Inside Lab Invest

The laboratory mouse Mus musculus is attractive as a model system because of an abundance of natural mutants as well as the fact that it is possible to genetically engineer a variety of activating and inactivating mutations into virtually any cell lineage. The model is increasingly being used to test various pharmacologic therapies that may eventually be used in humans. However, there are many differences between mice and humans that need to be taken into account when therapeutic interventions in mice are evaluated.

Cromolyn has been used clinically as an anti-asthma drug for more than 30 years. Its presumed mechanism of function is that it inhibits the release of mast cell granules that cause bronchospasm, leading to its characterization as a “mast cell stabilizer.” Recently, many groups have used cromolyn as a mast cell stabilizer in mice. However, there is evidence that cromolyn might not function the same in mice as it does in other mammals, leading Oka et al to compare the function of cromolyn in mice and rats.

The authors identified several differences between how rat and mouse cells responded to cromolyn, and even between how different mast cell populations within a single species responded. For instance, cromolyn was effective in inhibiting immunoglobulin E–dependent passive cutaneous anaphylaxis in rats but not in mice. Overall, these results highlight the need for caution when drugs with established effects in one mammalian system, such as the rat, are used in a different mammalian system, such as a mouse. As this study makes clear, one cannot assume that drugs will have the same effects in different mammalian model systems.

Osteoclasts are multinucleated cells that are formed from circulating mononuclear phagocyte precursors derived from the bone marrow. Because they play prominent roles in a variety of nonneoplastic and neoplastic diseases, it is important to understand what controls their formation and activity. It is well established that differentiation of osteoclasts from monocyte or macrophage precursors requires the activity of receptor activator of nuclear factor-κB ligand (RANKL). Macrophage–colony-stimulating factor (M-CSF) is also important in osteoclast development because mice with a loss-of-function mutation in the M-CSF gene (op/op mice) have very few osteoclasts. Interestingly, op/op mice undergo spontaneous agedependent recovery of osteoclastogenesis, suggesting that other factors can compensate for loss of M-CSF.

Taylor et al examined several possible factors. They found that vascular endothelial growth factor-A (VEGF-A), VEGF-D, FLT3 ligand, placental growth factor (PIGF), and hepatocyte growth factor (HGF) were all able to compensate—albeit to a lesser degree than M-CSF—by stimulating monocytes to form functional osteoclasts in a RANKL-dependent manner in vitro. Interestingly, the combination of M-CSF, VEGF-A, HGF, and RANKL stimulated the formation of hypernucleated and hyperresorptive osteoclasts as is seen in giant cell tumor of bone (GCTB), a benign bone tumor characterized by an admixture of osteoclasts and mononuclear cells. These growth factors were also identified in the mononuclear cells and osteoclasts in GCTB by immunohistochemistry. Overall, these results suggest that there are redundant growth factor–dependent pathways that can stimulate osteoclast formation from monocytes, and they are consistent with the hypothesis that the mononuclear cells of GCTB express growth factors that promote osteoclast formation.

Conventional light microscopy of formalinfixed, hematoxylin and eosin (H&E)-stained sections is the cornerstone of modern histopathology. It is inconceivable to most pathologists that it will ever be replaced. Previous work with in vivo microscopy yielded vague images that made most pathologists chuckle. However, technology advances in ways that most of us never imagined. Recent advances in technologies known as coherent Raman imaging, including coherent anti– Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering microscopy, enable chemical imaging based on intrinsic vibrational properties of molecules within tissue. Importantly, these technologies offer real-time, high-resolution, subcellular images that do not require tissue staining and can be obtained in vivo. In their most recent studies using CARS microscopy, Freudiger et al offer a state-of-the-art view of this fascinating technology.

Using fresh tissues from normal and tumor-laden mice, the authors produced amazing two- and three-dimensional images of various organs that approach the clarity of traditional H&E-stained images. Remarkably, the technique is based on imaging the vibrational properties of water, lipids, and proteins. They paid particular attention to the brain, for which the ability to image tissues in vivo could avoid unnecessary biopsies or resections of valuable tissue. The authors note that they are developing a handheld scanner for some applications that may be available in the near future but caution that the imaging of hollow organs inside a living patient will require further development of endoscopic instrumentation. Nonetheless, as the results of this study suggest, pathologists should start thinking about the reality of realtime, intraoperative in vivo histology.

