Tumor-associated macrophages (TAM) form a major component of the tumor stroma. However, important concepts such as TAM heterogeneity and the nature of the monocytic TAM precursors remain speculative. Here, we show for the first time that mouse mammary tumors contained functionally distinct subsets of TAMs and provide markers for their identification. Furthermore, in search of the TAM progenitors, we show that the tumor-monocyte pool almost exclusively consisted of Ly6ChiCX3CR1low monocytes, which continuously seeded tumors and renewed all nonproliferating TAM subsets. Interestingly, gene and protein profiling indicated that distinct TAM populations differed at the molecular level and could be classified based on the classic (M1) versus alternative (M2) macrophage activation paradigm. Importantly, the more M2-like TAMs were enriched in hypoxic tumor areas, had a superior proangiogenic activity in vivo, and increased in numbers as tumors progressed. Finally, it was shown that the TAM subsets were poor antigen presenters, but could suppress T-cell activation, albeit by using different suppressive mechanisms. Together, our data help to unravel the complexities of the tumor-infiltrating myeloid cell compartment and provide a rationale for targeting specialized TAM subsets, thereby optimally “re-educating” the TAM compartment. Cancer Res; 70(14); 5728–39. ©2010 AACR.

High rates of glucose metabolism and mitochondrial electron transport have been associated with increased mitochondrial production of reactive oxygen species (ROS). This mechanism was also proposed as a possible cause for dysfunction and death of pancreatic β cells exposed to high glucose levels. We examined whether high rates of glucose metabolism increase ROS production in purified rat β cells. Glucose up to 20 mm did not stimulate H₂O₂ or superoxide production, whereas it dose-dependently increased cellular NAD(P)H and FADH₂ levels with an EC₅₀ around 8 mm. On the contrary, glucose concentration-dependently suppressed H₂O₂ and superoxide formation, with a major effect between 0 and 5 mm, parallel to an increase in cellular NAD(P)H levels. This suppressive effect was more marked in β cells with higher NAD(P)H responsiveness to glucose; it was not observed in glucagon-containing α cells, which lacked a glucose-induced increase in NAD(P)H. Suppression was also induced by the mitochondrial substrates leucine and succinate. Experiments with electron transport chain inhibitors indicate a role of respiratory complex I in ROS production at low mitochondrial activity and low NADH levels. Superoxide production at low glucose is potentially cytotoxic, because scavenging by the superoxide dismutase mimetic agent manganese(III)tetrakis(4-benzoic acid)porphyrin was found to reduce the rate of β cell apoptosis. Analysis of islets cultured at 20 mm glucose confirmed that this condition does not induce ROS production in β cells as a result of their increased rates of glucose metabolism. Our study indicates the need of β cells for basal nutrients maintaining mitochondrial NADH production at levels that suppress ROS accumulation from an inadequate respiratory complex I activity and thus inhibit a potential apoptotic pathway.

In this study, we present a new method to obtain pure, viable, freshly isolated hepatic stellate cells. Stellate cells were purified by cell sorting using their high side scatter (SSC) of incident light. Purity of the cells was established by light and transmission electron microscopy (TEM). Starting from stellate cells that were 50% to 70% enriched by centrifugation in 11% Nycodenz, the cell purity after sorting was found to be 96.6% ± 2.9%. Viability of the sorted cells was 90.8% ± 2.2% as measured by the Trypan blue exclusion test and was confirmed by cell culturing. Per hour of sorting, 1.4 ± 0.4 million stellate cells were obtained. Sorting runs of up to 4 hours were practically feasible, resulting in yields of 5 to 6 million cells per rat liver. Cells attached to plastic substratum within 24 hours. Subsequently, they spread and underwent spontaneous transition into myofibroblast-like cells. The purity of sorted cells was documented by reverse-transcriptase polymerase chain reaction (RT-PCR) experiments using specific primer pairs for messenger RNA (mRNA) species that were only present in parenchymal (preproalbumin), endothelial (endothelial cell nitric oxide synthase [eNOS]), stellate (desmin), or Kupffer cells (77- to 88-kd fucose receptor). Contaminating mRNA species were absent in sorted stellate cells. Next, we examined freshly sorted stellate cells by Western blotting to confirm the presence of relevant cytoskeletal proteins. Cells were positive for vimentin, desmin, and glial fibrillary acidic protein (GFAP), but negative for α-smooth muscle actin (α-SMA). Sorted and cultured cells were immunophenotyped for the presence of collagen types I, III, and IV, laminin, and the cytoskeletal proteins, α-SMA, desmin, vimentin, and GFAP. At 90 hours in culture, cells expressed all the investigated extracellular matrix proteins. Desmin was present in 82%± 1%, vimentin in 96%± 2.5%, and GFAP in 91% ± 4.5% of cells. α-SMA was present in 91% ± 2% of cultured cells. We conclude that cell sorting based on SSC of incident light is a convenient method to obtain virtually pure stellate cells that can be used for direct analysis or for culturing. Although the yields obtained with this method are lower than with standard methods, and additional equipment is required, SSC-activated sorting offers the possibility of very pure cells when essential for analyses based on sensitive detection methods such as RT-PCR.

β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20–30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and β-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived β-cells at PT weeks 20–30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation.