Tissue requirements for the application of aortic valve neocuspidization - appropriate pericardium properties and homogeneity?

OBJECTIVE: Aortic valve neocuspidization (AVNeo) using autologous pericardium is a promising technique. Expected advantages are reduced immune response, appropriate biomechanics and lower treatment expenses. Nevertheless, autologous pericardium can be affected by patient's age and comorbidities. Usually, glutaraldehyde (GA) - fixed bovine pericardium is the basic material for aortic valve prostheses, easy available and carefully pre-examined in a standardized fabrication process. Aim of the study is the verification of autologous pericardial tissue homogeneity by analysing tissue thickness, biomechanics and extracellular matrix (ECM) composition. METHODS: Segments of human GA-fixed pericardium selected by the surgeon based on visual criteria for cusp pre-cut and remaining after surgical AV replacement were investigated in comparison to bovine standard tissue treated equivalently. Pericardium sampling was performed at up to three positions of each sutured cusp for histological or biomechanical analysis, according to tissue availability. RESULTS AND CONCLUSIONS: Human pericardia exhibited a higher heterogeneity in collagen content, density of vessel structures and elastic moduli. Thickness, vessel density and collagen and elastin content differed significantly between the species. In contrast, significant interindividual differences were detected in most properties investigated for human pericardial samples but only for tissue thickness in bovine tissues. Higher heterogeneity of human pericardium, differing vessel and collagen content compared to bovine state-of-the-art material might be detrimental for long term AV functionality or deterioration and have to be intensely investigated in patients follow up after autologous cusp replacement.

Challenges of aortic valve tissue culture - maintenance of viability and extracellular matrix in the pulsatile dynamic microphysiological system.

BACKGROUND: Calcific aortic valve disease (CAVD) causes an increasing health burden in the 21st century due to aging population. The complex pathophysiology remains to be understood to develop novel prevention and treatment strategies. Microphysiological systems (MPSs), also known as organ-on-chip or lab-on-a-chip systems, proved promising in bridging in vitro and in vivo approaches by applying integer AV tissue and modelling biomechanical microenvironment. This study introduces a novel MPS comprising different micropumps in conjunction with a tissue-incubation-chamber (TIC) for long-term porcine and human AV incubation (pAV, hAV). RESULTS: Tissue cultures in two different MPS setups were compared and validated by a bimodal viability analysis and extracellular matrix transformation assessment. The MPS-TIC conjunction proved applicable for incubation periods of 14-26 days. An increased metabolic rate was detected for pulsatile dynamic MPS culture compared to static condition indicated by increased LDH intensity. ECM changes such as an increase of collagen fibre content in line with tissue contraction and mass reduction, also observed in early CAVD, were detected in MPS-TIC culture, as well as an increase of collagen fibre content. Glycosaminoglycans remained stable, no significant alterations of α-SMA or CD31 epitopes and no accumulation of calciumhydroxyapatite were observed after 14 days of incubation. CONCLUSIONS: The presented ex vivo MPS allows long-term AV tissue incubation and will be adopted for future investigation of CAVD pathophysiology, also implementing human tissues. The bimodal viability assessment and ECM analyses approve reliability of ex vivo CAVD investigation and comparability of parallel tissue segments with different treatment strategies regarding the AV (patho)physiology.

Hemocompatibility tuning of an innovative glutaraldehyde-free preparation strategy using riboflavin/UV crosslinking and electron irradiation of bovine pericardium for cardiac substitutes.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.

Infrared Spectroscopic Verification of a α-Helical Collagen Structure in Glutaraldehyde-Free Crosslinked Bovine Pericardium for Cardiac Implants.

The degeneration of heart valve bioprostheses due to calcification processes is caused by the intercalation of calciumhydroxyapatite in pericardium collagen bundles. Variations of the protein secondary structure of biomaterials according to preparation are relevant for this mineralization process and thus the structural characterization of innovative bioprostheses materials is of great importance. The gold standard for prostheses preparation is glutaraldehyde (GA)-fixation of bovine pericardium that adversely promotes calcification. The novel GA-free SULEEI-treatment of bovine pericardium includes decellularization, UV-crosslinking, and electron beam sterilization. The aim of this study is the structural characterization of SULEEI-treated and GA-fixed bovine pericardium. IR spectroscopic imaging combined with multivariate data and curve fit analysis was applied to investigate the amide I and amide II regions of SULEEI-treated and GA-fixed samples. The spectroscopic images of GA-fixed pericardial tissue exhibited a generally high content of amine groups and side chains providing nucleation points for calcification processes. In contrast, in SULEEI-treated tissue, the typical α-helical structure was retained and was supposed to be less prone to deterioration.