Regenerating a functional human heart

As described in a recent letter in Nature, Shiba et al established a novel guinea pig model in which to examine functional aspects of human embryonic stem cell–derived cardiomyocytes (hESC-CMs) transplanted into damaged hearts. They found that the transplanted cells functioned well, attenuating left ventricular dilation after cryoinjury. Importantly, the transplanted hESC-CMs did not result in teratoma formation, and there was a decrease in sustained ventricular tachycardia. Furthermore, the hESC-CM grafts coupled electromechanically to the damaged myocardium, although coupling was heterogeneous. These results demonstrate the potential for using hESC-CMs to replace damaged myocardium, especially given that only 8.4 ± 1.5% of the scar area was remuscularized by hESC-CMs. This guinea pig model will provide a robust platform for refining hESC-CM grafting techniques that can be leveraged to provide hESC-CM-based therapies for human patients with cardiac damage.

Nature, published online 5 August 2012; doi:10.1038/nature11317

Characterization of chemotherapy-resistant glioblastoma cells

Glioblastoma multiforme is an incurable brain tumor due to local recurrence after initial surgery and chemotherapy. To work toward an effective therapy, it is important to identify and characterize the tumor cells that are responsible for resistance and that repopulate the tumor after therapy. As reported in a recent letter in Nature, Chen et al demonstrated that proliferating glioblastoma cells arise from neural stem cells (NSCs) that are able to repopulate the tumors after treatment. NSCs give rise to a relatively quiescent subpopulation of tumor cells that is resistant to temozolomide. Ablation of the glioblastoma cell population derived from NSCs delayed recurrence after therapy. The authors' results show that the NSC-derived glioblastoma cell population has many features that have been proposed for cancer stem cells. Further characterization of these cells should yield important insights into how to eradicate them therapeutically.

Nature, published online 1 August 2012; doi:10.1038/nature11287

Critical role of neutrophils in obesity-related insulin resistance

Adipose tissue inflammation contributes to insulin resistance in type 2 diabetes. A variety of immune cells, including lymphocytes, eosinophils, mast cells, and granulocytes, are known to contribute to insulin resistance. Neutrophils make up the largest fraction of white blood cells and are increased in adipose tissue in mice fed a high-fat diet. Hence, Talukdar et al, as described in a recent letter in Nature Medicine, sought to determine whether neutrophils and neutrophil elastase contribute to the etiology of inflammation-induced insulin resistance. They found that either genetic or pharmacologic depletion of neutrophil elastase decreased adipose tissue neutrophils, reversed insulin resistance, and decreased the adipose tissue proinflammatory milieu in mice with high-fat diet– induced obesity. Mechanistically, they demonstrated that neutrophil elastase activates Toll-like receptor 4, resulting in an increase in proinflammatory factors.

Nature Medicine, published online 5 August 2012; doi:10.1038/nm.2885

Tumor microenvironment contributes to chemotherapeutic resistance

Although cancer therapies are designed to target neoplastic cells, they also affect neighboring nonneoplastic cells in the tumor microenvironment. Because cells in the microenvironment are capable of producing proteins that interact with cancer cells, they have the potential to influence all facets of tumor biology. In a study recently published in Nature Medicine, Sun et al tested the hypothesis that treatment-associated DNA damage responses in nonneoplastic cells in the tumor microenvironment might promote therapeutic resistance. They demonstrated that DNA damage to fibroblasts in the microenvironment led to production and secretion of WNT16B in a nuclear factor-κB–dependent manner, which activated the β-catenin pathway in neoplastic cells in a paracrine fashion to promote cell survival. These experiments suggest that targeting the WNT16B secretion pathway in the tumor microenvironment might decrease therapeutic resistance to chemotherapy.

Nature Medicine, published online 5 August 2012; doi:10.1038/nm.2890