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Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.

Maletzki C, Huehns M, Knapp P, Waukosin N, Klar E, Prall F, Linnebacher M - PLoS ONE (2015)

Bottom Line: Initial DNA fingerprint analysis confirmed identity with the patient in all four cases.These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated).Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib).

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, University of Rostock, Rostock, Germany.

ABSTRACT
Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC) showing CpG island methylator phenotype (CIMP). Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated). Cell lines were of epithelial origin (EpCAM+) with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan). Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib). This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo-) therapeutics helps bringing forward the concept of personalized tumor therapy.

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Secretion profile, growth kinetics and invasive potential of CIMP+ cell lines.(A) Cytokine secretion pattern was quantified by ELISA. Cytokine concentrations were determined by comparison with a standard curve generated from serial dilutions of individual standards. Quantitative analysis of CEA, CA19-9 and IL8 secretion after one, three and six days of culture, respectively. (B) Growth kinetics of cells, counted every 24 hours for five consecutive days using a Neubauer chamber. (C) Cellular invasiveness was examined using a Matrigel®-based Boyden chamber assay. Quantification of cellular invasiveness was estimated by MTT assay. Data are expressed as percentage invasion versus HCT116 cells (= internal positive control). (A-C) Results show the mean + standard deviation of three independent experiments.
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pone.0143194.g005: Secretion profile, growth kinetics and invasive potential of CIMP+ cell lines.(A) Cytokine secretion pattern was quantified by ELISA. Cytokine concentrations were determined by comparison with a standard curve generated from serial dilutions of individual standards. Quantitative analysis of CEA, CA19-9 and IL8 secretion after one, three and six days of culture, respectively. (B) Growth kinetics of cells, counted every 24 hours for five consecutive days using a Neubauer chamber. (C) Cellular invasiveness was examined using a Matrigel®-based Boyden chamber assay. Quantification of cellular invasiveness was estimated by MTT assay. Data are expressed as percentage invasion versus HCT116 cells (= internal positive control). (A-C) Results show the mean + standard deviation of three independent experiments.

Mentions: All cell lines secreted high amounts of CEA in a time-dependent manner (Fig 5A). A comparable pattern was observed for CA19-9. However, this tumor marker was only detectable in supernatants of HROC40 and HROC43 cells (Fig 5A). By contrast, IL-8, an autocrine growth factor, was secreted by all four CIMP+ lines (Fig 5A). None of the cell lines secreted detectable amounts of IL-6, IL-10, TGF-β, or TNF-α.


Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.

Maletzki C, Huehns M, Knapp P, Waukosin N, Klar E, Prall F, Linnebacher M - PLoS ONE (2015)

Secretion profile, growth kinetics and invasive potential of CIMP+ cell lines.(A) Cytokine secretion pattern was quantified by ELISA. Cytokine concentrations were determined by comparison with a standard curve generated from serial dilutions of individual standards. Quantitative analysis of CEA, CA19-9 and IL8 secretion after one, three and six days of culture, respectively. (B) Growth kinetics of cells, counted every 24 hours for five consecutive days using a Neubauer chamber. (C) Cellular invasiveness was examined using a Matrigel®-based Boyden chamber assay. Quantification of cellular invasiveness was estimated by MTT assay. Data are expressed as percentage invasion versus HCT116 cells (= internal positive control). (A-C) Results show the mean + standard deviation of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664421&req=5

pone.0143194.g005: Secretion profile, growth kinetics and invasive potential of CIMP+ cell lines.(A) Cytokine secretion pattern was quantified by ELISA. Cytokine concentrations were determined by comparison with a standard curve generated from serial dilutions of individual standards. Quantitative analysis of CEA, CA19-9 and IL8 secretion after one, three and six days of culture, respectively. (B) Growth kinetics of cells, counted every 24 hours for five consecutive days using a Neubauer chamber. (C) Cellular invasiveness was examined using a Matrigel®-based Boyden chamber assay. Quantification of cellular invasiveness was estimated by MTT assay. Data are expressed as percentage invasion versus HCT116 cells (= internal positive control). (A-C) Results show the mean + standard deviation of three independent experiments.
Mentions: All cell lines secreted high amounts of CEA in a time-dependent manner (Fig 5A). A comparable pattern was observed for CA19-9. However, this tumor marker was only detectable in supernatants of HROC40 and HROC43 cells (Fig 5A). By contrast, IL-8, an autocrine growth factor, was secreted by all four CIMP+ lines (Fig 5A). None of the cell lines secreted detectable amounts of IL-6, IL-10, TGF-β, or TNF-α.

Bottom Line: Initial DNA fingerprint analysis confirmed identity with the patient in all four cases.These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated).Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib).

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, University of Rostock, Rostock, Germany.

ABSTRACT
Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC) showing CpG island methylator phenotype (CIMP). Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated). Cell lines were of epithelial origin (EpCAM+) with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan). Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib). This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo-) therapeutics helps bringing forward the concept of personalized tumor therapy.

Show MeSH
Related in: MedlinePlus