A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient - PubMed (original) (raw)
. 2021 Apr 23;13(9):2051.
doi: 10.3390/cancers13092051.
Annamaria Massa 2, Francesca Vita 3, Marco Basiricò 2 3, Chiara Raggi 4, Paola Bernabei 5, Paola Ostano 1, Laura Casorzo 6, Mara Panero 6, Francesco Leone 7, Giuliana Cavalloni 2, Massimo Aglietta 2 3
Affiliations
- PMID: 33922695
- PMCID: PMC8123022
- DOI: 10.3390/cancers13092051
A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient
Caterina Peraldo-Neia et al. Cancers (Basel). 2021.
Abstract
Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α-fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.
Keywords: intrahepatic cholangiocarcinoma; new cell line; patient-derived in vitro model.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Immunophenotypic analysis of 82.3 cells. Isotype control: cells incubated with antibody isotype as primary antibody (A). Epithelial cell markers CK19 (99.9%) (B), CK7 (94.5%) (C), and EPCAM (90.3%) (D).
Figure 2
Immunofluorescence staining of CCA markers in 82.3 cells. EPCAM (Alexa Fluor 488) (A), CK-7 (Alexa Fluor 647) (B), and CK-19 (Alexa Fluor 488) (C). Nuclei were counterstained in blue (DAPI) (magnification 40×).
Figure 3
Morphology of 82.3 cells. (A) Cells were able to grow in monolayer and exhibited epithelial morphology (4× magnification); (B) representative images of cholangiospheres of 82.3. Cells were seeded in ultra-low attachment plate and stem cell-serum-free medium. Sphere formation was monitored on days 7, 10, and 14 after seeding (4× magnification).
Figure 4
Expression of stem markers in 82.3 cells. Representative histograms of percentage of 82.3 positive cells for surface (CD24, CD44, CD133, CD49b, and Stro1), and intracellular stem markers (PAX6, FOXA2, OCTA3/4, PDX1, SOX2, and NANOG).
Figure 5
Growth curves of 82.3 cells. Viable cells were counted at 24, 48, 72, and 96 h after seeding (A). ATP production at 24, 48, 72, and 96 h of culture after seeding, obtained by CellTiter GLO® assay of 82.3 cells (B).
Figure 6
Transwell migration assay on 82.3 and HUH28 cells. Cells were seeded on the surface of the migration transwell chamber and separated by a porous membrane. After 48 h of incubation, the membranes were fixed with methanol and stained with crystal violet. The area of the cells that invaded the membrane was calculated using the ImageJ 2 software; five different fields were evaluated. Migration is expressed as the ratio of the mean ± SEM of the area of migrated cells to the area of the same number of cells plated (control). **** p < 0.00001.
Figure 7
Tumor formation in NOD/SCID mice inoculated with 82.3 cells. In vivo tumor growth curve of 82.3 cells (A). Representative images of tumors derived from the injection of 5 × 106 of 82.3 cells (B). Representative histochemistry and immunohistochemistry images for the CK7 expression of explanted tumors acquired at 10× and 20× magnification. Scale bar: 100 µm (C).
Figure 8
Chromosome analysis: Representative karyotype obtained by G-banding of 82.3 cells. Aberrations were described according to the International System for Human Cytogenetic Nomenclature, 2016.
Figure 9
Dose-effect graphs of gemcitabine (A), 5-FU (B), oxaliplatin (C), and carboplatin (D) in 82.3 cells. The cells were treated with the indicated scalar doses of the drugs, and cell growth was evaluated after 72 h of incubation using Cell Titer-Glo® (Promega, Milan, Italy). NT: not treated.
Figure 10
Evaluation of apoptosis after drug treatment in 82.3 and MT-CHC01 cells by Annexin V/Propidium Iodide assay. Cells were treated with the indicated doses of the drugs, and apoptosis was evaluated after 72 h. Flow charts report the percentage of early and late apoptosis (A). Graphs represent the percentage of all apoptotic events in MT-CHC01 (B) and 82.3 (C) cells. NT: not treated. * p < 0.01, ** p < 0.001 and **** p < 0.00001.
Figure 11
The first 20 GO biological processes (level 5) overrepresented within the list of up-regulated transcripts (A). Distribution of all significantly enriched biological processes (_p_-value < 0.01) (B).
Figure 12
The first 20 GO biological processes (level 5) overrepresented within the list of down-regulated transcripts (A). Distribution of all significantly enriched biological processes (_p_-value < 0.01) (B).
References
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