Fig 1.
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).