Sterilization and Cross-Linking Combined with Ultraviolet Irradiation and Low-Energy Electron Irradiation Procedure: New Perspectives for Bovine Pericardial Implants in Cardiac Surgery.

BACKGROUND: Bovine pericardium is the major natural source of patches and aortic valve substitutes in cardiac repair procedures. However, long-term tissue durability and biocompatibility issues lead to degeneration (e.g., calcification) that requires reoperation. Tissue preparation strategies, including glutaraldehyde fixation, are reasons for the deterioration of pericardial tissues. We describe a pretreatment procedure involving sterilization and cross-linking combined with ultraviolet (UV) irradiation and low-energy electron irradiation (SULEEI). This innovative, glutaraldehyde-free protocol improves the mechanical aspects and biocompatibility of porcine pericardium patches. METHODS: We adopted the SULEEI protocol, which combines decellularization, sterilization, and cross-linking, along with UV irradiation and low-energy electron irradiation, to pretreat bovine pericardium. Biomechanics, such as ultimate tensile strength and elasticity, were investigated by comparing SULEEI-treated tissue with glutaraldehyde-fixed analogues, clinical patch materials, and an aortic valve substitute. Histomorphological and cellular aspects were investigated by histology, DNA content analysis, and degradability. RESULTS: Mechanical parameters, including ultimate tensile strength, elasticity (Young's modulus), and suture retention strength, were similar for SULEEI-treated and clinically applied bovine pericardium. The SULEEI-treated tissues showed well-preserved histoarchitecture that resembled all pericardial tissues investigated. Fiber density did not differ significantly. DNA content after the SULEEI procedure was reduced to less than 10% of the original tissue material, and more than 50% of the SULEEI-treated pericardium was digested by collagenase. CONCLUSION: The SULEEI procedure represents a new treatment protocol for the preparation of patches and aortic valve prostheses from bovine pericardial tissue. The avoidance of glutaraldehyde fixation may lessen the tissue degeneration processes in cardiac repair patches and valve prostheses.

Brillouin confocal microscopy to determine biomechanical properties of SULEEI-treated bovine pericardium for application in cardiac surgery.

BACKGROUND: Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. OBJECTIVE: BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. METHODS: Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young's modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). RESULTS: SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young's modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. CONCLUSION: BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Use of MALDI Mass Spectrometry Imaging to Identify Proteomic Signatures in Aortic Aneurysms after Endovascular Repair.

Endovascular repair (EVAR) has become the standard procedure in treating thoracic (TAA) or abdominal aortic aneurysms (AAA). Not entirely free of complications, a persisting perfusion of the aneurysm after EVAR, called Endoleak (EL), leads to reintervention and risk of secondary rupture. How the aortic wall responds to the implantation of a stentgraft and EL is mostly uncertain. We present a pilot study to identify peptide signatures and gain new insights in pathophysiological alterations of the aortic wall after EVAR using matrix-assisted laser desorption or ionization mass spectrometry imaging (MALDI-MSI). In course of or accompanying an open aortic repair, tissue sections from 15 patients (TAA = 5, AAA = 5, EVAR = 5) were collected. Regions of interest (tunica media and tunica adventitia) were defined and univariate (receiver operating characteristic analysis) statistical analysis for subgroup comparison was used. This proof-of-concept study demonstrates that MALDI-MSI is feasible to identify discriminatory peptide signatures separating TAA, AAA and EVAR. Decreased intensity distributions for actin, tropomyosin, and troponin after EVAR suggest impaired contractility in vascular smooth muscle cells. Furthermore, inability to provide energy caused by impaired respiratory chain function and continuous degradation of extracellular matrix components (collagen) might support aortic wall destabilization. In case of EL after EVAR, this mechanism may result in a weakened aortic wall with lacking ability to react on reinstating pulsatile blood flow.

Optical coherence tomography and multiphoton microscopy offer new options for the quantification of fibrotic aortic valve disease in ApoE-/- mice.

