intra-iliac-artery-injection-for-efficient-selective-modeling

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Diabetic peripheral neuropathy (DPN) is a frequent and potentially traumatic complication in diabetic individuals. The chronic nature of diabetes and its associated hyperglycemic episodes initiate a complex and inter-related series of metabolic and vascular insults that contribute to the polygenic etiology of DPN. One contributing factor in DPN is an altered neurotrophism that results from changes in the synthesis and expression of neurotrophins, insulin-like growth factor, and various cytokine-like growth factors that can directly act upon distinct subpopulations of sensory and motor neurons. Although considerable effort has focused upon examining growth factor signaling in hyperglycemically stressed neurons, myelin-forming Schwann cells also undergo substantial degenerative changes in DPN. However, scant attention has been devoted to understanding the effect of hyperglycemia on the response of Schwann cells to growth factors critical to their function. Neuregulins are gliotrophic growth factors that signal through members of the Erb B receptor-tyrosine kinase family. The neuregulin/Erb B ligand-receptor cassette can differentially influence the response of Schwann cells throughout their development by regulating cell survival, mitogenesis, and differentiation. The activity of Erb B receptors may also be affected by their interaction with caveolin-1, a protein marker of caveolae ("little caves"). However, whether neuregulin signaling may be directly or indirectly altered under conditions of hyperglycemic stress and contribute to the physiological progression of DPN is unknown. This brief review will provide a perspective on a putative role of changes in the caveolar proteome of Schwann cells in contributing to an "altered neuregulinism" in DPN.

Altered neurotrophism in diabetic neuropathy: spelunking the caves of peripheral nerve.

Addition of exogenous ceramide causes a significant displacement of cholesterol in lipid raft model membranes. However, whether ceramide-induced cholesterol displacement is sufficient to alter the protein composition of caveolin-enriched lipid raft membranes is unknown. Therefore, we examined whether increasing endogenous ceramide levels with bacterial sphingomyelinase (bSMase) depleted cholesterol and changed the protein composition of caveolin-enriched membranes (CEMs) isolated from immortalized Schwann cells. bSMase increased ceramide levels severalfold and decreased the cholesterol content of detergent-insoluble CEMs by 25-50% within 2 h. To examine the effect of ceramide on the protein composition of the CEMs, we performed a quantitative proteomic analysis using stable isotope labeling of cells in culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although ceramide rapidly depleted lipid raft cholesterol, the levels of the cholesterol binding protein caveolin-1 (Cav-1) decreased by 25% only after 8 h. Importantly, replenishing the cells with cholesterol rapidly reversed the loss of Cav-1 from the CEMs. Ceramide-induced cholesterol depletion increased the association of 5'-nucleotidase and ATP synthase beta-subunit with the CEMs but had a minimal effect on changing the abundance of other lipid raft proteins, such as flotillin-1 and G-proteins. These results suggest that the ceramide-induced loss of cholesterol from CEMs may contribute to altering the lipid raft proteome.

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1.

Neuregulins (NRGs) are growth factors which bind to Erb receptor tyrosine kinases that localize to Schwann cells (SCs). Although NRGs can promote cell survival, mitogenesis, and myelination in undifferentiated SCs, they also induce demyelination of myelinated co-cultures of SCs and dorsal root ganglion (DRG) neurons. We have shown previously that Erb B2 activity increased in premyelinating SCs in response to hyperglycemia, and that this correlated with the downregulation of the protein caveolin-1 (Cav-1). As myelinated SCs undergo substantial degeneration in diabetic neuropathy, we used myelinated SC/DRG neuron co-cultures to determine if hyperglycemia and changes in Cav-1 expression could enhance NRG-induced demyelination. In basal glucose, NRG1 caused a 2.4-fold increase in the number of damaged myelin segments. This damage reached 3.8-fold under hyperglycemic conditions, and was also associated with a robust decrease in the expression of Cav-1 and compact myelin proteins. The loss of Cav-1 and compact myelin proteins following hyperglycemia and NRG treatment was not due to neuronal loss, since the axons remained intact and there was no loss of PGP 9.5, an axonal marker protein. To examine if changes in Cav-1 were sufficient to alter the extent of NRG-induced demyelination, SC/DRG neurons co-cultures were infected with antisense or dominant-negative Cav-1(P132L) adenoviruses. Either antisense-mediated downregulation or mis-localization of endogenous Cav-1 by Cav-1(P132L) resulted in a 1.5- to 2.4-fold increase in NRG-induced degeneration compared to that present in control cultures. These data support that hyperglycemia and changes in Cav-1 are sufficient to sensitize myelinated SC/DRG co-cultures to NRG-induced demyelination.

