Clinical significance of cancer stem cell markers in lung carcinoma

The identification of side population (SP) cells in several cancer studies has been proved to be involved in the treatment failure (chemotherapy) and tumor relapse. Here we have sorted 7% of side population (SP) cells from lung adenocarcinoma by Hoechst 33342 dye expulsion method. Further, the characterization of sorted SP cells showed cancer stem like properties such as transcriptional upregulation of stemness genes (OCT-4, SOX2 and NANOG), ATP binding cassette (ABC) transporter protein (ABCG2) and enhanced level of stem cell surface markers such as CD133 and CD44. Therefore, the aforesaid properties of lung adenocarcinoma SP cells play a significant and functional role in tumor invasion, metastasis, chemotherapeutic drug resistance and tumor recurrence in lung cancer.


INTRODUCTION
Cells that are present in small groups within a tumor bulk and have the ability to initiate cancer growth and metastasis (Clarke et al., 2006;Visvader et al., 2008;Rosen et al., 2009;) are known as cancer stem cells (CSCs). Studies in several tumors visualized the properties of CSCs, namely overexpression of cell surface proteins, ability to differentiate into different cell types, transcriptional upregulation of stemness genes and increased cell proliferate rate (Zhou et al., 2001;Haraguchi et al., 2006;Wu et al., 2008;Schoenhals et al., 2009).
Further, the CSCs are capable of pumping out the several chemotherapeutic drugs or DNA targeting drugs and become resistant as they express various membrane spanning ATP binding cassette proteins (ABC) such as ABCB1, ABCG1 and BCRP1. CSCs can be sorted either by CD133 overexpression or FACs based Hoechst 33342 dye efflux with lower intensity of fluorescence signal (Goodell et al., 1996;Kondo et al., 2004;Hirschmann et al., 2004;Patrawala et al., 2005). These small groups of cells, which can efficiently pump the dye out of the cell owing to the overexpression of ABC transporter proteins, are designated as Side Population (SP) cells (Goodell et al., 1996;Ho et al., 2007;Seo et al., 2007;Das et al., 2008;Salcido et al., 2010). The cancer stem cells-like properties of SP cells might play a major role in chemotherapy failure and tumor recurrence (Gao et al., 1998;Wang et al., 2007;Bourguignon et al., 2008). However, the precise signaling pathways and molecular mechanisms behind the side population cells mediated tumorigenesis are still unknown.
Worldwide, lung adenocarcinoma is one of the leading cause for cancer related deaths in all genders. Upon diagnosis, the average life span of lung cancer patients is about 5 years (Parkin et al., 2005). Different studies in lung cancer described that the occurrence of CSCs is responsible for inhibiting the uptake of different therapeutic drugs which leads cancer cells to resist apoptosis (Singh et al., 2004;Seo et al., 2007;Salnikov et al., 2010;Singh et al., 2010;Yue et al., 2014). As like in other solid tumors, 'lung CSCs' are also highly clonogenic, capable of self-renewal, and mutlidrug apoptosis-resistant (Salnikov et al., 2010;Singh et al., 2010). Accordingly, the sorting and further functional characterization of lung cancer SP cells (Das et al., 2008) will definitely pave the way for developing novel anti-cancer drugs that could effectively target CSCs. Therefore, we have performed functional characterization of lung adenocarcinoma SP cells.

Cancer samples and culture
All the procedures (sample collection and surgery) were followed according to the ethical rules approved by The Second Affiliated Hospital of Nantong University (License number: SEC013244). The cancer samples were obtained in agreement with patients oral consent. According to The Second Affiliated Hospital of Nantong University rules and regulations, we do not provide any data for the identification of patients in this manuscript.

Hoechst 33342 dye labeling
Study groups were assigned as Control: cells labelled with Hoechst dye alone (n=7) and Verapamil treatment (n=7): cells labelled with Hoechst dye and 75 µM of Verapamil drug as well. The propidium iodide staining and labelling of Hoechst 33342 dye are followed exactly as per (Liu et al., 2015). The method of sorting of side population cells by flow cytometry method was exactly followed by the procedure as described previously (Hirschmann et al., 2004;Liu et al., 2015).

