Zonal image analysis of tumour vascular perfusion, hypoxia, and necrosis

A number of laboratories are utilising both hypoxia and perfusion markers to spatially quantify tumour oxygenation and vascular distributions, and scientists are increasingly turning to automated image analysis methods to quantify such interrelationships. In these studies, the presence of regions of necrosis in the immunohistochemical sections remains a potentially significant source of error. In the present work, frozen MCa-4 mammary tumour sections were used to obtain a series of corresponding image montages. Total vessels were identified using CD31 staining, perfused vessels by DiOC7 staining, hypoxia by EF5/Cy3 uptake, and necrosis by haematoxylin and eosin staining. Our goal was to utilise image analysis techniques to spatially quantitate hypoxic marker binding as a function of distance from the nearest blood vessel. Several refinements to previous imaging methods are described: (1) hypoxia marker images are quantified in terms of their intensity levels, thus providing an analysis of the gradients in hypoxia with increasing distances from blood vessels, (2) zonal imaging masks are derived, which permit spatial sampling of images at precisely defined distances from blood vessels, as well as the omission of necrotic artifacts, (3) thresholding techniques are applied to omit holes in the tissue sections, and (4) distance mapping is utilised to define vascular spacing

Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response

Hypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer. TRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed. Breast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity. TRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxi

Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations

Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l−1 cell dry weight have been achieved with Chlorella, Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising technology

Systematic analysis of 18F-FDG PET and metabolism, proliferation and hypoxia markers for classification of head and neck tumors

Contains fulltext : 136904.pdf (publisher's version ) (Open Access)BACKGROUND: Quantification of molecular cell processes is important for prognostication and treatment individualization of head and neck cancer (HNC). However, individual tumor comparison can show discord in upregulation similarities when analyzing multiple biological mechanisms. Elaborate tumor characterization, integrating multiple pathways reflecting intrinsic and microenvironmental properties, may be beneficial to group most uniform tumors for treatment modification schemes. The goal of this study was to systematically analyze if immunohistochemical (IHC) assessment of molecular markers, involved in treatment resistance, and 18F-FDG PET parameters could accurately distinguish separate HNC tumors. METHODS: Several imaging parameters and texture features for 18F-FDG small-animal PET and immunohistochemical markers related to metabolism, hypoxia, proliferation and tumor blood perfusion were assessed within groups of BALB/c nu/nu mice xenografted with 14 human HNC models. Classification methods were used to predict tumor line based on sets of parameters. RESULTS: We found that 18F-FDG PET could not differentiate between the tumor lines. On the contrary, combined IHC parameters could accurately allocate individual tumors to the correct model. From 9 analyzed IHC parameters, a cluster of 6 random parameters already classified 70.3% correctly. Combining all PET/IHC characteristics resulted in the highest tumor line classification accuracy (81.0%; cross validation 82.0%), which was just 2.2% higher (p = 5.2x10-32) than the performance of the IHC parameter/feature based model. CONCLUSIONS: With a select set of IHC markers representing cellular processes of metabolism, proliferation, hypoxia and perfusion, one can reliably distinguish between HNC tumor lines. Addition of 18F-FDG PET improves classification accuracy of IHC to a significant yet minor degree. These results may form a basis for development of tumor characterization models for treatment allocation purposes