Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.     

Design Maps for the Hyperthermic Treatment of Tumors with Superparamagnetic Nanoparticles

A plethora of magnetic nanoparticles has been developed and investigated under different alternating magnetic fields (AMF) for the hyperthermic treatment of malignant tissues. Yet, clinical applications of magnetic hyperthermia are sporadic, mostly due to the low energy conversion efficiency of the metallic nanoparticles and the high tissue concentrations required. Here, we study the hyperthermic performance of commercially available formulations of superparamagnetic iron oxide nanoparticles (SPIOs), with core diameter of 5, 7 and 14 nm, in terms of absolute temperature increase ΔT and specific absorption rate (SAR). These nanoparticles are operated under a broad range of AMF conditions, with frequency f varying between 0.2 and 30 MHz; field strength H ranging from 4 to 10 kA m⁻¹; and concentration c_MNP varying from 0.02 to 3.5 mg ml⁻¹. At high frequency field (∼30 MHz), non specific heating dominates and ΔT correlates with the electrical conductivity of the medium. At low frequency field (<1 MHz), non specific heating is negligible and the relaxation of the SPIO within the AMF is the sole energy source. We show that the ΔT of the medium grows linearly with c_MNP, whereas the SAR_MNP of the magnetic nanoparticles is independent of c_MNP and varies linearly with f and H². Using a computational model for heat transport in a biological tissue, the minimum requirements for local hyperthermia (T_tissue >42°C) and thermal ablation (T_tissue >50°C) are derived in terms of c_MNP, operating AMF conditions and blood perfusion. The resulting maps can be used to rationally design hyperthermic treatments and identifying the proper route of administration – systemic versus intratumor injection – depending on the magnetic and biodistribution properties of the nanoparticles.     

A Computational Model for Predicting Nanoparticle Accumulation in Tumor Vasculature

Vascular targeting of malignant tissues with systemically injected nanoparticles (NPs) holds promise in molecular imaging and anti-angiogenic therapies. Here, a computational model is presented to predict the development of tumor neovasculature over time and the specific, vascular accumulation of blood-borne NPs. A multidimensional tumor-growth model is integrated with a mesoscale formulation for the NP adhesion to blood vessel walls. The fraction of injected NPs depositing within the diseased vasculature and their spatial distribution is computed as a function of tumor stage, from 0 to day 24 post-tumor inception. As the malignant mass grows in size, average blood flow and shear rates increase within the tumor neovasculature, reaching values comparable with those measured in healthy, pre-existing vessels already at 10 days. The NP vascular affinity, interpreted as the likelihood for a blood-borne NP to firmly adhere to the vessel walls, is a fundamental parameter in this analysis and depends on NP size and ligand density, and vascular receptor expression. For high vascular affinities, NPs tend to accumulate mostly at the inlet tumor vessels leaving the inner and outer vasculature depleted of NPs. For low vascular affinities, NPs distribute quite uniformly intra-tumorally but exhibit low accumulation doses. It is shown that an optimal vascular affinity can be identified providing the proper balance between accumulation dose and uniform spatial distribution of the NPs. This balance depends on the stage of tumor development (vascularity and endothelial receptor expression) and the NP properties (size, ligand density and ligand-receptor molecular affinity). Also, it is demonstrated that for insufficiently developed vascular networks, NPs are transported preferentially through the healthy, pre-existing vessels, thus bypassing the tumor mass. The computational tool described here can effectively select an optimal NP formulation presenting high accumulation doses and uniform spatial intra-tumor distributions as a function of the development stage of the malignancy.     

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil.     

Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress

Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.     

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.     

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus)

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (N_e) are much smaller than those expected in a fishes with large population census sizes (N_c). Estimates of N_e indicate low effective population sizes, even before the population collapse. The ratio N_e/N_e ranged between 10⁻⁶ and 10⁻⁸, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.