A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

p38 MAPK activation promotes denervated Schwann cell phenotype and functions as a negative regulator of Schwann cell differentiation and myelination.

Physical damage to the peripheral nerves triggers Schwann cell injury response in the distal nerves in an event termed Wallerian degeneration: the Schwann cells degrade their myelin sheaths and dedifferentiate, reverting to a phenotype that supports axon regeneration and nerve repair. The molecular mechanisms regulating Schwann cell plasticity in the PNS remain to be elucidated. Using both in vivo and in vitro models for peripheral nerve injury, here we show that inhibition of p38 mitogen-activated protein kinase (MAPK) activity in mice blocks Schwann cell demyelination and dedifferentiation following nerve injury, suggesting that the kinase mediates the injury signal that triggers distal Schwann cell injury response. In myelinating cocultures, p38 MAPK also mediates myelin breakdown induced by Schwann cell growth factors, such as neuregulin and FGF-2. Furthermore, ectopic activation of p38 MAPK is sufficient to induce myelin breakdown and drives differentiated Schwann cells to acquire phenotypic features of immature Schwann cells. We also show that p38 MAPK concomitantly functions as a negative regulator of Schwann cell differentiation: enforced p38 MAPK activation blocks cAMP-induced expression of Krox 20 and myelin proteins, but induces expression of c-Jun. As expected of its role as a negative signal for myelination, inhibition of p38 MAPK in cocultures promotes myelin formation by increasing the number as well as the length of individual myelin segments. Altogether, our data identify p38 MAPK as an important regulator of Schwann cell plasticity and differentiation.

Soluble neuregulin-1 has bifunctional, concentration-dependent effects on Schwann cell myelination.

Members of the neuregulin-1 (Nrg1) growth factor family play important roles during Schwann cell development. Recently, it has been shown that the membrane-bound type III isoform is required for Schwann cell myelination. Interestingly, however, Nrg1 type II, a soluble isoform, inhibits the process. The mechanisms underlying these isoform-specific effects are unknown. It is possible that myelination requires juxtacrine Nrg1 signaling provided by the membrane-bound isoform, whereas paracrine stimulation by soluble Nrg1 inhibits the process. To investigate this, we asked whether Nrg1 type III provided in a paracrine manner would promote or inhibit myelination. We found that soluble Nrg1 type III enhanced myelination in Schwann cell-neuron cocultures. It improved myelination of Nrg1 type III(+/-) neurons and induced myelination on normally nonmyelinated sympathetic neurons. However, soluble Nrg1 type III failed to induce myelination on Nrg1 type III(-/-) neurons. To our surprise, low concentrations of Nrg1 type II also elicited a similar promyelinating effect. At high doses, however, both type II and III isoforms inhibited myelination and increased c-Jun expression in a manner dependent on Mek/Erk (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) activation. These results indicate that paracrine Nrg1 signaling provides concentration-dependent bifunctional effects on Schwann cell myelination. Furthermore, our studies suggest that there may be two distinct steps in Schwann cell myelination: an initial phase dependent on juxtacrine Nrg1 signaling and a later phase that can be promoted by paracrine stimulation.

Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis.

Peripheral nerve injury is followed by a wave of Schwann cell proliferation in the distal nerve stumps. To resolve the role of Schwann cell proliferation during functional recovery of the injured nerves, we used a mouse model in which injury-induced Schwann cell mitotic response is ablated via targeted disruption of cyclin D1. In the absence of distal Schwann cell proliferation, axonal regeneration and myelination occur normally in the mutant mice and functional recovery of injured nerves is achieved. This is enabled by pre-existing Schwann cells in the distal stump that persist but do not divide. On the other hand, in the wild type littermates, newly generated Schwann cells of injured nerves are culled by apoptosis. As a result, distal Schwann cell numbers in wild type and cyclin D1 null mice converge to equivalence in regenerated nerves. Therefore, distal Schwann cell proliferation is not required for functional recovery of injured nerves.