway elicits local and systemic anti-tumor immune responses in a lymphocyte poor sarcoma model and deserves further evaluation as an adjunctive local and systemic treatment for sarcomas.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

PNKP is required for maintaining the integrity of progenitor cell populations in adult mice.

DNA repair proteins are critical to the maintenance of genomic integrity. Specific types of genotoxic factors, including reactive oxygen species generated during normal cellular metabolism or as a result of exposure to exogenous oxidative agents, frequently leads to "ragged" single-strand DNA breaks. The latter exhibits abnormal free DNA ends containing either a 5'-hydroxyl or 3'-phosphate requiring correction by the dual function enzyme, polynucleotide kinase phosphatase (PNKP), before DNA polymerase and ligation reactions can occur to seal the break. Pnkp gene deletion during early murine development leads to lethality; in contrast, the role of PNKP in adult mice is unknown. To investigate the latter, we used an inducible conditional mutagenesis approach to cause global disruption of the Pnkp gene in adult mice. This resulted in a premature aging-like phenotype, characterized by impaired growth of hair follicles, seminiferous tubules, and neural progenitor cell populations. These results point to an important role for PNKP in maintaining the normal growth and survival of these murine progenitor populations.

A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.

Nano-Delivery of a Novel Inhibitor of Polynucleotide Kinase/Phosphatase (PNKP) for Targeted Sensitization of Colorectal Cancer to Radiation-Induced DNA Damage.

Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) or solubilized with the aid of Cremophor EL: Ethanol (CE/A83) in human HCT116 colorectal cancer (CRC) models. Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc+/HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [18F]-fluoro-3'-deoxy-3'-L:-fluorothymidine ([18F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [18F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC.

Is Use of BMP-2 Associated with Tumor Growth and Osteoblastic Differentiation in Murine Models of Osteosarcoma?

BACKGROUND: The putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent. QUESTIONS/PURPOSES: (1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma. METHODS: In this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H2O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation. RESULTS: Constitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm versus 1316 ± 387.4 mm, mean difference 1009 mm [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm versus 326.1 ± 72.8 mm, mean difference 326.1 mm [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10 ± 8.3x10 photons/s versus 9.3 x 10 ± 1.5 x 10 photons/s, mean difference 8.6 x 10 photons/s [95% CI 5.1 x 10 to 1.2 x 10]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm versus 1335 ± 102.7 mm, mean difference 840.3 mm [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions. CONCLUSIONS: As demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted. CLINICAL RELEVANCE: Our results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.

Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis.

The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.

Stimulator of interferon genes (STING) activation exacerbates experimental colitis in mice.

Detection of cytoplasmic DNA by the host's innate immune system is essential for microbial and endogenous pathogen recognition. In mammalian cells, an important sensor is the stimulator of interferon genes (STING) protein, which upon activation by bacterially-derived cyclic dinucleotides (cDNs) or cytosolic dsDNA (dsDNA), triggers type I interferons and pro-inflammatory cytokine production. Given the abundance of bacterially-derived cDNs in the gut, we determined whether STING deletion, or stimulation, acts to modulate the severity of intestinal inflammation in the dextran sodium sulphate (DSS) model of colitis. DSS was administered to Tmem173gt (STING-mutant) mice and to wild-type mice co-treated with DSS and a STING agonist. Colitis severity was markedly reduced in the DSS-treated Tmem173gt mice and greatly exacerbated in wild-type mice co-treated with the STING agonist. STING expression levels were also assessed in colonic tissues, murine bone marrow derived macrophages (BMDMs), and human THP-1 cells. M1 and M2 polarized THP-1 and murine BMDMs were also stimulated with STING agonists and ligands to assess their responses. STING expression was increased in both murine and human M1 polarized macrophages and a STING agonist repolarized M2 macrophages towards an M1-like subtype. Our results suggest that STING is involved in the host's response to acutely-induced colitis.

Simple and robust polymer-based sensor for rapid cancer detection using serum.

We report a polymer-based sensor that rapidly detects cancer based on changes in serum protein levels. Using three ratiometric fluorescence outputs, this simple system identifies early stage and metastatic lung cancer with a high level of accuracy exceeding many biomarker-based assays, making it an attractive strategy for point-of-care testing.

Recombinant human PRG4 (rhPRG4) suppresses breast cancer cell invasion by inhibiting TGFß-Hyaluronan-CD44 signalling pathway.

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFß) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFß-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFß-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFß has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFß has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFß actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.

Expression of a constitutively active human STING mutant in hematopoietic cells produces an Ifnar1-dependent vasculopathy in mice.

STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disorder characterized by blood vessel occlusions, acral necrosis, myositis, rashes, and pulmonary inflammation that are the result of activating mutations in the STimulator of Interferon Genes (STING). We generated a transgenic line that recapitulates many of the phenotypic aspects of SAVI by targeting the expression of the human STING-N154S-mutant protein to the murine hematopoietic compartment. hSTING-N154S mice demonstrated failure to gain weight, lymphopenia, progressive paw swelling accompanied by inflammatory infiltrates, severe myositis, and ear and tail necrosis. However, no significant lung inflammation was observed. X-ray microscopy imaging revealed vasculopathy characterized by arteriole occlusions and venous thromboses. Type I interferons and proinflammatory mediators were elevated in hSTING-N154S sera. Importantly, the phenotype was prevented in hSTING-N154S mice lacking the type I interferon receptor gene (Ifnar1). This model, based on a mutant human STING protein, may shed light on the pathophysiological mechanisms operative in SAVI.