Hyperglycemia and downregulation of caveolin-1 enhance neuregulin-induced demyelination.

Hyperglycemia-induced mitochondrial dysfunction contributes to sensory neuron pathology in diabetic neuropathy. Although Schwann cells (SCs) also undergo substantial degeneration in diabetic neuropathy, the effect of hyperglycemia on the SC mitochondrial proteome and mitochondrial function has not been examined. Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantify the temporal effect of hyperglycemia on the mitochondrial proteome of primary SCs isolated from neonatal rats. Of 317 mitochondrial proteins identified, about 78% were quantified and detected at multiple time points. Pathway analysis indicated that proteins associated with mitochondrial dysfunction, oxidative phosphorylation, the TCA cycle, and detoxification were significantly increased in expression and over-represented. Assessing mitochondrial respiration in intact SCs indicated that hyperglycemia increased the overall rate of oxygen consumption but decreased the efficiency of coupled respiration. Although a glucose-dependent increase in superoxide production occurs in embryonic sensory neurons, hyperglycemia did not induce a substantial change in superoxide levels in SCs. This correlated with a 1.9-fold increase in Mn superoxide dismutase expression, which was confirmed by immunoblot and enzymatic activity assays. These data support that hyperglycemia alters mitochondrial respiration and can cause remodeling of the SC mitochondrial proteome independent of significant contributions from glucose-induced superoxide production.

Hyperglycemia alters the schwann cell mitochondrial proteome and decreases coupled respiration in the absence of superoxide production.

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice.

Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human data set analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases.

The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.

In breast cancer, the most frequent site of metastasis is bone. Disseminated tumor cells (DTCs) can be detected in the bone marrow of patients by their expression of epithelial oroncogenic markers [1], and the presence and frequency of these DTCs are associated with poor prognosis. However, many of the details behind this process remain elusive, including the biological properties and fates of these apparently indolent cancer cells. To provide pre-clinical models of DTCs, we have developed a procedure that allows for controlled and enhanced delivery of tumor cells to the bone in animal experiments via injection into the iliac artery of the hind limb [2]. To our surprise, we found that most cancer cells became integrated into the solid bone matrix shortly after arriving in the bone, and only a minority can be flushed out with the bone marrow.Here we describe a method that helps to retrieve DTCs homing to the bone in which we achieve an improved recovery of those tumor cells closely associated with the bone microenvironment. In our view it is especially important to analyze these tumor cell subpopulations, as they may take full advantage of growth-, survival- and immune-protective signals provided by neighbor cells. We also show a pilot study on how this approach may be applied to the analysis of cancer dormancy. Our study suggests that the detection and retrieval of DTCs in clinical studies are incomplete because they are conducted exclusively with bone marrow aspirates.

Retrieval of Disseminated Tumor Cells Colonizing the Bone in Murine Breast Cancer Metastasis Models.

We recently discovered that bone micrometastases of breast cancer predominantly reside in the microenvironment termed the "osteogenic niche". The heterotypic adherens junctions between cancer cells and osteogenic cells promote early-stage bone colonization by activating the mTOR pathway in cancer cells. Here, we discuss a few questions raised by these findings.

Article	Year
Altered neurotrophism in diabetic neuropathy: spelunking the caves of peripheral nerve. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 15608075	2005
Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1. Journal of lipid research\| PubMed ID: 15863835	2005
Hyperglycemia and downregulation of caveolin-1 enhance neuregulin-induced demyelination. Glia\| PubMed ID: 18338795	2008
Hyperglycemia alters the schwann cell mitochondrial proteome and decreases coupled respiration in the absence of superoxide production. Journal of proteome research\| PubMed ID: 19905032	2010
Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice. ASN neuro\| PubMed ID: 20711301	2010
The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells. Cancer cell\| PubMed ID: 25600338	2015
Retrieval of Disseminated Tumor Cells Colonizing the Bone in Murine Breast Cancer Metastasis Models. Journal of mammary gland biology and neoplasia\| PubMed ID: 26490041	2015
Devil's Wake: Early-stage bone colonization by breast cancer. Molecular & cellular oncology\| PubMed ID: 27314064	2016

Cuijuan Yu

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