Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2

Abstract

In vitro studies of a disease are key to any in vivo investigation in understanding the disease and developing new therapy regimens. Immortalized cancer cell lines are the best and easiest model for studying cancer in vitro. Here, we report the establishment of a naturally immortalized highly tumorigenic and triple-negative breast cancer cell line, KAIMRC2. This cell line is derived from a Saudi Arabian female breast cancer patient with invasive ductal carcinoma. Immunocytochemistry showed a significant ratio of the KAIMRC2 cells' expressing key breast epithelial and cancer stem cells (CSCs) markers, including CD47, CD133, CD49f, CD44, and ALDH-1A1. Gene and protein expression analysis showed overexpression of ABC transporter and AKT-PI3Kinase as well as JAK/STAT signaling pathways. In contrast, the absence of the tumor suppressor genes p53 and p73 may explain their high proliferative index. The mice model also confirmed the tumorigenic potential of the KAIMRC2 cell line, and drug tolerance studies revealed few very potent candidates. Our results confirmed an aggressive phenotype with metastatic potential and cancer stem cell-like characteristics of the KAIMR2 cell line. Furthermore, we have also presented potent small molecule inhibitors, especially Ryuvidine, that can be further developed, alone or in synergy with other potent inhibitors, to target multiple cancer-related pathways.

Keywords: KAIMRC2; breast cancer; cell line; characterization; drug treatment; metastatic; stem cells; triple negative.

Figure 1

Microscopic characterization of the KAIMRC2 cell line. (a) Schematic representation of the workflow of isolation and culture of cancerous cells. Briefly, collected tissue sections were minced into approximately 1 cm pieces, followed by several weeks of growth in normal cell culture media. Large fibroblastlike cells were passaged for almost 15 times. Afterward, smaller sized, cuboidal, and epithelial-like KAIMRC2 cells started to make colonies. (c) SEM image of the KAIMRC2 cell line with a large-sized lipid droplet formation on its surface. Scale bar = 5 µm. (b) Enlarged image of lipid droplet on the surface of the cell membrane. Scale bar = 1 µm. (d,e) TEM images of KAIMRC2 cells. Large lipid droplets are apparent inside and outside the cell in the image, d. Scale bar = 2 µm. (f) The transmitted light image of KAIMRC2 cells before the transformation. Elongated and fibroblastlike cells are visible. Scale bar = 400 µm. (g,h) Cuboidal cells after transformation. Scale bar = 400µm. (i) Cell size shrinks to 4–5 um, and cells started to make small round colonies (black arrowheads), a typical cancer cell characteristic. Multinucleated giant cells are also visible (white arrowheads), suggesting another subpopulation of cells, probably CSCs. Scale bar = 100 µm. (j–o) Transmitted light time-series images of adhesion of KAIMRC2 cells on a glass surface. White and black arrows show the formation and circular movement of lipid droplets during cells to the glass surface adhesion process. To the best of our knowledge, this is a very novel mechanism of cell adhesion that has not been shown before. A video of this process is available in the Supplementary Materials (Supplementary Video S1).

Figure 2

Growth characterization of the KAIMRC2 Cell line. (a–g) Wound healing assay of the KAIMRC2 cell line. Live-cell time-lapse images of the wound healing assay were acquired. A confluent layer of the cells was scratched (the area between the two parallel dotted lines), and transmitted light time-lapse imaging was performed for 24 h. Images show cells after 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h. Scale bar = 200 μm. (h–m) Colony formation assay of KAIMRC2 Cell Line. Formation of colonies was visualized microscopically, and transmitted light images were acquired three times a week. Scale bar = 200 μm except m = 400 μm. (o) Colonies were stained with Hematoxylin and Eosin on day 10, and images were acquired using Molecular Devices EVOS FL Auto system. Scale bar = 200 μm (n) The average growth curves of the cell line are shown. Cells were counted in triplicate for 24 days. The doubling time of KAIMRC2 cell line was approximately 16 h. (p) Immunocytochemistry images of a panel of markers to perform a comprehensive proofing of the KAIMRC2 cell line. Scale bars = 100 μm.

Figure 3

(a,b) Protein profiling of KAMRC2 cell line. (a) Western blot analysis of comparison of several breast carcinoma cell lines revealed that KAIMRC2 has constitutively active AKT and mTOR. Strongly positive Integrin β4 and E-cadherin are indicative of cancer stemlike subtype. (b) Several protein profiler arrays showed noticeable insulin receptor expression, GATA-4, PDX-1, E-cadherin, and Serpin E1. Interestingly, high expression of Cyclin D1 was found in the starvation condition in KAIMRC2 cells. (c) Our proposed model of the KAIMRC2 cell line survival pathway.

Figure 4

Gene Expression Analysis of breast cancer KAIMRC2 cell line. (a) Relative up- or downregulation of key genes of KAIMRC2 cells is presented. Each column represents a single gene and represents data from duplicates. (b,c) Segregation of the identified genes into up- and downregulated genes. The web-based freely available pathway analysis tool, Reactome Pathway database (reactome.org), was used to identify the pathways affected by these genes.

Figure 5

Treatment of KAIMRC2 cell line with kinase inhibitors, stem cell modulators, and epigenetic regulators. Cell viability was assessed by MTT assay. (a) Initially, compound panels were tested at 10 µM concentration. Cytotoxic compounds with a % cell survival of less than 50% were selected (Black bars). (b) IC₅₀ calculation of the selected compounds.

Figure 6

Determination of tumorigenicity potential of the KAIMRC2 cell line. (a) Image of nude mice after 18 days of injection. A noticeable tumor lump is visible. (b) Image of the same mice on the 26th day. A big lump of the tumor is visible. The mouse was sacrificed on the same day, and the tumor was isolated and stained. (c,d) H&E staining of a histological section of the isolated tumor. Cells exhibit all the hallmarks of malignancy, i.e., nuclear pleomorphism, hyperchromasia, clumped nuclear chromatin, and mitoses (yellow arrows). Focal tumor necrosis is also present (Yellow 4-point star). Extensive tumor coagulative necrosis and the concentration of viable cancer cells can be seen around the blood vessels, i.e., Peritheliomatous necrosis (red arrows), a hallmark of the high-grade tumor. Scale bar = 50 µm. (e) Flow cytometric analysis of selected breast cancer and CSCs markers expressed on the KAIMRC2 cell line. (f) Representative karyograph of KAIMRC2 cell line. It revealed the presence of an abnormal, complex, and composite karyotype including chromosome range of 58–79; hexasomy of chromosome 9, tetrasomy of chromosomes 1, 2, 5, 6, 7, 11,12, 17, 18 and 21; trisomy of chromosomes 3, 8, 14, 15, 16 and 19. Genomic instability and the existence of heterogeneity in the KAIMRC2 cell line is represented by these chromosomal abnormalities.