Aortic valve sclerosis is characterized as the thickening of the aortic valve without obstruction of the left ventricular outflow. It has a prevalence of 30% in people over 65 years old. Aortic valve sclerosis represents a cardiovascular risk marker because it may progress to moderate or severe aortic valve stenosis. Thus, the early recognition and management of aortic valve sclerosis are of cardinal importance. We examined the aortic valve geometry and structure from healthy C57Bl6 wild type and age-matched hyperlipidemic ApoE-/- mice with aortic valve sclerosis using optical coherence tomography (OCT) and multiphoton microscopy (MPM) and compared results with histological analyses. Early fibrotic thickening, especially in the tip region of the native aortic valve leaflets from the ApoE-/- mice, was detectable in a precise spatial resolution using OCT. Evaluation of the second harmonic generation signal using MPM demonstrated that collagen content decreased in all aortic valve leaflet regions in the ApoE-/- mice. Lipid droplets and cholesterol crystals were detected using coherent anti-Stokes Raman scattering in the tissue from the ApoE-/- mice. Here, we demonstrated that OCT and MPM, which are fast and precise contactless imaging approaches, are suitable for defining early morphological and structural alterations of sclerotic murine aortic valves.

Microenvironmentally-driven Plasticity of CD44 isoform expression determines Engraftment and Stem-like Phenotype in CRC cell lines.

Theranostic biomarkers for putative cancer stem-like cells (CSC) in colorectal cancer (CRC) are of particular interest in translational research to develop patient-individualized treatment strategies. Surface proteins still under debate are CD44 and CD133. The structural and functional diversity of these antigens, as well as their plasticity, has only just begun to be understood. Our study aimed to gain novel insight into the plasticity of CD133/CD44, thereby proving the hypothesis of marker-associated tumorigenic and non-tumorigenic phenotypes to be environmentally driven. Methods: CD133/CD44 profiles of 20 CRC cell lines were monitored; three models with distinct surface patterns in vitro were systematically examined. CD133/CD44 subpopulations were isolated by FACS and analyzed upon in vitro growth and/or in limiting dilution engraftment studies. The experimental setup included biomarker analyses on the protein (flow cytometry, Western blotting, immunofluorescence) and mRNA levels (RT-/qPCR) as well as CD44 gene sequencing. Results: In general, we found that (i) the in vitro CD133/CD44 pattern never determined engraftment and (ii) the CD133/CD44 population distributions harmonized under in vivo conditions. The LS1034 cell line appeared as a unique model due to its de novo in vivo presentation of CD44. CD44v8-10 was identified as main transcript, which was stronger expressed in primary human CRC than in normal colon tissues. Biomarker pattern of LS1034 cells in vivo reflected secondary engraftment: the tumorigenic potential was highest in CD133+/CD44+, intermediate in CD133+/CD44- and entirely lost in CD133-/CD44- subfractions. Both CD44+ and CD44- LS1034 cells gave rise to tumorigenic and non-tumorigenic progeny and were convertible - but only as long as they expressed CD133 in vivo. The highly tumorigenic CD133+/CD44(v8-10)+ LS1034 cells were localized in well-oxygenated perivascular but not hypoxic regions. From a multitude of putative modulators, only the direct interaction with stromal fibroblasts triggered an essential, in vivo-like enhancement of CD44v8-10 presentation in vitro. Conclusion: Environmental conditions modulate CD133/CD44 phenotypes and tumorigenic potential of CRC subpopulations. The identification of fibroblasts as drivers of cancer-specific CD44 expression profile and plasticity sheds light on the limitation of per se dynamic surface antigens as biomarkers. It can also explain the location of putative CD133/CD44-positive CRC CSC in the perivascular niche, which is likely to comprise cancer-associated fibroblasts. The LS1034 in vitro/in vivo model is a valuable tool to unravel the mechanism of stromal-induced CD44v8-10 expression and identify further therapeutically relevant, mutual interrelations between microenvironment and tumorigenic phenotype.

Treatment with XAV-939 prevents in vitro calcification of human valvular interstitial cells.

The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the ß-catenin complex and dephosphorylation of ß-catenin. The dephosphorylated active form of ß-catenin (non-phospho-ß-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1µM XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, ß-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-ß-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of ß-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-ß-catenin. Although XAV-939 does not downregulate active ß-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

Subclinical Endocarditis Might be a Hidden Trigger of Early Prosthetic Valve Calcification: A Histological Study.

