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. 2009 Sep 14;28(1):127.
doi: 10.1186/1756-9966-28-127.

Characterization of human breast cancer epithelial cells (HBCEC) derived from long term cultured biopsies

Ralf Hass  1 , Catharina Bertram
Affiliations

Characterization of human breast cancer epithelial cells (HBCEC) derived from long term cultured biopsies

Ralf Hass et al. J Exp Clin Cancer Res. .

Abstract

Introduction: For a more individualized therapeutic approach we explored a protease-free method to culture primary cells from breast cancer biopsies.

Methods and results: Tumor tissue from breast cancer patients after surgery was cultured ex vivo without enzymatic digestion for more than one year and revealed the continuous outgrowth of adherent and proliferating primary cell populations. Immunofluorescence staining of these human breast cancer-derived epithelial cells (HBCEC) and quantification by flow cytometry revealed nearly exclusively cytokeratin-expressing cells. Analysis of surface markers during long term tumor culture of primary HBCEC (more than 476d) demonstrated a prominent expression of CD24, CD44 and MUC1 (CD227). According to aging markers, expression of senescence-associated beta-galactosidase revealed little if any positive staining in a primary tumor-derived HBCEC population after 722d in culture, whereas the majority of normal human mammary epithelial cells (HMEC) demonstrated senescent cells already after a culture period of 32d. In this context, HBCEC populations derived from a tumor culture after 152d and 308d, respectively, exhibited a significant telomerase activity, suggesting continuous proliferative capacity. Treatment with several chemotherapeutic compounds and their combinations revealed distinct cytotoxic effects among HBCEC from different breast cancer patients, indicating an individualized response of these tumor-derived primary cells.

Conclusion: The protease-free outgrowth of primary HBCEC offers a patient-specific approach to optimize an individually-designed cancer therapy. Moreover, HBCEC from long term breast tumor tissue cultures resemble tumor cell-like properties by an intact ECM formation and a stable cell surface protein expression providing a reproducible screening platform to identify new biomarkers and to test new therapeutics in individual tumor samples.

