Introduction
Cancer is a complex disease featuring uncontrolled cellular growth resulting in the accumulation of abnormal cells [1]. Cancer is a leading cause of death worldwide with an estimated 9.6 million deaths in 2018. Globally, breast cancer (BC) is the most common cancer in women with 2.1 million new cases were estimated in 2018 and it is the primary cause of cancer death among them as well [2]. In Egypt, 32% of the total cancer incidence rates among females were attributable to BC in 2013 which represented the most prevailing cancer across Egyptian women [3].
The great progress that has been made in cancer therapy is supposed to bring this cancer burden down, however, a considerable number of patients still experience relapse [4]. Albeit conventional therapies as chemotherapy and radiotherapy suppress tumor growth, the success of these agents is limited by therapy-resistant cells which consequently recur the disease [5]. Accumulating evidence has suggested that tumors are made of a heterogeneous population of cells which include cancer stem cells (CSCs), a minority subpopulation of undifferentiated cells that generate the differentiated progeny that comprise the bulk of the tumor. CSCs are supposed to be the main culprit for tumor initiation, metastasis, chemotherapeutics resistance, and disease relapse [6].
In 2003, CSCs have been identified in a solid form of human breast cancer (BC) by M. Al-Hajj and M.S. Wicha [7]. Breast cancer stem cells (BCSCs) have been found to be characterized by CD44+CD24-/low the epithelial surface antigen (ESA+) phenotype [8]. As few as hundred CD44+/CD24−/low/ESA+breast cancer cells were able to initiate tumor in vivo , unlike CD44-/CD24+ breast cancer cells [6].
Unfortunately, standard therapies as chemotherapy, radiotherapy, and surgery are not only incapable of eradicating BCSCs, strikingly, BCSCs were further enriched after treatment of different breast cancer cell lines with different chemotherapeutic agents [1, 9]. Similarly, there was an increase in the CD44+CD24-/low subpopulation in breast tumors harvested from doxorubicin-treated mice [9]. This entails looking for therapeutic agents that could target CSCs to completely eradicate cancer and prevent disease relapse.
Recently, nonsteroidal anti-inflammatory drugs (NSAIDs) have gained a big reputation for their chemopreventive effects against several cancer types including colorectal [10], breast [11], glioblastoma [12], esophageal [13], and gastric cancers [14]. Indomethacin, among them, has displayed to act as a chemosensitizer in resistant BC as well as reduce metastasis from human BC cells [15, 16]. Since CSCs are linked to chemoresistance and metastasis, we postulated that the beneficial effects of indomethacin in BC could dwell in a CSC-suppressing effect. Herein, we investigated the anti-BCSC activity of indomethacin, particularly on chemotherapy-enriched CSCs.
Materials and Methods
In vitro study
Cells and cell culture
The murine mammary adenocarcinoma Ehrlich ascites carcinoma (EAC) cells were derived from Ehrlich ascites bearing female BALB/c mice (Pharmacology and Experimental Oncology Unit of the National Cancer Institute (NCI), Cairo, Egypt). The ascitic fluid was collected, suspended in PBS (Lonza, Verviers, Belgium), and centrifuged at 1200 rpm for 10 minutes at 4 0C (ThermoFisher Scientific, Germany). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS (Biowest, Nuaillé, France), 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine (Lonza, Verviers, Belgium), and 7 µL/mL amphotericin B (Lonza, Walkersville, MD USA). All cells were maintained in a 5% CO2 incubator at 37oC (Nuaire, USA). To enrich CSCs, cultured cells were incubated till it reached 80% confluency and then exposed to a single concentration of either doxorubicin (DOX) (2, 4, 6 µg/mL), paclitaxel (PTX) (8, 12, 16 µg/mL), or cisplatin (CDDP) (20, 40, 50 µg/mL) for 72 hours. These concentrations were selected to broadly span concentrations above and below the IC50 of each drug in EAC cells which are as follows; DOX IC50 = 3 µg/mL [17], PTX IC50 = 12 µg/mL [18], and CDDP IC50 = 32 µg/mL [19].
Drugs and antibodies
Indomethacin was gifted from Sigma pharmaceutical company (Cairo, Egypt). CDDP, DOX, and PTX were purchased from Mylan (Pallini, Attiki, Greece), EIMC United Pharmaceuticals (Badr city, Cairo, A.R.E.), and Bristol-Myers Squibb (Latina, Italy), respectively. Antibodies used for flow cytometry were FITC-conjugated anti-mouse/human CD44 and phycoerythrin (PE)-conjugated anti-mouse CD24 (BioLegend, San Diego, California, United States).
Flow cytometric analysis
To determine the concentration of chemotherapy that enriched CSCs the most, chemotherapy-treated EAC cells were detached by trypsin-EDTA (Biowest, Nuaillé, France) and suspended in PBS for flow cytometric analysis. FITC conjugated anti-CD44 and PE-conjugated anti-CD24 were added and incubated for 30 minutes in dark according to the manufacturer’s instructions. Samples were then washed with PBS and analyzed using BD FACSCanto II flow cytometer (BD Biosciences, USA) coupled with a computer with BD FACS Diva software for data analysis.