Aged polymorphonuclear leukocytes cause fibrotic interstitial lung disease in the absence of regulation by B cells.

Pulmonary immunity requires tight regulation, as interstitial inflammation can compromise gas exchange and lead to respiratory failure. Here we found a greater number of aged CD11bhiL-selectinloCXCR4+ polymorphonuclear leukocytes (PMNs) in lung vasculature than in the peripheral circulation. Using pulmonary intravital microscopy, we observed lung PMNs physically interacting with B cells via ß2 integrins; this initiated neutrophil apoptosis, which led to macrophage-mediated clearance. Genetic deletion of B cells led to the accumulation of aged PMNs in the lungs without systemic inflammation, which caused pathological fibrotic interstitial lung disease that was attenuated by the adoptive transfer of B cells or depletion of PMNs. Thus, the lungs are an intermediary niche in the PMN lifecycle wherein aged PMNs are regulated by B cells, which restrains their potential to cause pulmonary pathology.

Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.

Characterization of a murine model of metastatic human non-small cell lung cancer and effect of CXCR4 inhibition on the growth of metastases.

Despite successful preclinical testing carried out through the use of subcutaneous xenografted tumors, many anti-cancer agents have gone on to fail in human trials. One potential factor accounting for this discrepancy may relate to the inadequacy of the commonly employed preclinical models to recapitulate the human disease, particularly when it comes to discovery of agents that are effective against advanced disease. Herein, we report the characterization of a NSCLC model and an exploration of the impact that a CXCR4 inhibitor, AMD3100, had on NCI-H1299-derived metastasis. These cells express a variety of metastasis-promoting factors, hence we selected them for a study of their metastatic colonization potential. To accomplish this, luciferase-expressing H1299 (H1299-luc2) cells were inoculated into athymic mice via the intracardiac route. This strategy produced adrenal, bone, ovarian, and pancreatic metastases, sites commonly involved in human metastatic NSCLC. Notably, micro-computed tomography and histological evaluation of the skeletal lesions revealed the presence of extensive osteolysis. To investigate the potential role of CXCR4 in mediating metastatic colonization of tissues, AMD3100 was administered to mice inoculated with H1299-luc2 cells. While this treatment did not appreciably alter the frequency of metastatic colonization, it was able to slow the growth of macrometastases. This model, recapitulating some of the events seen in late-stage human NSCLC, may prove useful in the evaluation of new therapies targeting metastatic disease.

DNA mismatch repair deficiency accelerates lung neoplasm development in K-ras(LA1/+) mice: a brief report.

Inherited as well as acquired deficiencies in specific DNA mismatch repair (MMR) components are associated with the development of a wide range of benign and malignant neoplasms. Loss of key members such as MSH2 and MLH1 severely cripples the ability of the cell to recognize and correct such lesions as base:base mismatches and replicative DNA polymerase errors such as slippages at repetitive sequences. Genomic instability resulting from MMR deficiency not only predisposes cells to malignant transformation but may also promote tumor progression. To test the latter, we interbred Msh2(-/-) mice with the K-ras(LA1/+) transgenic line that spontaneously develops a range of premalignant and malignant lung lesions. Compared to K-ras(LA1/+) mice, K-ras(LA1/+); Msh2(-/-) mice developed lung adenomas and adenocarcinomas at an increased frequency and also demonstrated evidence of accelerated adenocarcinoma growth. Since MMR defects have been identified in some human lung cancers, the mutant mice may not only be of preclinical utility but they will also be useful in identifying gene alterations able to act in concert with Kras mutants to promote tumor progression.

Mind the gap: genetic manipulation of basicranial growth within synchondroses modulates calvarial and facial shape in mice through epigenetic interactions.

Phenotypic integration patterns in the mammalian skull have long been a focus of intense interest as a result of their suspected influence on the trajectory of hominid evolution. Here we test the hypothesis that perturbation of cartilage growth, which directly affects only the chondrocranium during development, will produce coordinated shape changes in the adult calvarium and face regardless of mechanism. Using two murine models of cartilage undergrowth that target two very different mechanisms, we show that strong reduction in cartilage growth produces a short, wide, and more flexed cranial base. This in turn produces a short, wide face in both models. Cranial base and face are already correlated early in ontogeny, and the relationship between these modules gains structure through postnatal growth and development. These results provide further evidence that there exist physical interactions between developing parts of the phenotype that produce variation at a distance from the actual locus upon which a particular selective pressure is acting. Phenotypic changes observed over the course of evolution may not all require adaptationist explanations; rather, it is likely that a substantial portion of observed phenotypic variation over the history of a clade is not directly adaptive but rather a secondary consequence of some local response to selection.