Biochemistry
Cell lysates extraction, protein preparation and separation by SDS-PAGE were performed as described previously (Yue et al., 2014). Primary rabbit antibodies (rabbit anti-human ABCG2, CD133 and CD44) and secondary antibody anti-rabbit-IgG HRP conjugated (Santa Cruz) were used. Protein signals were detected by ECL kit with Biorad densitometer.

Real-time PCR
Using Reverse Transcriptase kit (Fermentas), the total RNA was extracted, complementary DNA was synthesized and subsequently RT-qPCR was performed in iCycler (Biorad). The sequences of the primers and the signal quantification were performed as described previously (Yue et al., 2014). The PCR parameters applied for this study were as follows: initial denaturation, 93°C for 40 sec; followed by 40 cycles of denaturation, 93°C for 5 min.; annealing, 58°C for 45 sec, and extension, 72°C for 30s. Final extension was executed at 72°C for 10 min. The amplified PCR products were electrophoresed on 1.2% agarose gel and stained with ethidium bromide. The band intensity was measured using ImageJ software and the relative RNA expression level was adjusted with GAPDH level, which was used as housekeeping gene.

In vitro assays
Matrigel invasion assay. D six-well matrigel invasion chambers (BD Biosciences) were applied for this assay. 10 4 cells were plated per insert in serum free medium while the insert outlets contained DMEM-F12 supplemented with 10% FBS as chemoattractant. Cells were grown for 48 hours and further subjected to washing and wiping the top layer of Matrigel with cotton swabs. The membrane containing invading cells was stained with hematoxylin for 5 minutes and then visualized under a light microscope using a 60× objective for counting the number of cells invaded. The quantitative graph represents the average values from three independent experiments.
Cell proliferation assay. This experiment was performed in triplicate. Both SP and non-SP cells were seeded in a 96-well plate at a density of 2×10 6 cells/ well. The optical density (OD) at 450 nm was measured to determine cell proliferation activity. This measurement was taken for 7 days continuously and the growth graph presented the average value of three different experiments. Differentiation and Matrigel assays protocols were followed as per (Ho et al., 2007). Similarly, the sphere formation assay was carried out exactly by the protocol descried in the literature (Yanamoto et al., 2011).

Statistical analysis
Student's t-test was performed for comparing the significance of differences between two different groups and the values denoted in the quantification charts are mean ± S.D. The values *P<0.05 and **P<0.01 are considered as significant.

Isolation of side population cells from lung adenocarcinoma
By Hoechst dye exclusion assay we have found that side population cells accounted for 7% (Fig. 1A) of lung adenocarcinoma samples and that their rate was significantly diminished to 0.5% by subsequent treatment of verapamil, which inhibits the function of ABC transporters (Fig. 1B). Hence, this result confirms the presence of ABC transporter proteins and its exclusive function of drug expulsion of SP cells.

Expression profile of SP and non-SP cells from lung adenocarcinoma
Expression profile of stemness and ABC transporter genes were analyzed by RT-qPCR. The transcriptional regulation of stemness and ABC transporter genes such as OCT-4, NANOG, SOX2 and ABCG2 were found to be significantly accelerated in SP cells ( Fig. 2A). Subsequently, we have performed western blot analysis to monitor the expression pattern of cell surface proteins (CD133 and CD44) and ABCG2 as well. Interestingly, SP cells overexpressed CD133, CD44 and ABCG2 while the parental population cells did not (Fig. 2B). In addition, we observed that the number of total spheres formed by SP cells increased much faster and reached higher values than that formed by non-SP cells (Fig. 3A). The SP cells grew faster, formed tumor spheres more rapidly and more efficiently -spheres produced by SP cells were all augmented in size compared to non-SP cells (Fig. 3B). Therefore, the SP cells can be regarded as able to self- renew, highly tumorigenic, as well as crucial for tumor recurrence and metastasis.