In vivo mouse experiments
Tumor models
25-30 g female BALB/c mice were purchased from the National Research Center (Cairo, Egypt) and acclimated for 10 days. Animals were maintained and treated following the Research Ethics Committee-approved guidelines for the care and use of laboratory animals (College of Pharmacy, Tanta University, Egypt).
Based on the results of flow cytometry surface staining of chemotherapy-treated cells, 50 µg/mL CDDP was the concentration that induced the highest CSCs enrichment. Accordingly, this concentration was used for the subsequent in vivo experiment. The viable CDDP-treated cells were considered as being CDDP-resistant cells. Either CDDP-resistant EAC cells or parent EAC cells were suspended in saline and counted via a Neubauer haemacytometer (Marienfeld, Germany) by the trypan blue (Lonza, Walkersville, MD USA) exclusion method under the EVOS XL Core inverted microscope AMEX1000 (Life Technologies, Carlsbad, California, USA).
CSCs in EAC were enriched by 50 µg/mL CDDP then 250 × 103 of CDDP-resistant cells were injected subcutaneously into the back of each mouse for a total of 28 mice, meanwhile, parent EAC cells were injected into another 28 mice (250 × 103 EAC cells for each mouse). When palpable tumors developed, mice in each group were then randomly divided into 4 subgroups (control, CDDP, indomethacin, and the combination of the same dose and frequency of both drugs).
A single intraperitoneal dose of CDDP (7.5 mg/kg body weight) was administered on the first day of treatment [20]. Indomethacin (1.0 mg/kg body weight) was administered every day for 16 days as an oral suspension in a 0.5% carboxymethylcellulose (CMC) (Isochem, France) solution while on the same day both the control and CDDP alone-treated mice groups were given the same volume of 0.5% CMC solution without indomethacin [5].
Tumor size was measured by a Vernier caliper (APT, China) and tumor volume was calculated based on the formula: volume = (length × width2)/2. The tumor growth rate was computed using the supplementary excel file provided by Gregory Hather, et al[21]. All mice were sacrificed on day 17 since the commencement of treatment and blood samples were collected in EDTA tubes for flow cytometric analysis of CSC and immunological markers. The tumor masses were dissected, washed with saline and weighed. Some of the excised tumors were immediately fixed in 10% buffered formalin solution for histopathological examination whereas the others were placed in -80oC for PCR analysis.
Flow cytometric analysis
To investigate the CSC-suppressing effect as well as the immunomodulatory influence of indomethacin, flow cytometric analysis of CSC markers and the hematological and immunological markers was performed on the collected blood samples. Two panels of surface staining were made. Panel one included FITC-conjugated anti-CD44 along with PE-conjugated rat anti-mouse stem cell antigen-1 (Sca-1). In panel two, PE-conjugated anti-CD24, FITC-conjugated rat anti-mouse CD117, PerCP-conjugated rat anti-mouse CD4, and allophycocyanin (APC)-conjugated anti-mouse CD62L were added together. The samples were incubated in dark for 30 min before the lysing solution was added and then incubated for another 15 min in dark. PBS was added to wash the samples before the acquisition. Data analysis was made by BD FACS Diva software. The anti-SCa-1, anti-CD117, and anti-CD4 antibodies were all purchased from BD Biosciences (USA). The vendor for the anti-CD62L antibody was BioLegend (USA).
Histopathology
The formalin-fixed tumors were routinely processed in ascending grades of alcohol then xylene. The tissues were then embedded in paraffin blocks, serially sectioned to 3-5 µm thick before being stained with Hematoxylin and Eosin (H&E, Sigma pharmaceutical company, Egypt). All stained tissue sections were examined under a light microscope (Olympus BX 51, Olympus America, Melville, USA) coupled with a digital camera (Olympus DP11) for photographing.
Quantitative reverse transcription PCR (qRT-PCR)
For microRNAs (miRNAs) expression assays, the tumor tissues were disrupted and homogenized using a TissueLyser II (QIAGEN, Germany) following the manufacturer’s protocols. Total RNA was extracted using the RNeasy spin column and the concentration and purity of it were determined using a nanodrop (DS-11+ Spectrophotometer, Denovix, USA).
Using the TaqMan® MicroRNA Reverse Transcription Kit, cDNA was reverse transcribed using MultiScribeReverse Transcriptase by the thermal cycler (GeneAmp PCR System 9700, Applied Biosystems, Japan) following the TaqMan microRNA assays protocol. The PCR amplification step was done using TaqMan® 2X Universal PCR Master Mix and TaqMan MicroRNA Assay (20X) in a QuantStudio 5 (Applied Biosystems, Thermo Fisher Scientific, Singapore). As an endogenous control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize the expression levels of target genes using the comparative CT method. Genes primers were purchased from OriGene Technologies (USA) except the reverse primer of miR-7 which was obtained from QIAGEN. The primers sequences are shown in Table 1 .