Ehrlich ascites carcinoma EAC tumor cell line was purchased
Ehrlich ascites carcinoma (EAC) tumor cell line was purchased from National Research Center, Cairo, Egypt. EAC cell line is an aggressive and malignant breast carcinoma cell line isolated originally from female CD1 mice with a high transplantable capability and rapid proliferation which is considered as an appropriate model for the evaluation of ex-perimental cancer treatments (Khedr and Khalil, 2015). Ehrlich carci-noma (EC) cell line has two subtypes: ascites and solid according to the method of tumor cells inoculation (Aysan et al., 2013). The EC cell line was maintained in our laboratory by weekly transplantation of 2.5 × l06 viable tumor cells in 0.2 ml of sterile saline by intraperitoneal in-jection into female Swiss albino mice. The cell viability was assessed using trypan blue exclusion assay and counted by hemocytometer be-fore injection into naïve CD1 mice for solid tumor induction (Osman et al., 2015).
2.2. Preparation of CSCs - enriched EC cell cultures by chemotherapeutic drug resistance selection method
Drug surviving cells (DSCs) were allowed to grow for additional
48 h in drug- free medium. Then, all of the media was removed and adherent tumor cells were washed with PBS and harvested using 0.25% trypsin-EDTA detachment solution and subsequently stained with trypan blue dye for cell viability and counting. The number of cells per flask were counted using a haemocytometer under digital inverted microscope. DSCs were washed twice with PBS and stored at – 80 ⁰C until use. The highest concentration of chemotherapy in which cells could survive and grow was used to select cells with drug resistant phenotype.
2.3. Flow cytometric analysis of the expression of CSCs surface markers (CD44+/CD24−) in DSCs populations We assessed the percentage of CSCs (CD44+/CD24−) in DSC po-pulations using flow cytometric analysis of cultures from parental and drug-treated EC cells. Single cell suspensions of the harvested DSCs were prepared and counted as previously mentioned and then in-cubated with fluorescent-conjugated monoclonal Latrunculin A anti-mouse CD44-FITC and anti-mouse CD24-PE (Miltenyi Biotec Gmb, Germany) at 4 ⁰C for 30 min. Cells were washed twice with PBS, harvested after centrifugation and then re-suspended in 300 μL FACS buffer and ana-lyzed by BD FACSCanto™ II flow cytometer (BD biosciences, USA). The obtained data were analyzed by the FlowJo software package (Tree star, Ashland, USA). DSCs were analyzed using flow cytometry to de-termine the suitable chemotherapy and the optimal dose that could enrich cells populations with the highest percentage of CD44+/CD24-cells to be used in further experiments.
2.4. Preparation of CSC- enriched cell lysate
CSC lysates were prepared from selected DSCs with the highest percentage of CSC surface markers as previously described. In brief, cells were re-suspended in sterile PBS at a concentration of 5 × 106 cells per mL. Cells were lysed by four rapid freeze and thaw cycles (37 ⁰C water bath and – 80 ⁰C), Cell lysates were centrifuged at 2000 rpm for 7 min at 4 ⁰C (Salem et al., 2016a). The supernatants were collected in sterile vials and stored at – 80 ⁰C until use.
2.5. Generation of mice bone marrow – derived dendritic cells in vitro and identification of their phenotypical characteristics using flow cytometry DCs were generated from bone-marrow (BM) derived mononuclear cells of healthy CD1 mice. BM cells were flushed from the marrows of femur and tibia bones of 13 male mice under aseptic conditions. The harvested BM cells was depleted of RBCs by incubation with ammo-nium chloride potassium lysis buffer and were then cultured at a
concentration of 1 × 106 / mL in complete medium of RPMI-1640 supplemented with 10% heat inactivated FBS, 1% P/S, 2 mM L-gluta-mine in addition to 20 ng/mL GM-CSF and 20 ng/mL IL-4. On day 4, the cultures were fed with freshly prepared complete medium containing 20 ng/mL of GM-CSF and IL-4. On day 6, immature DCs were supple-mented with either PBS (unloaded DCs) or loaded with previously prepared CSC-lysate (loaded DCs) at ratio 1:1 (Li et al., 2014) and in-cubated overnight at 37 ⁰C and 5% CO2. Maturation stimulus TNF-α was added to the cultures at a concentration of 20 ng/mL on day 7.