SP cells are highly proliferative and resist DNA targeting drugs
To evaluate the in vivo tumorigenesis potential, we have performed in vitro cell proliferation assay for lung adenocarcinoma SP cells. The FACs sorted SP cells were proliferating much faster on the third day itself (D3) and reached 90% confluence on the seventh day. The growing rate and confluence abilities of non-SP cells were significantly lower than that of SP cells (Fig. 4A). Similarly, the cell differentiation assays revealed that the morphology of SP cells was remarkably changed. SP cells lost their original identity and were extended to form filamentous structures similar to ones present in fibroblasts (Fig. 4B). In addition, the SP cells became resistant to anti-cancer drugs and had prolonged cell survival rate once treated with drugs such as Paclitaxel, Etoposide, Cisplatin, Gemcitabine, Oxaliplatin and 5-Flurouracil (5-FU) (Fig. 5A). The number of SP cells able to invade through the matrigel was significantly higher when compared to non-SP cells (Fig. 5B). The matrigel invasion assay data suggest SP cells do have higher invasion potential. Hence, all our findings suggest that lung adenocarcinoma SP cells play an essential role in the resistance to chemotherapeutic targets that might be involved in treatment failure, recurrence of tumor and invasion.

DISCUSSION
In the present study, we have identified a small, rare group of cancer initiating cells from lung adenocarcinoma samples called "side population (SP)" cells from lung adenocarcinoma samples, which showed the properties of CSCs. In line with previous findings (Ho et al., 2007;Nakatsugawa et al., 2011), our results also demonstrated the properties of SP cells which facilitate the en-    hanced cell proliferation rate, self-renewal capacity and metastasis of lung adenocarcinoma. We have found that approximately 7% of the cells from the tumor of lung adenocarcinoma samples were SP cells, which efflux the Hoechst 33342 dye by the over expression of ABCG2 pumps. Once treated with verapamil (ABCG2 inhibitor), the drug efflux activity was compromised and therefore the proportion of SP decreased to 0.5%. High expression profile of ABCG2 in SP cells contributes to severe resistance to several anti-cancer drugs and promotes prolonged cell survival as well. Therefore, these results confirm the presence and active role of ABC transporter protein in drug resistance of cancer stem cells which leads to the attenuation of apoptosis in cancer cells.
The FACs assorted SP cells were characterized phenotypically and functionally. They exhibited high cell proliferation rate and self-renewing capacity (Chiou et al., 2010;Nakatsugawa et al., 2011;Singh et al., 2012). They were highly capable of forming several-fold higher numbers of tumor spheres all of which were significantly bigger in size over the time course when compared to parental non-SP cells. The high potent sphere formation efficiency is regulated and driven by up-regulation of stemness genes such as OCT-4, SOX2 and NANOG, and is crucial for metastasis of lung adenocarcinoma patients (Salnikov et al., 2010;Nakatsugawa et al., 2011;Singh et al., 2012;Yue et al., 2014). Similar studies in lung cancers reported that the transcriptional upregulation of embryonic stemness genes is highly tumorigenic, may lead to epithelial mesenchymal transition (EMT) like features and might also involve high differentiation properties of SP cells. The possible explanation could be that SOX2 upregulation is due to elevated EGFR signaling mechanism whereas the OCT-4 and NANOG are upregulated by different independent pathways, which remains to be elucidated (Amann et al., 2005;Gazdar et al., 2008;Salnikov et al., 2010;Singh et al., 2012). Recent reports also showed that overexpression of OCT-4 and NANOG increases the tumor initiating property of A549 cells (Chiou et al., 2010).
Further, the enhanced expression of exclusive markers for CSCs such as CD133 and CD44 in SP cells are responsible for chemo-and radiotherapy resistance (Honeth et al., 2008). These cell surface proteoglycans play a major role in cancer relapse, progression, and metastasis in several tumors (Honeth et al., 2008). Consequently, we identified that SP cells are highly invasive in matrigel assay. However, the complexity of mechanisms and receptor signaling pathways involved in SP cells function still needs to be studied in more detail. Taken together, our findings strengthen the hypothesis that elevated levels of ABC transporters and stemness genes promote stem like properties of SP cells which are significantly sufficient for tumor initiating, self-renewing, tumor invasion, metastasis, chemotherapy resistance and tumor recurrence. Further, more research is needed to elucidate the pathways and mechanisms involved in stemness properties and tumorigenic nature of SP cells, as this might lead to the development of a unique chemotherapeutic drug which eradicating CSCs to preclude the tumor recurrence.