Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

Abstract

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development.

Fig 1. Scheme of culture method.

Schematic illustration of the method of obtaining mixed culture from the gross tumor containing all cell types present in the breast tissue and further purification with differential trypsinization.

Fig 2. Morphology of primary mixed cell cultures.

A-C. Mixed primary culture (organoid) derived from mixed carcinoma, 100x magnification. D-E. Epithelial cells derived by differential trypsinization from adenoma, cultured in Advanced MEM supplemented with mammary epithelial growth supplement (MEGS), 100x magnification. F. Mesenchymal cells (fibroblasts) derived by differential trypsinization from mixed carcinoma, cultured in Advanced MEM supplemented with 10% FBS, 200x magnification. G-I. Organoid derived from normal breast tissue by enzymatic dissociation with collagenase and hyaluronidase IV, 100x magnification.

Fig 3. Cell cycle analysis for cells derived from canine mammary gland lesions.

Cell cycle phases sub G1, G1, S and G2/M and sub G1 are represented. Table below shows values indicating the percentage of the cell population for each phase of the cell cycle. Complex Adenoma (CAd), Mixed Adenoma (MAd), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2), Mixed Carcinoma 1 (MCa1), Mixed Carcinoma 2 (MCa2).

Fig 4. Expression level of mRNA in primary mixed cell cultures obtained from tissue tumor samples.

A. MYH11 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2, ++++ P < 0.05 versus MCa1. B. MUC1 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2, ++++ P < 0.05 versus MCa1. Normal Mammary Epithelium (NME), Complex Adenoma (CAd), Mixed Adenoma (MAd), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2), Mixed Carcinoma 1 (MCa1), Mixed Carcinoma 2 (MCa2).

Fig 5. Expression level of mRNA in primary mixed cell cultures obtained from tissue tumor samples.

A. ERRα where * P < 0.05 versus CAd, ** P < 0.05 versus MAd, *** P < 0.05 versus SCa, + P < 0.05 versus CCa1, ++ P < 0.05 versus MCa2. B. ERRβ where * P < 0.05 versus CAd, ** P < 0.05 versus MAd, *** P < 0.05 versus SCa, + P < 0.05 versus CCa1, ++ P < 0.05 versus CCa2, +++ P < 0.05 versus MCa1, ++++ P < 0.05 versus MCa2. C. ERRγ where * P < 0.05 versus CAd, ** P < 0.05 versus MAd, *** P < 0.05 versus SCa, + P < 0.05 versus CCa1, ++ P < 0.05 versus CCa2, +++ P < 0.05 versus MCa1, ++++ P < 0.05 versus MCa2. D. ERα where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2. Normal Mammary Epithelium (NME), Complex Adenoma (CAd), Mixed Adenoma (MAd), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2), Mixed Carcinoma 1 (MCa1), Mixed Carcinoma 2 (MCa2).

Fig 6. Expression level of mRNA in primary mixed cell cultures obtained from tissue tumor samples.

A. BRCA1 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2. B. BRCA2 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2. C. GATA3 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa2. D. FGFR2 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1. Normal Mammary Epithelium (NME), Complex Adenoma (CAd), Mixed Adenoma (MAd), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2), Mixed Carcinoma 1 (MCa1), Mixed Carcinoma 2 (MCa2).

Fig 7. Proliferation assays in primary mixed cell cultures.

DES and genistein treatment effect on the basal proliferation and proliferation in the presence of CoCl₂ cells derived from NME (A), SCa (B), CCa1 (C) and CCa2 (D). Proliferation was assessed 48 h after treatments. The graphics represent an independent experiment with the mean and standard deviation of triplicates. A. * P < 0.05 versus control, ** P < 0.05 versus CoCl₂, *** P < 0.05 versus DES 10μM, + P < 0.05 versus genistein 0.1μM, ++ P < 0.05 versus genistein + CoCl₂. B. * P < 0.05 versus control. C. * P < 0.05 versus control, ** P < 0.05 versus DES 10μM. D. * P < 0.05 versus control, ** P < 0.05 versus DES 10μM. Normal Mammary Epithelium (NME), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2).