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

Dittfeld, Claudia; Dietrich, Antje; Peickert, Susann; Hering, Sandra; Baumann, Michaël; Grade, Marian; Ried, Thomas; Kunz-Schughart, Leoni A.

doi:10.1016/j.radonc.2009.10.010

Cited by 30 publications

(30 citation statements)

References 69 publications

(96 reference statements)

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“…Routine verification of cell line purity and clonality was detailed earlier for HCT-116 cells. 21 Authenticity of cell lines was confirmed by microsatellite analyses at the Institute of Legal Medicine (Dresden University of Technology) using the commercial multiplex PCR kits Mentype NonaplexQS Twin (Biotype, Germany) and PowerPlex 16 (Promega Corporation, USA). Amplicons were detected by capillary electrophoresis in denaturing polymer POP4 in an ABI 310 sequencer (Perkin-Elmer, USA) according to the manufacturer's instructions.…”

Section: Materials and Methods Cell Lines And Culture Routinesmentioning

confidence: 99%

“…An advanced anti-CD133 staining protocol was applied as described earlier. 21 Briefly, cells were incubated for 30 min in the dark at 41C with a PE (phycoerythrin)-conjugated anti-CD133 antibody (CD133/1-PE or isotype, Miltenyi Biotec) diluted 1:100 in PBS containing 0.5% FCS and 2 mM EDTA followed by signal enhancement via a two-step FASER series (Fluorescence Amplification by Sequential Employment of Reagents) according to the manufacturer's instructions (Miltenyi Biotec). Cell suspensions originated from xenograft tumors were also co-stained with an anti-human CD326-FITC (fluorescein isothiocyanate) antibody (dilution 1:25; Miltenyi Biotec) to discriminate human epithelial and mouse stromal cells.…”

Section: Materials and Methods Cell Lines And Culture Routinesmentioning

confidence: 99%

“…In principle, HT29 cells were seeded and cultured as described in 2-D assays and HCT-116 cells were processed as depicted earlier. 21 For drug treatment, however, cells (400 HT29 or 500 HCT-116) were inoculated in only 1 ml culture medium per well and allowed to adhere over a period of 4 h under standard conditions before 1 ml of conditioned DMEM containing double concentrated chemotherapeutics (Sigma-Aldrich, Germany) was added resulting in final concentrations of 0.1-25 mM for 5-Fluorouracil and 1-50 mM for Oxaliplatin. After an incubation time of 72 h, drugs were removed by 100% medium exchange and cultures were kept in the incubator for the time indicated and processed as detailed previously.…”

Section: Evaluation Of Drug and Radiation Responsementioning

confidence: 99%

“…21 In contrast, HT29 were described by others to show a CD133-related CSC behavior based on small cohort animal experiments 22 and characteristics consistent with those of CSCs are also stated for the CD133 þ subfraction of SW620 cells, although no difference in tumor formation rate but in tumor size was observed. 23 HT29 and SW620 are aneuploid or hyperdiploid, respectively, with a microsatellite-stable, chromosome instable (MSS/CIN) phenotype, which is seen in the majority (about 85%) of spontaneous CRC.…”

mentioning

confidence: 94%

“…20 Interestingly though, established CRC cell lines routinely grown as adherent monolayers under serum-containing conditions show a highly variable CD133 surface expression. 21 The argument that cell lines no longer reflect the original in vivo tumor cell geno-and/or phenotype is legitimate but challenges all previous and ongoing functional studies based on such cancer cell lines notwithstanding that they are tumorigenic in mouse models and have indeed led to numerous insights in cancer cell biology. In addition, primary and secondary anti-tumor treatment testings are not routinely performed with primary cells from patients' material but most frequently relies on established cell lines.…”

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confidence: 99%

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

Peickert

Waurig

Dittfeld

et al. 2012

Laboratory Investigation

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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 þ and CD133 À /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. CD133À /low HT29 cells showed a significantly lower clonogenic survival and reduced spheroid formation capacity than their CD133 þ 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 À /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 þ 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 CD133À -sorted population. And even SW480 cells, classified as a CD133 À 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.

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Section: Materials and Methods Cell Lines And Culture Routinesmentioning

confidence: 99%

Section: Materials and Methods Cell Lines And Culture Routinesmentioning

confidence: 99%

Section: Evaluation Of Drug and Radiation Responsementioning

confidence: 99%

mentioning

confidence: 94%

mentioning

confidence: 99%

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

Peickert

Waurig

Dittfeld

et al. 2012

Laboratory Investigation

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Flow cytometric cell‐based assay to preselect antibody constructs for radionuclide conjugation

et al. 2012

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Radiolabeled antibodies (Abs) are an attractive tool for targeting and delivering particle emitters for therapy or imaging applications. The labeling of Abs with metal radionuclides requires chelating agents and can cause loss of binding to their ligands. The aim of the present approach was to design an easy-handling flow cytometric cell-based assay to evaluate Ab-binding capacity of conjugates of the therapeutic Ab Cetuximab and to verify the most promising candidate in a competitive radioactive binding experiment. The final setup for flow cytometric assessment of cellular binding capacities of epidermal growth factor receptor (EGFR)/ErbB1-directed Ab conjugates is based on (a) the selection of a robust cell line model (b) the definition of nonsaturated staining concentrations for the unconjugated reference Ab Cetuximab plus implementation of a reasonable isotype control, and (c) the calculation of relative Ab affinities based on the flow cytometric data. Two (FaDu, SAS) out of the three cell lines with different total and cell surface expression levels of EGFR turned out to be adequate models but the application of one cell line was sufficient to estimate reduced binding capacities of conjugates relative to Cetuximab. Only 1/11 conjugate Abs exhibited a fluorescence signal comparable to unconjugated Cetuximab and was applied for radiolabeling with Yttrium-90. Unaltered binding affinity of this conjugate was proven in a competitive radioactive Ab-binding study. We conclude that the flow cytometric assay is reliable and that the relative binding capacity of

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

Vynnytska-Myronovska

Bobak

Garbe

et al. 2011

Intl Journal of Cancer

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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...

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

Cited by 30 publications

References 69 publications

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

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

Flow cytometric cell‐based assay to preselect antibody constructs for radionuclide conjugation

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

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