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Figures

Figure 1
Figure 1
Characterization of primary human breast cancer epithelial cells (HBCEC). A. Scanning electron micrographs of human breast cancer-derived cell cultures. The cells are squamous with many short and thin processes and grow upon each other. B. Ultrathin sections of two human breast cancer-derived cells, which partially overlap and are connected by desmosomes. The cells contain bundles of intermediate filaments and cytoplasmic vacuoles, whereas organelles are almost absent. In the right transmission micrograph, two squamous cell processes are connected by desmosomes and bundles of intermediate filaments are orientated in parallel to the cell surface. C. Immunofluorescence of intermediate filaments. Nuclei became visual using DAPI and the intermediate filament proteins cytokeratin (green) and vimentin (red) were detected by FITC-conjugated mouse anti-cytokeratin and mouse anti-vimentin antibody, respectively. D. Quantification of cytokeratin, vimentin and desmin expression by flow cytometric analysis. About 99% of the HBCEC population stained positive for cytokeratin, whereof some were positive for both, cytokeratin and vimentin intermediate filament proteins. Expression of desmin intermediate filaments remained undetectable. The FITC-labeled IgG control and the secondary antibody control served as background staining balance.
Figure 2
Figure 2
Surface marker expression, SA-β-gal staining and telomerase activity in HBCEC. A. Determination of the percentage of cell surface marker expression in HBCEC at different ages. Expression of the surface marker proteins CD24, CD44, CD227 was maintained during long term culture of HBCEC. Whereas CD24 and CD44 were similarly expressed after 176d and 462d, CD227 increased from 52% to 88% in HBCEC 462d. The flow cytometry measurements varied by about 8%. B. SA-β-gal staining of primary HBCEC and HMEC cultures. Staining for SA-β-gal of a HBCEC population after 722d in culture revealed little if any positive cell. Normal HMEC in passage 16, however, displayed already predominantly enlarged senescent cells after 32d, demonstrated by the dark-green stain (bar = 200 μm). C. Telomerase (TRAP-)assay of primary cultures from breast cancer biopsies. Telomerase activity was analyzed according to the Telomeric Repeat Amplification Protocol (TRAP). HBCEC populations demonstrated telomerase activity independent of the age of the culture and the harvest method. The human embryonic kidney (HEK) 293T cell line was used as a positive control and 1× CHAPS buffer served as a negative control. Quantification was performed using densitometric analysis.
Figure 3
Figure 3
Chemotherapeutic effects on HBCEC from breast cancer patients. HBCEC derived from a 40 year-old (HBCEC 366) (Fig. 3A) and a 63 year-old (HBCEC 367) (Fig. 3B) woman both with ductal breast carcinoma, the breast cancer cell lines MCF-7 (Fig. 4A) and MDA-MB-231 (Fig. 4B), and normal HMEC in passage 16 (Fig. 5) were incubated with a single dose of 1 μM (blue bars) and 125 nM (red bars) of appropriated chemotherapeutic compounds (Taxol, Epothilone A, Epothilone B, Epirubicin, Doxorubicin) and certain anthracyclin combinations (Epirubicin/Taxol, Epirubicin/Epothilone A, Epirubicin/Epothilone B) for 6d, respectively. Alternatively, the drugs were replaced after 3d, resulting in a similar 6d (= 2× 3d) incubation of the same compounds, using concentrations of 1 μM (yellow bars) and 125 nM (turquoise bars), respectively. Whereas the higher concentration of 1 μM was generally more effective, this was further promoted by a sequential treatment. Moreover, the HBCEC populations revealed distinct effects to the anticancer drugs Epothilone A and B, suggesting an individual responsiveness specific for the appropriate patient (Fig. 3A, B). Similarly, Epothilone A and B exhibited different effects on the two breast carcinoma cell lines. Furthermore, the non-metastatic MCF-7 cell line displayed an overall increased sensitivity to the administered drugs or drug combinations as compared to the highly metastatic MDA-MB-231 cells (Fig. 4A, B). HMEC (P16) demonstrated reduced cytotoxic effects of the chemotherapeutics as compared to the HBCEC cultures (Fig. 5). Data represent the mean +s.d. (n = up to 5 replicates). P values were calculated by the unpaired T-test according to the appropriate untreated control cells (Control). Results were considered as statistically significant when P value was < 0.5 (*P < 0.5; **P < 0.05; ***P < 0.005).
Figure 4
Figure 4
Chemotherapeutic effects on HBCEC, breast cancer cell lines. HBCEC derived from a 40 year-old (HBCEC 366) (Fig. 3A) and a 63 year-old (HBCEC 367) (Fig. 3B) woman both with ductal breast carcinoma, the breast cancer cell lines MCF-7 (Fig. 4A) and MDA-MB-231 (Fig. 4B), and normal HMEC in passage 16 (Fig. 5) were incubated with a single dose of 1 μM (blue bars) and 125 nM (red bars) of appropriated chemotherapeutic compounds (Taxol, Epothilone A, Epothilone B, Epirubicin, Doxorubicin) and certain anthracyclin combinations (Epirubicin/Taxol, Epirubicin/Epothilone A, Epirubicin/Epothilone B) for 6d, respectively. Alternatively, the drugs were replaced after 3d, resulting in a similar 6d (= 2× 3d) incubation of the same compounds, using concentrations of 1 μM (yellow bars) and 125 nM (turquoise bars), respectively. Whereas the higher concentration of 1 μM was generally more effective, this was further promoted by a sequential treatment. Moreover, the HBCEC populations revealed distinct effects to the anticancer drugs Epothilone A and B, suggesting an individual responsiveness specific for the appropriate patient (Fig. 3A, B). Similarly, Epothilone A and B exhibited different effects on the two breast carcinoma cell lines. Furthermore, the non-metastatic MCF-7 cell line displayed an overall increased sensitivity to the administered drugs or drug combinations as compared to the highly metastatic MDA-MB-231 cells (Fig. 4A, B). HMEC (P16) demonstrated reduced cytotoxic effects of the chemotherapeutics as compared to the HBCEC cultures (Fig. 5). Data represent the mean +s.d. (n = up to 5 replicates). P values were calculated by the unpaired T-test according to the appropriate untreated control cells (Control). Results were considered as statistically significant when P value was < 0.5 (*P < 0.5; **P < 0.05; ***P < 0.005).
Figure 5
Figure 5
Chemotherapeutic effects on normal human mammary epithelial cells in passage 16 (HMEC P16). HBCEC derived from a 40 year-old (HBCEC 366) (Fig. 3A) and a 63 year-old (HBCEC 367) (Fig. 3B) woman both with ductal breast carcinoma, the breast cancer cell lines MCF-7 (Fig. 4A) and MDA-MB-231 (Fig. 4B), and normal HMEC in passage 16 (Fig. 5) were incubated with a single dose of 1 μM (blue bars) and 125 nM (red bars) of appropriated chemotherapeutic compounds (Taxol, Epothilone A, Epothilone B, Epirubicin, Doxorubicin) and certain anthracyclin combinations (Epirubicin/Taxol, Epirubicin/Epothilone A, Epirubicin/Epothilone B) for 6d, respectively. Alternatively, the drugs were replaced after 3d, resulting in a similar 6d (= 2× 3d) incubation of the same compounds, using concentrations of 1 μM (yellow bars) and 125 nM (turquoise bars), respectively. Whereas the higher concentration of 1 μM was generally more effective, this was further promoted by a sequential treatment. Moreover, the HBCEC populations revealed distinct effects to the anticancer drugs Epothilone A and B, suggesting an individual responsiveness specific for the appropriate patient (Fig. 3A, B). Similarly, Epothilone A and B exhibited different effects on the two breast carcinoma cell lines. Furthermore, the non-metastatic MCF-7 cell line displayed an overall increased sensitivity to the administered drugs or drug combinations as compared to the highly metastatic MDA-MB-231 cells (Fig. 4A, B). HMEC (P16) demonstrated reduced cytotoxic effects of the chemotherapeutics as compared to the HBCEC cultures (Fig. 5). Data represent the mean +s.d. (n = up to 5 replicates). P values were calculated by the unpaired T-test according to the appropriate untreated control cells (Control). Results were considered as statistically significant when P value was < 0.5 (*P < 0.5; **P < 0.05; ***P < 0.005).
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