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Establishment and characterization of HROC69 - a Crohn´s related colonic carcinoma cell line and its matched patient-derived xenograft.

Kuehn F, Mullins CS, Krohn M, Harnack C, Ramer R, Krämer OH, Klar E, Huehns M, Linnebacher M - Sci Rep (2016)

Bottom Line: They show strong invasive but in comparison weak migratory activity.The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient.They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

View Article: PubMed Central - PubMed

Affiliation: University Medicine Rostock, Department of General-, Thoracic-, Vascular- and Transplantation Surgery, Rostock, Germany.

ABSTRACT
Colitis-associated colorectal cancer (CAC) seems to be a rather unique entity and differs in its genetic alterations, tumour formation capacities, and clinical features from sporadic colorectal carcinoma. Most descriptions about tumour biology of CAC refer to ulcerative colitis; data about Crohn´s colitis related carcinomas are scarce. The majority of patients with Crohn´s disease are under immunosuppression which generates a different environment for tumour growth. We first describe the clinical case of a fast growing CAC in a long-term immunosuppressed patient with Crohn´s disease and successful establishment and characterization of carcinoma cell lines along with their corresponding patient-derived xenograft. Subsequently, these tumor models were molecularly and functionally analysed. Beside numerous chromosomal alterations, mutations in TP53, APC, PTEN and SMAD3 were identified. The cell lines express numerous cancer testis antigens, surface molecules involved in immune evasion but low levels of HLA class I molecules. They show strong invasive but in comparison weak migratory activity. The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient. They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

No MeSH data available.


Related in: MedlinePlus

Migratory potential and invasiveness of HROC69 cells.Invasion and migration of the PDX-derived cell line HROC69 T0 M2 and the patient-derived cell line HROC69 in comparison to the reference cell line HCT116 was analysed. Cells were subjected to migration assay (Migration, black bars) and matrigel invasion assay (Invasion, white bars). For invasion and migration assays, cells were used at a density of 2 × 105 cells per upper Boyden chamber in a 24-well-plate format in 500 μl serum-free DMEM, respectively. In Boyden chamber assays DMEM containing 10% FCS was used as chemoattractant in the companion plate. Subsequently, cells were incubated for 72 hours. Values are means ± SEM of n = 4. ***P < 0.001, vs. HCT116 FL of the respective assay, ANOVA plus post hoc Bonferroni-test.
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f3: Migratory potential and invasiveness of HROC69 cells.Invasion and migration of the PDX-derived cell line HROC69 T0 M2 and the patient-derived cell line HROC69 in comparison to the reference cell line HCT116 was analysed. Cells were subjected to migration assay (Migration, black bars) and matrigel invasion assay (Invasion, white bars). For invasion and migration assays, cells were used at a density of 2 × 105 cells per upper Boyden chamber in a 24-well-plate format in 500 μl serum-free DMEM, respectively. In Boyden chamber assays DMEM containing 10% FCS was used as chemoattractant in the companion plate. Subsequently, cells were incubated for 72 hours. Values are means ± SEM of n = 4. ***P < 0.001, vs. HCT116 FL of the respective assay, ANOVA plus post hoc Bonferroni-test.

Mentions: A cell line from the PDX could again easily be established (HROC69 T0 M2). Both the patient- and PDX-derived cell lines express moderate to high levels of common epithelial cell markers: CD66acde <CD44 < CD15 (Lewis X) <CD26 < CD326 (Fig. 2). With the exception of sialyl-lewis x (CD15), the expression did not differ from that of a sporadic CRC patient-derived cell line (Supplementary figure 1). Some of these markers alone or in variable combinations have repeatedly been described as indicating stem cell related properties2324. Invasion and migration analyses revealed a remarkable difference in the infiltrative activity (when comparing the patient-derived cell line HROC69 and the PDX-derived cell line HROC69 T0 M2; p = 0.0254). Compared to HCT116, which has repeatedly been described as relatively highly-invasive, the PDX-derived HROC69 T0 M2 was slightly less invasive whereas the patient-derived HROC69 tended to be slightly more invasive (Fig. 3). Both PDX-derived HROC69 T0 M2 and the patient-derived HROC69 revealed significantly less migratory activity (through uncoated Boyden chambers) than the HCT116 cell line. The fact that the HROC69 cell lines are relatively immobile but highly invasive implies that they must be very active secretors of proteolytic enzymes.


