Breast Cancer Tissue Explants: An Approach to Develop Personalized Therapy in Public Health Services

Abstract

Breast cancer is one of the main causes of death worldwide. Lately, there is great interest in developing methods that assess individual sensitivity and/or resistance of tumors to antineoplastics to provide personalized therapy for patients. In this study we used organotypic culture of human breast tumor slices to predict the experimental effect of antineoplastics on the viability of tumoral tissue. Samples of breast tumor were taken from 27 patients with clinically advanced breast cancer; slices were obtained and incubated separately for 48 h with paclitaxel, docetaxel, epirubicin, 5-fluorouracil, cyclophosphamide, and cell culture media (control). We determined an experimental tumor sensitivity/resistance (S/R) profile by evaluating tissue viability using the Alamar Blue^® metabolic test, and by structural viability (histopathological analyses, necrosis, and inflammation). These parameters were related to immunohistochemical expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The predominant histological type found was infiltrating ductal carcinoma (85.2%), followed by lobular carcinoma (7.4%) and mixed carcinoma (7.4%). Experimental drug resistance was related to positive hormone receptor status in 83% of samples treated with cyclophosphamide (p = 0.027). Results suggest that the tumor S/R profile can help to predict personalized therapy or optimize chemotherapeutic treatments in breast cancer.

Keywords: breast cancer; chemotherapy; ex vivo; explants; tissue slices; treatment response.

Figure 1

Representative photographs showing the preparation of breast tumor explants with precise thickness. (a) Tumor biopsy obtained during surgery. (b) Breast tumor after removing surrounding fat. (c) Tumor tissue cores (8–10 mm diameter). (d) Precision-cut breast tumor slices (250–300 μm thickness) prepared with a Krumdieck^® tissue slicer. (e) Breast tumor explants (4 mm diameter and 250–300 μm thickness) were manually prepared from precision cut breast tumor slices and collected in KB buffer (f) Breast tumor tissues after treatment culture and Alamar Blue assay. (A) untreated viable tissues (pink), (B) resistant tissues (pink) and (C) sensitive tissues (blue) (48 h, 37 °C, 5%CO₂/95%O₂, 30 rpm). (g) Tumor viability analysis by the Alamar Blue assay after antineoplastic treatment (C) cyclophosphamide 1 mg/mL, (E) epirubicin 3 μg/mL, (F) 5-fluororacil 50 μg/mL, and (P) paclitaxel 20 μg/mL. * p < 0.05 compared to the control (CTRL). (h) Histopathological analysis. Scale bar: (a–f), 5 mm; (h), 100 μm.

Figure 2

Ex vivo tissue culture viability. (A) Metabolic viability of untreated tissue explants after 48 h of incubation. Viability percentages remained without significant changes through the culture time. N = 27 samples by triplicate. T student test, p < 0.05 compared to viability of uncultured tissues. (B) Representative photographs of untreated viable invasive ductal carcinoma tissue samples from patient 5 and 22 after 48 h incubation. H&E staining (a,b,e,f) and high (>20%) Ki67 expression (c,d,g,h). Scale bar: 100 μm.

Figure 3

Metabolic and histological viability of explants cultivated with antineoplastics. (A) Resistance/sensitivity of breast tumor explants based on the Alamar Blue assay (values are expressed as mean ± standard deviation, n = 3–5 explants per treatment, * p < 0.05 compared to the control). (B) Structural viability by histopathological analysis. Necrotic areas are delimited by dotted lines. Values are presented as the average of 5–6 explants per treatment. Tumor viability was evaluated 48 h after treatment with 1 mg/mL cyclophosphamide (C), 3 μg/mL epirubicin (E), 50 μg/mL 5-fluororacil (F), and 20 μg/mL paclitaxel (P). Scale bar: 100 μm.