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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.

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.Single amino acid arginine starvation is a new promising approach in the metabolic anticancer therapy. Phase I/II clinical studies showed that pharmacological deprivation of arginine with the recombinant arginine-degrading enzyme, arginine deiminase, was well tolerated by the organism and had antiproliferative effect against some types of tumors, e.g., human hepatocellular carcinomas 1-3 and metastatic melanomas. 4 Another arginine-degrading enzyme, recombinant human arginase I, is currently undergoing Phase I/II clinical trials (http://cme.cancer.gov/drugdictionary/?CdrID¼657226). However, the in vivo approbation and clinical studies of the proposed enzymotherapies revealed that this antitumor approach is not free of drawbacks. The limited spectrum of sensitive tumors seems one of them. It was shown that only tumors deficient in argininosuccinate synthetase (ASS), a rate-limiting enzyme of citrulline to arginine conversion in urea cycle, were sensitive to the treatment with recombinant arginine-degrading enzymes. [5][6][7][8][9] Another obstacle in the arginine deprivation-based anticancer approach is reappearance of therapy-resistant tumor clones due to the derepression of ASS. 10,11 It is assumed that these drawbacks can be overcome by the development of more e...

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.

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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.

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.

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