TiHo-0906: a new feline mammary cancer cell line with molecular, morphological, and immunocytological characteristics of epithelial to mesenchymal transition

Abstract

Feline mammary carcinomas (FMCs) with anaplastic and malignant spindle cells histologically resemble the human metaplastic breast carcinoma (hMBC), spindle-cell subtype. hMBCs display epithelial-to-mesenchymal transition (EMT) characteristics. Herein we report the establishment and characterization of a cell line (TiHoCMglAdcar0906; TiHo-0906) exhibiting EMT-like properties derived from an FMC with anaplastic and malignant spindle cells. Copy-number variations (CNVs) by next-generation sequencing and immunohistochemical characteristics of the cell line and the tumour were compared. The absolute qPCR expression of EMT-related markers HMGA2 and CD44 was determined. The growth, migration, and sensitivity to doxorubicin were assessed. TiHo-0906 CNVs affect several genomic regions harbouring known EMT-, breast cancer-, and hMBCs-associated genes as AKT1, GATA3, CCND2, CDK4, ZEB1, KRAS, HMGA2, ESRP1, MTDH, YWHAZ, and MYC. Most of them were located in amplified regions of feline chromosomes (FCAs) B4 and F2. TiHo-0906 cells displayed an epithelial/mesenchymal phenotype, and high HMGA2 and CD44 expression. Growth and migration remained comparable during subculturing. Low-passaged cells were two-fold more resistant to doxorubicin than high-passaged cells (IC50: 99.97 nM, and 41.22 nM, respectively). The TiHo-0906 cell line was derived from a poorly differentiated cellular subpopulation of the tumour consistently displaying EMT traits. The cell line presents excellent opportunities for studying EMT on FMCs.

Figure 1

Histopathology. Tumour paraffin sections, H&E. The neoplasm, of which the cell line TiHo-0906 is derived, was characterized by (a) tubular, (b) solid anaplastic, and (c) spindle areas.

Figure 2

Cellular morphology. (a) TiHo-0906 P76 cell culture at subconfluence, inverted microscopy. Bipolar to multipolar shaped cells (arrows). (b) TiHo-0906 P76 cell culture at confluence, inverted microscopy. Monolayer of epithelial-like cells characterized by polygonal (circles) to bipolar morphology (arrows). (c) TiHo-0906 P79 pellet; paraffin sections, H&E. Round to polygonal shaped cells characterised by marked anisokaryosis and anisocytosis, atypical mitotic figure (arrow) and large multinucleated syncytia (arrowheads).

Figure 3

Immunohistochemical characteristics of the tumour. (a) E-cad; tubular epithelial cells strongly positive (arrows). (b) E-cad; polygonal cells in solid anaplastic areas of the tumour are negative. (c) E-cad; well-differentiated spindle cells negative. (d) pan-CK; intense cytoplasmic immunostaining by the neoplastic tubular cells (arrows). (e) pan-CK; polygonal cells moderately positive. (f) pan-CK; spindle cells negative. (g) Vim; moderate immunolabeling of neoplastic tubular epithelial cells (arrows). (h) Vim; intense cytoplasmic immunolabeling of the polygonal cells. (i) Vim; spindle cells moderately positive. (j) CD44; neoplastic tubular cells negative (arrows), polygonal cells moderately to intensely positive (arrowheads). (k) CD44; moderate to intense membrane staining of polygonal cells. (l) CD44; spindle cells moderately positive.

Figure 4

Immunohistochemical characteristics of the TiHo-0906 cell line. (a) E-cad, low passage (P8); most cells display a weak cytoplasmic and membranous labelling. (b) E-cad, high passage (P80); weak cytoplasmic and membranous labelling in most cells, some cells display a more intense reaction. (c) pan-CK, low passage (P8); moderate cytoplasmic labelling, some cells display a more intense reaction. (d) pan-CK, high passage (P80); moderate cytoplasmic labeling. (e) Vim, low passage (P8); strong cytoplasmic labelling and numerous positive cells. (f) Vim, high passage (P80); most of the cells are moderately positive, some cells are strongly positive. (g) p63, low passage (P8); moderate nuclear labelling in most of the cells, some nuclei are strongly positive. (h) CD44, low passage (P8); cellular membranes are moderately positive. (i) CD44, high passage (P80); moderate membranous labelling in most of the cells, some cells are strongly positive.

Figure 5

Comparative Circos plots of the original tumour and TiHo-0906 cells at low and high passages. Outer multicolor ring: chromosome location. Inner rings from the outside-in: original tumour, TiHo-0906 P7, and TiHo-0906 P76. Green lines indicate CNGs, and red lines indicate CNLs.

Figure 6

HMGA2 and CD44 qPCR-based expression. Comparative expression of (a) HMGA2 and (b) CD44 in TiHo-0906 cells at low (P8) and high (p80) passage versus selected reference tissues (feline testis, and healthy mammary feline tissue, respectively). Data are displayed as mean (SD); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 7

Cell proliferation, growth curves and scratch assay. (a) BrdU cell proliferation assay of TiHo-0906 at low and high passages, absorbance values expressed as Max V [delta 370–492]. (b) Growth curves of TiHo-0906 at low and high passages, data are shown as mean (SD). (c–j) Scratch assay, TiHo-0906 P76 cell culture at inverted microscopy (10×).

Figure 8

Influence of doxorubicin on metabolic activity of TiHo-0906 cells using MTS-test. (a) Doxorubicin resistance analysis of TiHo-0906 cells at low and high passage. Data are displayed as mean (SD) of metabolically active cells (%). Significance was calculated by comparing doxorubicin-treated versus untreated cells; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Doxorubicin dose-response curves of (b) TiHo-0906 cells at low passage, IC50: 99.97 nM and (c) TiHo-0906 cells at high passage, IC50: 41.22 nM.

Figure 9

Flow cytometric assessment of doxorubicin effects on TiHo-0906 cells. (a) Percentage of intact cells (1^st and 2^nd column, low and high passage; respectively), and cell debris (3^rd and 4^th column, low and high passage; respectively) after incubation with doxorubicin. Cellular debris increases in proportion to the concentration of doxorubicin while intact cells decrease. Notice the higher amount of intact cells compared with cell debris at all doxorubicin concentrations tested. (b) In both passages tested viable cells decrease in a dose-dependent manner. Cells at low passage were more resistant to higher concentrations of doxorubicin (significance bars), except for 1000 nM. (c) The amount of apoptotic cells rises in parallel to the concentration of doxorubicin. Doxorubicin-induced Apoptosis was higher in cells at high passages in almost all concentrations tested (significance bars). (d) Incubation with doxorubicin was not able to give a significant increase of dead cells. Data are displayed as mean (SD); *p < 0.05, **p < 0.01, and ***p < 0.001.