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Establishment of primary cell cultures from canine mammary gland malignant tumours: a preliminary study

Patŕicia Petrouškova

Patŕicia Petrouškova

Department of Epizootiology, Parasitology and Protection of One Health, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
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Petrouškova, Patŕicia
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Nikola Hudáková

Nikola Hudáková

Small Animal Clinic, University Veterinary Hospital, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
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Hudáková, Nikola
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Viera Almášiová

Viera Almášiová

Department of Morphological Disciplines, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
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Almášiová, Viera
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Alexandra Valenčáková

Alexandra Valenčáková

Small Animal Clinic, University Veterinary Hospital, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
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Valenčáková, Alexandra
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L’ubica Horňáková

L’ubica Horňáková

Small Animal Clinic, University Veterinary Hospital, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
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Horňáková, L’ubica
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Mykhailo Huniadi

Mykhailo Huniadi

Small Animal Clinic, University Veterinary Hospital, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
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Huniadi, Mykhailo
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Daša Čížková

Daša Čížková

  • Small Animal Clinic, University Veterinary Hospital, University of Veterinary Medicine and Pharmacy in KošiceKošice, Slovak Republic
  • Institute of Neuroimmunology, Slovak Academy of Sciences v.v.i.Slovak Republic
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Čížková, Daša
  
01. März 2025
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Online veröffentlicht: 01. März 2025
Seitenbereich: 159 - 168
Eingereicht: 17. Juli 2024
Akzeptiert: 10. Feb. 2025
DOI: https://doi.org/10.2478/jvetres-2025-0007
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© 2025 Patŕicia Petrouškova et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Fig. 1.

Microphotographs of mammary gland tumours. A – magnification 200×, scale bar 50 μm; B – magnification 400×, scale bar 20 μm
Microphotographs of mammary gland tumours. A – magnification 200×, scale bar 50 μm; B – magnification 400×, scale bar 20 μm

Fig. 2.

Primary cell cultures derived from mammary gland solid adenocarcinoma and carcinosarcoma. A and G – cellular debris and blood cells present directly after plating the cells on day in vitro (DIV) 0; B and H – cells before washing on DIV 1; C and I – cells after washing on DIV 1. Washing the cells effectively removed the majority of cellular debris and non-adherent cells; D and J – enlarged irregular and star-shaped cells on DIV 3; E and K – larger polygonal and irregularly shaped cells on DIV 5; F and L – cells with a range of morphologies from spindle-shaped to polygonal-shaped on DIV 7. Magnification 200×, scale bar 50 μm
Primary cell cultures derived from mammary gland solid adenocarcinoma and carcinosarcoma. A and G – cellular debris and blood cells present directly after plating the cells on day in vitro (DIV) 0; B and H – cells before washing on DIV 1; C and I – cells after washing on DIV 1. Washing the cells effectively removed the majority of cellular debris and non-adherent cells; D and J – enlarged irregular and star-shaped cells on DIV 3; E and K – larger polygonal and irregularly shaped cells on DIV 5; F and L – cells with a range of morphologies from spindle-shaped to polygonal-shaped on DIV 7. Magnification 200×, scale bar 50 μm

Fig. 3.

Immunofluorescence staining to show mucin 1 (MUC1), cytokeratins 8 and 18 (CK8/18) and Kiel 67 (Ki-67) expression in canine mammary gland tumour cells. A and B – solid adenocarcinoma-derived and carcinosarcoma-derived cells expressing MUC1; C – solid adenocarcinoma-derived cells showing higher expression of CK8/18 than carcinosarcoma-derived cells; D – carcinosarcoma-derived cells showing lower expression of CK8/18 than solid adenocarcinoma-derived cells; E – solid adenocarcinoma-derived cells showing lower expression of Ki-67 than carcinosarcoma-derived cells; F – carcinosarcoma-derived cells showing higher expression of Ki-67 than solid adenocarcinoma-derived cells; G and H – 400× magnification of details of E and F. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification 100×, scale bar 100 μm
Immunofluorescence staining to show mucin 1 (MUC1), cytokeratins 8 and 18 (CK8/18) and Kiel 67 (Ki-67) expression in canine mammary gland tumour cells. A and B – solid adenocarcinoma-derived and carcinosarcoma-derived cells expressing MUC1; C – solid adenocarcinoma-derived cells showing higher expression of CK8/18 than carcinosarcoma-derived cells; D – carcinosarcoma-derived cells showing lower expression of CK8/18 than solid adenocarcinoma-derived cells; E – solid adenocarcinoma-derived cells showing lower expression of Ki-67 than carcinosarcoma-derived cells; F – carcinosarcoma-derived cells showing higher expression of Ki-67 than solid adenocarcinoma-derived cells; G and H – 400× magnification of details of E and F. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification 100×, scale bar 100 μm

Fig. 4.

Immunocytochemistry controls verifying the specificity of primary antibody staining. A – non-cancerous cells isolated from canine mammary glands demonstrating weak positive staining for mucin 1 (MUC1) (I) and cytokeratins 8 and 18 (CK8/18) (II), and minimal Kiel 67 (Ki-67) staining (III). (IV) is a 400× magnification of details of (III); B – negative controls where primary antibodies (MUC1 (I), CK8/18 (II) and Ki-67 (III)) were excluded from the assay. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification ×100, scale bar 100 μm
Immunocytochemistry controls verifying the specificity of primary antibody staining. A – non-cancerous cells isolated from canine mammary glands demonstrating weak positive staining for mucin 1 (MUC1) (I) and cytokeratins 8 and 18 (CK8/18) (II), and minimal Kiel 67 (Ki-67) staining (III). (IV) is a 400× magnification of details of (III); B – negative controls where primary antibodies (MUC1 (I), CK8/18 (II) and Ki-67 (III)) were excluded from the assay. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification ×100, scale bar 100 μm

Specific primary and secondary antibodies used for immunocytochemistry

Primary antibodies
Marker Primary antibody Type, clone Host, isotype Reactivity Dilution Catalogue No. Supplier
mucin 1 MUC1 mAb, MUC1/955 mouse, IgG human, mouse 1 : 150 NBP2-44658 Novus Biologicals, Centennial, CO, USA
cytokeratin 8 cytokeratin 18 Cytokeratins 8/18 mAb, 5D3 mouse, IgG human, mouse 1 : 100 NB120-17139 Novus Biologicals
Ki-67 Anti-Ki-67 pAb, N/A rabbit, IgG human, mouse 1 : 300 ab15580 Abcam, Cambridge, UK
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