OBJECTIVE: Despite various improvements in valve prosthetics, early valve deterioration still occurs, leading to prosthetic failure. Studying the early phase of this deterioration is quite difficult, as the prosthesis to be examined is almost always explanted only after extensive deterioration. The objective of this research is to study the pathology of early valve deterioration in an early stage in order to reveal the possible trigger of the process. METHODS: Three cusps of the same type of bovine pericardium valve prosthesis underwent comparative examination. Two cusps (cusps 1 and 2) were retrieved from a valve prosthesis explanted three months post-implantation, and the third cusp was from a non-implanted valve prosthesis and used as a reference cusp (ref. cusp). The examination included macroscopic examination, Non-linear Optical Microscopy using a multiphoton microscope, and histological examination with staining, using Hematoxylin and Eosin, Movat Pentachrome stain, Von-Kossa stain, and Alizirin-Red stain. Parallel sections were decalcified using Osteosoft® solution prior to Von-Kossa and Alizirin-Red staining to exclude false positive results. RESULTS: Macroscopically, cusp 1 showed early deterioration, and cusp 2 showed endocarditic vegetations. Histologically, cusp 1 showed calcifications in acellular deposits on the surface of the cusp, with pathological signs of subacute/healed endocarditis and intact cusp tissue. The examination did not show calcifications of the cellular remnants within the valve tissue. Cusp 2 showed florid endocarditis, with microscopic destruction of the valve tissue. CONCLUSION: Early prosthetic valve deterioration can exist as early as three months post-implantation. Subacute or subclinical endocarditis can be the cause for early valve calcification and deterioration.

Movat Pentachrom stain reveals unexpected high osteogenesis rate in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Aortic valve (AV) stenosis is the most common valvular heart disease with an incidence of 3% for people ≥ 65years in the industrialized world with indication for a surgical or transcatheter valve replacement. Researchers suppose osteogenic processes as key mechanisms in calcific aortic valve stenosis. Recently, Torre et al. published impressive histological analyses and detected osseous and/or chondromatous metaplasia in 15.6% of 6685 native calcified aortic valves. Therefore one HE section per valve originated from the area with the greatest extent of calcification was analyzed. Aim of our experimental setup was to identify regions of neo-osteogenesis and to determine the rate of specimens with active mineralization in human aortic valve tissue by Movat Pentachrom staining of sections of lager tissue segments. METHODS: Operational replaced aortic valves of 35 patients, 15 female and 20 male with an average age of 66.2 years were formalin fixed and decalcified using Osteosoft®-solution. Tissue samples were cut and 2µm specimens were stained with Movat Pentachrom to visualize osteogenic regions. Instead of screening a large number of sections, tissue samples were cut up to five times with at least 100µm space each if no region of osseous and/or chondromatous metaplasia was visible. RESULTS/CONCLUSIONS: Using this setup, a region of osseous metaplasia was detected in 25 (71.4%) of 35 samples analyzed. In some cases, these regions were small sized and only visible due to the bright color of Movat Pentachrom stain. This leads to the suggestion that a higher rate of calcified aortic valve samples would be classified as cusps with areas of neo-osteogenesis after staining with Movat Pentachrom stain and by the systematic analysis of larger parts of the tissue blocks.

Arginine starvation in colorectal carcinoma cells: Sensing, impact on translation control and cell cycle distribution.

Tumor cells rely on a continued exogenous nutrient supply in order to maintain a high proliferative activity. Although a strong dependence of some tumor types on exogenous arginine sources has been reported, the mechanisms of arginine sensing by tumor cells and the impact of changes in arginine availability on translation and cell cycle regulation are not fully understood. The results presented herein state that human colorectal carcinoma cells rapidly exhaust the internal arginine sources in the absence of exogenous arginine and repress global translation by activation of the GCN2-mediated pathway and inhibition of mTOR signaling. Tumor suppressor protein p53 activation and G1/G0 cell cycle arrest support cell survival upon prolonged arginine starvation. Cells with the mutant or deleted TP53 fail to stop cell cycle progression at defined cell cycle checkpoints which appears to be associated with reduced recovery after durable metabolic stress triggered by arginine withdrawal.

Three-dimensional environment renders cancer cells profoundly less susceptible to a single amino acid starvation.

