Immunolocalisation of Ki-67 gene in Immortalised Human Breast Cancer Cell Line (s) Using Immunocytochemical Methods.
Module Leader: Dr.
Principles & Practice of Cellular Science
Module Number:
Word count: 947
Ki-67 is a nuclear protein that is not associated with histones and is easily altered. It is closely associated with the cell cycle as the G1 phase of the cell cycle is characterized by moderate expression of Ki-67, which increases as the cells proliferate in the S and G2 phases.5 The highest expression (peak) levels of Ki-67 are noted in the M phase before undergoing rapid catabolism to undetectable levels in resting cells (G0 and early G1).5 Cell proliferation in cancer is a phenomenon that is effectively studied by analysing the expression levels of Ki-67. Moreover, this marker is important for categorizing breast cancer according to prognosis, that is, poor or good. Additionally, it is an important factor in evaluating the clinical response of breast cancer to chemotherapy.5 Through immunocytochemical analysis of Ki-67, clinicians and oncologists can determine the presence or absence of breast cancer based on the proportion of cells expressing the biomarker. This information can also be refined further to determine whether the cancer is benign or malignant. For example, 4it can detect a higher concentration of Ki-67 in malignant and high-grade lesions compared to low-grade benign lesions in bone tumours.
Section 2: Aim of the Practical
To detect the expression of Ki-67 in MDA and Mcf7 breast cancer cell lines through immunocytochemical analysis of different slides and to investigate the link between Ki-67 expression levels and clinicopathology of human breast cancer.
Section 3: Presentation of Data
Figure 1: Representative image of immunolocalised Ki-67 expression in MDA and Mcf7 breast cancer cell lines using x400 magnification.
As illustrated in Fig. 1 above, the slide staining produced varying degrees of staining across the three fields of view (FOV). In FOV A, there was an observably higher level of stained cells compared to B and C. Subsequently, the FOV C displayed the lowest degree of staining. The differences in the staining intensity matched with expression intensity of Ki-67 biomarker. In essence, FOV A represents a high expression of Ki-67 and thus a probability of these cells being in G2 or M phases of cell division. Additionally, the cells in FOV B were moderately stained indicating a moderate expression of Ki-67 and cells in transition between G1 and S phase in the cell cycle.  In FOV C, the cells were weakly stained indicating a much lower expression of Ki-67 and a high chance of these cells being in the G1 phase of the cell cycle.
Table 1: Proportion of cells exhibiting strong/weak/negative expression of Ki-67 in three fields of view (A, B, C) respectively.

Field of View   Individual data & Mean
A Total cells=125
Stained cells=70
Proportion: 0.56×100
Strong positive
B Total cells=125
Stained cells=32
Proportion: 0.256×100
Weak positive
C Total cells=125
Stained cells=23
Proportion: 0.184×100

The representative image of immunolocalised Ki-67 expression in Fig. 1 can be analysed in more details based on Table 1 results. Moreover, FOV A in Fig. 1 corresponds with strong positive results as calculated in Table 1. In other words, the majority of cells (56%) in FOV A stained more strongly than in B (25.6%) and C (18.4%). As such, the corresponding observations are strong positive, weak positive, and negative.
Section 4: Discussion (word count: 403)
It is recommended that all women who have reached menopause and those in the child-bearing bracket should undergo a regular breast cancer screening. The assessment entails the study of cell proliferation using immunohistochemical and immunocytochemical techniques.1 While Ki-67 as a breast cancer biomarker has been extensively studied, the cutoffs and staining thresholds that apply have largely been arbitrary.5 Therefore, the lack of universally standardized staining thresholds is still a major issue. For example, a staining threshold of 20 percent for Ki-67 has been used to define strong positive results3 while other researchers have advocated for Ki-67 staining thresholds; <20% (low or negative expression); ≥20% (high expression); 14-19% (moderate/intermediate expression).2
The results of the practical as illustrated in Fig. 1 and the corresponding numerical computation in Table 1 show that Ki-67 is a gene that is differentially expressed in human breast cancer cell lines. Consequently, the applied immunocytochemical protocol enabled the researcher to determine Ki-67 both qualitatively and quantitatively. In the FOV A in Fig. 1, the high intensity of immunostaining is indicative of high localization of Ki-67 due to the high rate of cell proliferation in this group of cells. Correspondingly, FOV A in Table 1 produced the highest percentage of stained cells (56%); an indication of malignancy or highly cancerous cells. Similar work in the past showed that malignant and high-grade lesions in bone tumours displayed a higher concentration of Ki-67 compared with low-grade benign lesions.4 Additionally, the cells in FOV A could also be indicative of a cell line that is in the advanced stages of cell division.
While there is a significant difference in the Ki-67 expression between FOV A and FOV B, the difference is much smaller between FOV B and FOV C (25.6% vs. 18.4%). These observations depict the possibility of B and C belonging to the same cell line while A belongs to a different cell line. Apart from belonging to the same cell line, the weak positive and negative immunostaining of cells in B and C respectively may represent a good prognosis of breast cancer.
In the same practical session, two other students, Sluiman and Yasser recorded comparable observations. Sluiman obtained 56%, 29.2%, and 14.8% in fields of view A, B, and C respectively. The results compared with those of Yasser who obtained 53%, 32.7%, 14.3% in fields of view A, B, and C respectively. Therefore, the overall data had notable levels of reproducibility, reliability, and accuracy.

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  2. Maisonneuve, P. et al. Proposed new clinicopathological surrogate definitions of luminal A and luminal B (HER2-negative) intrinsic breast cancer subtypes. Breast Cancer Res. 16 (2014).
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