Enrichment and characterization of cancer stem-like cells in ultra-low concentration of serum and non-adhesive culture system

Abstract

Cancer stem cells (CSCs) play important roles in tumor initiation, metastasis, and progression. They are also mainly responsible for high treatment failure rates. Identification and characterization of CSCs are crucial for facilitating the detection, prevention, or therapy of cancer. Great efforts have been paid to develop an effective method and the ideal method for CSCs research is still in the going. In our study, we created an ultra-low concentration of serum and non-adhesive (ULCSN) culture system to enrich CSCs from murine lewis lung cancer cell line LL/2 with cell spheres structure and characterize the LL/2 CSCs properties. Their characteristics were investigated through colony formation, spheres formation, chemoresistance, flow cytometry for putative stem cell markers, such as CD133, CD34 and CD45, immunofluorescence staining and tumor initiation capacity in vivo. Tumor spheres were formed within 7-10 days under the condition of ULCSN culture system. Compared with adherent parental LL/2 cells, the colony capacity, chemo-resistance, and expression of stem cell markers increased significantly in addition to tumor-initiating capability in the tumor sphere cells. Using the ULCSN culture system, an available isolation method of lewis lung CSCs was established, which is simple, effective, and inexpensive compared with the cytokines attachment serum free culture method. The stem cell properties of the tumor sphere LL/2 cells reflected the CSCs phenotypes. We developed a useful CSCs model for basic and pre-clinical studies for lung cancer and other types of cancer.

Keywords: Cancer stem cells; cancer; characterization; marker.

Figure 1

Morphology of the parental adherent monolayer cells and tumor sphere LL/2 cells. (A) Parental LL/2 cells cultured in DMEM medium with 10% FBS grew as an adherent monolayer. (B, C) Tumor sphere LL/2 cells derived from the parental LL/2 cells cultured under an ULCSN culture system formed the first generation (B) and the third generation (C) tumor spheres.

Figure 2

Colony formation of parental and tumor sphere LL/2 cells. Parental and tumor sphere LL/2 cells were seeded onto 6-well plates at 500 or 1000 per well. The number of colony was counted under a dissection microscope. (A and C) represent 500 or 1000 per well for parental LL/2 cells while (B and D) represent 500 or 1000 per well for tumor sphere LL/2 cells. (E and F) Colony formation rate for parental and tumor sphere LL/2 cells, represent 500 or 1000 per well, respectively. **P<0.01, vs. control. The results are expressed as the mean percentage ± standard deviation of three independent experiments.

Figure 3

Cell viability analysis of the parental adherent cells and tumor sphere LL/2 cells response to chemotherapy drugs. A. Cell viability analysis of the parental adherent cells and tumor sphere LL/2 cells response to epirubicin. B. Cell viability assays of the parental adherent monolayer cells and tumor sphere LL/2 cells response to cisplatin. *P<0.05 and **P<0.01, vs. control. The results are expressed as the mean ± standard deviation of three independent experiments.

Figure 4

mRNA expression of key protein of embryonic stem cells for Oct4 and Sox2 in tumor sphere LL/2 cells. (A, B) RT-PCR analysis for Oct4 and Sox2 in tumor sphere LL/2 cells compared with parental LL/2 cells. (C, D) Statistical results for (A and B) were showed, respectively. OCT4, octamer-binding transcription factor 4; Sox2, SRY-box 2.

Figure 5

Expression of lung CSCs markers by LL/2 parental cells and spheres. Cells were incubated with Abs against CD133, CD34 or CD45, respectively. (A) Average fluorescence intensity of CD133 in LL/2 parental cells (solid line) and spheres (imaginary line). The above picture showed the expression of CD133 in the first generation of LL/2 sphere cells, middle picture represented the expression of CD133 in the second generation of LL/2 sphere cells and the picture at the bottom showed the third generation, respectively. (B, C) Average fluorescence intensity of CD34 or CD45 in LL/2 parental cells (solid line) and spheres (imaginary line) for the same generation with (A), respectively.

Figure 6

Tumor sphere LL/2 cells expressed high levels of the putative stem cell markers, CD133 and CD34. Immunofluorescence staining to assess the cellular levels of CD133 and CD34 in tumor sphere LL/2 cells. Red, expression levels of CD133 and CD34; blue, DAPI-stained cell nuclei.