Increased amino acid requirement of malignant cells is exploited in metabolic antitumor therapy, e.g., enzymotherapies based on arginine or methionine deprivation. However, studies on animal models and clinical trials revealed that solid tumors are much less susceptible to single amino acid starvation than could be expected from the in vitro data. We conducted a comparative analysis of the response of several tumor cell lines to single amino acid starvation in 2-D monolayer versus 3-D spheroid culture. We revealed for the first time that in comparison with monolayer culture tumor cells, spheroids are much less susceptible to the deprivation of individual amino acids (i.e., arginine, leucine, lysine or methionine). Accordingly, even after prolonged (up to 10 days) starvation, spheroid cells could readily resume proliferation when appropriate amino acid was resupplemented. In the case of arginine deprivation, similar apoptosis induction was detected both in 2-D and 3-D culture, suggesting that this process does not determine the level of tumor cell sensitivity to this kind of treatment. It was also observed that spheroids much better mimic the in vivo ability of tumor cells to utilize citrulline as arginine precursor for growth in amino acid deficient environment. We conclude that 3-D spheroid culture better reflects in vivo tumor cell response to single amino acid starvation than 2-D monolayer culture and should be used as an integral model in the studies of this type of antitumor metabolic targeting.

CD133 as a biomarker for putative cancer stem cells in solid tumours: limitations, problems and challenges.

The cancer stem cell (CSC) hypothesis, despite the limitations of the currently available models and assays, has ushered in a new era of excitement in cancer research. The development of novel strategies for anti-tumour therapy relies on the use of biomarkers to identify, enrich, and/or isolate the cell population(s) of interest. In this context, various cell characteristics and antigen expression profiles are discussed as surrogate markers. The cell surface expression of the human prominin-1 (CD133) antigen, in particular of the AC133 epitope, is among those that have been most frequently studied in solid cancers, although no mechanism has yet been proposed to link CD133 expression with the CSC phenotype. Some inconsistencies between published data can be ascribed to different analytical tools as well as methodological limitations and pitfalls, highlighted in the present review. Therefore, a comprehensive overview on the current state of knowledge in this growing and exciting field with an emphasis on the most recent studies is presented. We highlight the link between the tumour microenvironment, tumour cell plasticity, and CD133 expression, and evaluate the utility of CD133 expression as a prognostic marker.

Rapid re-expression of CD133 protein in colorectal cancer cell lines in vitro and in vivo.

Studies related to the cancer stem cell hypothesis are challenging because of the imperfect tools to identify cell populations of interest and controversy on the usefulness of established cancer cell lines. We previously found CD133 to not be selective for a tumor-propagating or radioresistant population in a near-diploid, microsatellite-instable colorectal carcinoma (CRC) cell line. Because of discrepant literature data, we herein systematically analyzed the behavior of microsatellite-stable cell line subpopulations reflecting the more frequent carcinogenesis pathway in spontaneous CRC. CD133⁺ and CD133(-/low) populations were isolated by fluorescence-activated cell sorting and further processed. HT29 and SW620 cells were studied in detail in monolayer and/or spheroid culture assays and upon subcutaneous injection in NMRI (nu/nu) mice using a limiting dilution approach. CD133(-/low) HT29 cells showed a significantly lower clonogenic survival and reduced spheroid formation capacity than their CD133⁺ counterparts. However, the cell populations neither differed in growth kinetics and response to treatment in vitro nor in tumor formation capacity when injecting as low as 10 cells. CD133(-/low) HT29 cells rapidly re-expressed CD133 protein in vitro and in vivo as shown by flow cytometry and/or western blot analyses, and they also showed a particular survival benefit under tissue normoxic conditions. In contrast, CD133 protein in the CD133⁺ population was quite stable throughout culturing. The observation of CD133 re-expression and lack of difference in tumor take rate of subpopulations was confirmed in SW620 cells. Here, we found cell density to affect CD133 re-expression in the CD133(-)-sorted population. And even SW480 cells, classified as a CD133⁻ cell line, presented some CD133 protein on their surface upon in vivo engraftment. We conclude that (i) CD133 protein expression shows high plasticity in CRC cell lines, and (ii) in vitro CD133 status on the cell surface neither determines tumorigenic potential nor CD133 profile in vivo.

The SARAH Domain of RASSF1A and Its Tumor Suppressor Function.