Establishment and characterization of HROC69 - a Crohn´s related colonic carcinoma cell line and its matched patient-derived xenograft.

Kuehn F, Mullins CS, Krohn M, Harnack C, Ramer R, Krämer OH, Klar E, Huehns M, Linnebacher M - Sci Rep (2016)

Migratory potential and invasiveness of HROC69 cells.Invasion and migration of the PDX-derived cell line HROC69 T0 M2 and the patient-derived cell line HROC69 in comparison to the reference cell line HCT116 was analysed. Cells were subjected to migration assay (Migration, black bars) and matrigel invasion assay (Invasion, white bars). For invasion and migration assays, cells were used at a density of 2 × 105 cells per upper Boyden chamber in a 24-well-plate format in 500 μl serum-free DMEM, respectively. In Boyden chamber assays DMEM containing 10% FCS was used as chemoattractant in the companion plate. Subsequently, cells were incubated for 72 hours. Values are means ± SEM of n = 4. ***P < 0.001, vs. HCT116 FL of the respective assay, ANOVA plus post hoc Bonferroni-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834534&req=5

f3: Migratory potential and invasiveness of HROC69 cells.Invasion and migration of the PDX-derived cell line HROC69 T0 M2 and the patient-derived cell line HROC69 in comparison to the reference cell line HCT116 was analysed. Cells were subjected to migration assay (Migration, black bars) and matrigel invasion assay (Invasion, white bars). For invasion and migration assays, cells were used at a density of 2 × 105 cells per upper Boyden chamber in a 24-well-plate format in 500 μl serum-free DMEM, respectively. In Boyden chamber assays DMEM containing 10% FCS was used as chemoattractant in the companion plate. Subsequently, cells were incubated for 72 hours. Values are means ± SEM of n = 4. ***P < 0.001, vs. HCT116 FL of the respective assay, ANOVA plus post hoc Bonferroni-test.
Mentions: A cell line from the PDX could again easily be established (HROC69 T0 M2). Both the patient- and PDX-derived cell lines express moderate to high levels of common epithelial cell markers: CD66acde <CD44 < CD15 (Lewis X) <CD26 < CD326 (Fig. 2). With the exception of sialyl-lewis x (CD15), the expression did not differ from that of a sporadic CRC patient-derived cell line (Supplementary figure 1). Some of these markers alone or in variable combinations have repeatedly been described as indicating stem cell related properties2324. Invasion and migration analyses revealed a remarkable difference in the infiltrative activity (when comparing the patient-derived cell line HROC69 and the PDX-derived cell line HROC69 T0 M2; p = 0.0254). Compared to HCT116, which has repeatedly been described as relatively highly-invasive, the PDX-derived HROC69 T0 M2 was slightly less invasive whereas the patient-derived HROC69 tended to be slightly more invasive (Fig. 3). Both PDX-derived HROC69 T0 M2 and the patient-derived HROC69 revealed significantly less migratory activity (through uncoated Boyden chambers) than the HCT116 cell line. The fact that the HROC69 cell lines are relatively immobile but highly invasive implies that they must be very active secretors of proteolytic enzymes.

Bottom Line: They show strong invasive but in comparison weak migratory activity.The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient.They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

View Article: PubMed Central - PubMed

Affiliation: University Medicine Rostock, Department of General-, Thoracic-, Vascular- and Transplantation Surgery, Rostock, Germany.

ABSTRACT
Colitis-associated colorectal cancer (CAC) seems to be a rather unique entity and differs in its genetic alterations, tumour formation capacities, and clinical features from sporadic colorectal carcinoma. Most descriptions about tumour biology of CAC refer to ulcerative colitis; data about Crohn´s colitis related carcinomas are scarce. The majority of patients with Crohn´s disease are under immunosuppression which generates a different environment for tumour growth. We first describe the clinical case of a fast growing CAC in a long-term immunosuppressed patient with Crohn´s disease and successful establishment and characterization of carcinoma cell lines along with their corresponding patient-derived xenograft. Subsequently, these tumor models were molecularly and functionally analysed. Beside numerous chromosomal alterations, mutations in TP53, APC, PTEN and SMAD3 were identified. The cell lines express numerous cancer testis antigens, surface molecules involved in immune evasion but low levels of HLA class I molecules. They show strong invasive but in comparison weak migratory activity. The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient. They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

No MeSH data available.


Related in: MedlinePlus