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Establishment and characterization of six canine hepatocellular carcinoma cell lines (Transcriptional changes induced by drug treatment with toceranib or sorafenib)

ABSTRACT: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor in dogs. Although surgical resection is a major treatment option for canine HCC, there are no distinct strategies for unresectable tumor subtypes or adjuvant chemotherapy for tumors with positive margins. We aimed to establish and characterize novel HCC cell lines from canine patients. The cellular morphology, general growth features and tumorigenicity of the established cell lines were evaluated. We also examined the sensitivity of the cell lines to multi-target tyrosine kinase inhibitors (TKIs). Toceranib, a veterinary TKI that targets vascular endothelial growth factor (VEGFR)/platelet-derived growth factor receptor (PDGFR)/c-kit, effectively inhibited the mitogen-activated protein kinase pathway and induced apoptosis. The established canine HCC cell lines showed greater sensitivity to toceranib than to sorafenib, a first-line treatment for human HCC targeting RAF/VEGFR/PDGFR. Sorafenib showed improved anti-tumor effects when co-treated with SCH772984, an extracellular signal-regulated kinase inhibitor. Our study suggests new therapeutic strategies for canine HCC, and these cell lines are valuable research materials for understanding HCC tumor biology in both humans and dogs.

ORGANISM(S): Canis lupus familiaris

PROVIDER: GSE267624 | GEO | 2024/05/20

REPOSITORIES: GEO

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Project description:Proliferation, dedifferentiation, loss of cell-cell contacts and angiogenesis are among the first steps of the metastasis cascade. The complex molecular pathways associated with these events in canine mammary tumors are mostly unknown and the value of this spontaneous canine tumor as a comparative model for human breast cancer is therefore still under debate. Messenger RNA profiles of lymph-node positive canine mammary carcinomas and normal mammary glands of the same dogs were compared by microarray analysis to elucidate molecular pathways associated with metastatic progression. Differential gene expression was analyzed by gene set enrichment and pathway analysis and compared with the gene expression data of human breast cancer. Metastatic canine carcinomas had 1,312 significantly differentially expressed genes when compared to the corresponding normal mammary gland. This expression profile included a significantly increased expression of cell division and extracellular matrix invasion genes (MMP1, MMP11, MMP13, SERPINE1, TFPI2, TIMP3). In contrast, genes associated with epithelial differentiation (EGF, EGFR, KRT17, MAP2K6, STAT5), cell adhesion (CLDN5, CLDN8, CTNNAL1, MCAM, MUC1, PECAM1) and angiogenesis (ANGPT2, ANGPTL1, ANGPTL2, ANGPTL4, FGFR1, FIGF, TIE1) were mostly down-regulated. Interestingly, tumors had a significant decrease in membrane receptors and growth factor pathway gene expression (EGFR, FGFR1, GHR, LYVE1, PDGFR, PDGFR, TGFBR, TIE1), indicating a potential independence from these proliferative stimuli. Several of the identified deregulated pathways overlap with gene expression profiles of human breast cancer. Gene expression profiling of metastatic carcinomas therefore identified complex molecular pathways and functional gene families that are deregulated during malignant progression in the tumors. This study provides new insights into canine mammary tumor metastasis and suggests that canine mammary tumors may serve as a valuable model for the human disease. 13 simple mammary carcinomas with lymph node metastases at the time of tumor resection were compared with the non-neoplastic mammary gland of the same dogs. Sufficient amounts of normal mammary gland were not available for dogs no. 1 (822) and 9 (61). In addition, one normal sample without matching tumor sample was analyzed (no. 8; 2609). All patients had no radiographically detectable pulmonary metastases at the time of tumor resection. Postoperative survival was analyzed by bi-yearly telephone interviews with the owners or the attending veterinarian. Distant metastases as the cause of death were determined postoperatively by radiographic detection of metastases (nos. 1-7, 9-12) or necropsy (nos. 13 and 14). Necropsy was not authorized by the owners of dogs nos. 1-7 and 9-12. Tissue specimens of tumors and lymph node metastases were fixed in neutral-buffered 4% formalin or snap frozen in liquid nitrogen within 15 minutes after resection and stored at -80ºC until further use. Formalin-fixed tumor tissue samples were routinely embedded in paraffin and sections of 2-μm thickness were mounted on adhesive glass slides and stained with hematoxylin and eosin. Tumors and lymph nodes were evaluated histologically independently by two board-certified pathologists, following the criteria of the WHO classification of canine mammary tumors (Misdorp et al. 1999).

Project description:OBJECTIVE: Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterize the role of tumor-initiating cells (T-ICs) and signaling pathways involved in sorafenib resistance. DESIGN: HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: 1) Role of T-ICs by in vitro sphere formation and in vivo tumorigenesis assays using NOD/SCID mice, 2) Activation of alternative signaling pathways and 3) Efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, qRT-PCR) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in 2 independent cohorts. RESULTS: Sorafenib-acquired resistance tumors showed significant enrichment of T-ICs (164 cells needed to create a tumor) vs. sorafenib-sensitive tumors (13400 cells) and non-treated tumors (1292 cells), p<0.001. Tumors with sorafenib-acquired resistance were enriched with IGF and FGF signaling cascades (FDR<0.05). In vitro, cells derived from sorafenib-acquired resistant tumors and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumor growth and improved survival in sorafenib-resistant tumors. A sorafenib-resistance 175-gene signature was characterized by enrichment of progenitor-cell features, aggressive tumoral traits and predicted poor survival in 2 cohorts (n=442 HCC patients). CONCLUSION: Acquired resistance to sorafenib is driven by tumor initiating cells with enrichment of progenitor markers and activation of IGF and FGF signaling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression. Transcriptomic profile of subcutaneous Huh7 cells-derived tumors treated with sorafenib that developed acquired resistance to the drug (n=4), remain responsive to sorafenib (n=3) or were treated with brivanib after development of resistance (n=3). Gene profiling of hepatospheres generated from tumors with acquired resistance to sorafenib (n=3) and non-treated tumors (n=3) was also analyzed.

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Establishment and characterization of six canine hepatocellular carcinoma cell lines (Transcriptional changes induced by drug treatment with toceranib or sorafenib)

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