The Ras association domain family 1A (RASSF1A) tumor suppressor encodes a Sav-RASSF-Hpo domain (SARAH), which is an interaction domain characterized by hWW45 (dSAV) and MST1/2 (dHpo). In our study, the interaction between RASSF1A and RASSF1C with MST1 and MST2 was demonstrated and it was shown that this interaction depends on the SARAH domain. SARAH domain-deleted RASSF1A had a similar growth-reducing effect as full-length RASSF1A and inhibited anchorage independent growth of the lung cancer cell lines A549 significantly. In cancer cells expressing the SARAH deleted form of RASSF1A, reduced mitotic rates (P = 0.001) with abnormal metaphases (P < 0.001) were observed and a significantly increased rate of apoptosis was found (P = 0.006) compared to full-length RASSF1A. Although the association with microtubules and their stabilization was unaffected, mitotic spindle formation was altered by deletion of the SARAH domain of RASSF1A. In summary, our results suggest that the SARAH domain plays an important role in regulating the function of RASSF1A.

Single amino acid arginine starvation efficiently sensitizes cancer cells to canavanine treatment and irradiation.

Single amino acid arginine deprivation is a promising strategy in modern metabolic anticancer therapy. Its potency to inhibit tumor growth warrants the search for rational chemo- and radio-therapeutic approaches to be co-applied. In this report, we evaluated, for the first time, the efficacy of arginine deprivation as anticancer therapy in three-dimensional (3D) cultures of human tumor cells, and propose a new combinatorial metabolic-chemo-radio-treatment regime based on arginine starvation, low doses of arginine natural analog canavanine and irradiation. A sophisticated experimental setup was designed to evaluate the impact of arginine starvation on four human epithelial cancer cell lines in 2D monolayer and 3D spheroid culture. Radioresponse was assessed in colony formation assays and by monitoring spheroid regrowth probability following single dose irradiation using a standardized spheroid-based test platform. Surviving fraction at 2 Gy (SF(2Gy)) and spheroid control dose(50) (SCD(50) ) were calculated as analytical endpoints. Cancer cells in spheroids are much more resistant to arginine starvation than in 2D culture. Spheroid volume stagnated during arginine deprivation, but even after 10 days of starvation, 100% of the spheroids regrew. Combination treatment, however, was remarkably efficient. In particular, pretreatment of cancer cells with the arginine-degrading enzyme arginase combined with or without low concentration of canavanine substantially enhanced cell radioresponse reflected by a loss in spheroid regrowth probability and SCD(50) values reduced by a factor of 1.5-3. Our data strongly suggest that arginine withdrawal alone or in combination with canavanine is a promising antitumor strategy with potential to enhance cancer cure by irradiation.

Genome and transcriptome profiles of CD133-positive colorectal cancer cells.

Colorectal carcinomas (CRC) might be organized hierarchically and contain a subpopulation of tumorigenic, putative cancer stem cells that are CD133 positive. We studied the biological and genetic characteristics of such cells in CRC cell lines and primary tumors. Three CRC cell lines were sorted in CD133 positive and negative fractions. The respective genetic aberration profiles were studied using array comparative genomic hybridization (aCGH) and expression profiling. Tumorigenicity for each cellular population was tested by injection into nude mice. Additionally, we compared CD133+ and CD133- cells of 12 primary colorectal tumors using laser capture microdissection and aCGH. Three of five CRC cell lines displayed both CD133+ and CD133- cells, but tumorigenicity of these subfractions did not differ significantly and aCGH revealed essentially identical genomic imbalances. However, 96 genes were differentially expressed between the two populations. Array comparative genomic hybridization analysis after laser capture microdissection of CD133+ and CD133- areas in primary colorectal tumors revealed genetic differences in 7 of 12 cases. The use of cell lines for studying genomic alterations that define cancer stem cell characteristics, therefore, seems questionable. In contrast, CD133+ cells in primary cancer samples showed a unique genomic aberration profile. In conclusion, our data suggest that CD133 positivity defines a genetically distinct cellular compartment in primary CRC, which potentially includes tumor initiating cells.

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell line HCT-116.

BACKGROUND AND PURPOSE: CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133+/- subpopulations of one cell line of interest. MATERIALS AND METHODS: Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133+ and CD133- HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice. RESULTS AND CONCLUSIONS: CRC cell lines could be classified into three groups: (i) CD133-, (ii) CD133+ and (iii) those with two distinct CD133+ and CD133- subpopulations. Isolated CD133+/- HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133- HCT-116 showed a striking enrichment in the CD133+ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell line. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.