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.

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.

An MSI tumor specific frameshift mutation in a coding microsatellite of MSH3 encodes for HLA-A0201-restricted CD8+ cytotoxic T cell epitopes.

BACKGROUND: Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI(+) colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. METHODOLOGY/PRINCIPAL FINDINGS: FSP-specific CD8(+) T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI(+) colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. CONCLUSIONS: We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were (386)-FLLALWECSL (FSP18) and (387)-LLALWECSL (FSP19) as well as (403)-IVSRTLLLV (FSP23) and (402)-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI(+)-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or--even more important--preventive purposes.

Semiallogenic fusions of MSI(+) tumor cells and activated B cells induce MSI-specific T cell responses.

BACKGROUND: Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells.Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. METHODS: We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. RESULTS: The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4(+), activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. CONCLUSION: Cellular fusions of MSI(+) carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and this approach should be further analyzed in preclinical as well as clinical trials. Moreover, this is the first report on the induction of frameshift-specific T cell responses without the use of synthetic peptides.

Serum antibodies against frameshift peptides in microsatellite unstable colorectal cancer patients with Lynch syndrome.

High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer or Lynch syndrome. In MSI-H CRC, mismatch repair deficiency leads to insertion/deletion mutations at coding microsatellites and thus to the translation of frameshift peptides (FSPs). FSPs are potent inductors of T cell responses in vitro and in vivo. The present study aims at the identification of FSP-specific humoral immune responses in MSI-H CRC and Lynch syndrome. Sera from patients with history of MSI-H CRC (n = 69), healthy Lynch syndrome mutation carriers (n = 31) and healthy controls (n = 52) were analyzed for antibodies against FSPs using peptide ELISA. Reactivities were measured against FSPs derived from genes frequently mutated in MSI-H CRCs, AIM2, TGFBR2, CASP5, TAF1B, ZNF294, and MARCKS. Antibody reactivity against FSPs was significantly higher in MSI-H CRC patients than in healthy controls (P = 0.036, Mann-Whitney) and highest in patients with shortest interval between tumor resection and serum sampling. Humoral immune responses in patients were most frequently directed against FSPs derived from mutated TAF1B (11.6%, 8/69) and TGFBR2 (10.1%, 7/69). Low level FSP-specific antibodies were also detected in healthy mutation carriers. Our results show that antibody responses against FSPs are detectable in MSI-H CRC patients and healthy Lynch syndrome mutation carriers. Based on the high number of defined FSP antigens, measuring FSP-specific humoral immune responses is a highly promising tool for future diagnostic application in MSI-H cancer patients.