Limits...
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.

Show MeSH

Related in: MedlinePlus

Morphology and phenotype of individual CIMP+ cell lines.(A) Light microscopy of freshly established tumor cell lines (all Passage 15). Cell lines were directly established from patients’ tumor material as described in material and methods. Original magnification ×100. (B) Phenotyping was conducted by flow cytometry using fluorochrome-labeled mAbs as given on the x-axis. (C) MHC class II expression as assessed by flow cytometry with and without IFN-γ (200 IU/mL for 48 hours) pre-treatment. Results are given as the mean % of positive cells + standard deviation of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664421&req=5

pone.0143194.g002: Morphology and phenotype of individual CIMP+ cell lines.(A) Light microscopy of freshly established tumor cell lines (all Passage 15). Cell lines were directly established from patients’ tumor material as described in material and methods. Original magnification ×100. (B) Phenotyping was conducted by flow cytometry using fluorochrome-labeled mAbs as given on the x-axis. (C) MHC class II expression as assessed by flow cytometry with and without IFN-γ (200 IU/mL for 48 hours) pre-treatment. Results are given as the mean % of positive cells + standard deviation of three independent experiments.

Mentions: Light microscopy revealed tight adherence to the bottom of the flasks (Fig 2A). In the early cell culture (< 3 passages), several different epithelial-like cellular clones were observed. At later passage, one cell clone dominated the culture in all cases. Morphologically, all cultures appeared as multi-cellular islands forming aggregates or polygonal cell clusters. All but one of the cell cultures were dominated by a phenotypically small cell clone. The exception is HROC60. In this cell line, flat-shaped adhesive cells with large nuclei, having an irregular size and silhouette were present. HROC60 cells were the only one that grew to complete confluence.


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)

Morphology and phenotype of individual CIMP+ cell lines.(A) Light microscopy of freshly established tumor cell lines (all Passage 15). Cell lines were directly established from patients’ tumor material as described in material and methods. Original magnification ×100. (B) Phenotyping was conducted by flow cytometry using fluorochrome-labeled mAbs as given on the x-axis. (C) MHC class II expression as assessed by flow cytometry with and without IFN-γ (200 IU/mL for 48 hours) pre-treatment. Results are given as the mean % of positive cells + standard deviation of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143194.g002: Morphology and phenotype of individual CIMP+ cell lines.(A) Light microscopy of freshly established tumor cell lines (all Passage 15). Cell lines were directly established from patients’ tumor material as described in material and methods. Original magnification ×100. (B) Phenotyping was conducted by flow cytometry using fluorochrome-labeled mAbs as given on the x-axis. (C) MHC class II expression as assessed by flow cytometry with and without IFN-γ (200 IU/mL for 48 hours) pre-treatment. Results are given as the mean % of positive cells + standard deviation of three independent experiments.
Mentions: Light microscopy revealed tight adherence to the bottom of the flasks (Fig 2A). In the early cell culture (< 3 passages), several different epithelial-like cellular clones were observed. At later passage, one cell clone dominated the culture in all cases. Morphologically, all cultures appeared as multi-cellular islands forming aggregates or polygonal cell clusters. All but one of the cell cultures were dominated by a phenotypically small cell clone. The exception is HROC60. In this cell line, flat-shaped adhesive cells with large nuclei, having an irregular size and silhouette were present. HROC60 cells were the only one that grew to complete confluence.

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