α2δ1 may be a potential marker for cancer stem cell in laryngeal squamous cell carcinoma
Abstract
Cancer stem cells (CSCs) have the ability to dictate tumor initiation, recurrence, and metastasis. Here, we examined the expression of a
1.Background
Laryngeal cancer is the second most common head and neck malignancy. Globally, approximately 110,000 to 130,000 new cases are diagnosed each year [1]. In China, the crude incidence rate of laryngeal cancer was 1.86/100,000 between 2008 and 2012 and the crude mortality was 1.01/100,000 [2]. Mainstay treatments for laryngeal cancer include radiotherapy, chemotherapy and surgical resection; however, management of advanced laryngeal cancer is complex and a consensus therapeutic strategy has not yet emerged.
Cancer stem cells (CSCs) are implicated in tumor initiation and differentiation, and are of high self-renewal properties, and can drive the tumorigenic process. The ability of CSCs to drive cancer initiation and progression also make them prime targets for treatments [4]. Evidence suggests that a minor population of CSCs in laryngeal cancer is extremely tumorigenic and possesses the potential to differentiate into cells that are responsible for tumor propagation and relapse [4]. Laryngeal CSCs are characterized by the expression of aldehyde dehydrogenase isoform 1 (ALDH1) [5], CD133 [6], and CD44 [7, 8]. However, laryngeal CSCs appear to differ considerably and may contribute to the heterogeneity of laryngeal cancer [9]. The calcium channel
Currently, no study is available on the role of
2.Materials and methods
2.1General design
The current study included 3 major parts. In the first part that involved clinical samples,
2.2Tissue specimen acquisition
Archived surgically resected laryngeal squamous cell carcinoma (LSCC) tissue specimens were obtained from 16 treatment-naive male patients and snap-frozen in liquid nitrogen. The study protocol was approved by the institute ethics committee of Beijing Friendship Hospital, Capital Medical University (no. 2017-P2-187-01) and written informed consent was obtained from all the study subjects.
2.3Immunofluorescence staining
Frozen tissues were sectioned with acryostat and fixed with methanol for 30 seconds. After blocking with 5% nonfat milk in PBS, slides were incubated with
2.4Cells
Human LSCC cell lines TU212 and TU686 were obtained from Shanghai Huiying Biological Technology (Shanghai, China), and were cultured in RPMI 1640 medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen) at 37
Table 1
Primer sequences for quantitative PCR in the study
Gene | Sense | Anti-sense |
---|---|---|
BMI1 | 5-AGCAGCAATGACTGTGATGC-3 | 5-CAGTCTCAGGTATCAACCAG-3 |
SOX2 | 5-ACATGAACGGCTGGAGCAAC-3 | 5-AGGAAGAGGTAACCACAGGG-3 |
EPCAM | 5-CTGAATTCTCAATGCAGGGTC-3 | 5-CCCATCTCCTTTATCTCAGCC-3 |
OCT-4 | 5-TGGGCTCGAGAAGGATGTGGTC-3 | 5-AGTTTGAATGCATGGGAGAGC-3 |
CTNNB | 5-TGATGGAGTTGGACATGGCC-3 | 5-CTCATACAGGACTTGGGAGG-3 |
KLF4 | 5-AAGCCAAAGAGGGGAAGAC-3 | 5-CATCTGAGCGGGCGAATTTC-3 |
NANOG | 5-TGCCTCACACGGAGACTGTC-3 | 5-TGCTATTCTTCGGCCAGTTG-3 |
ABCG2 | 5-GGAGGCCTTGGGATACTTTGAA-3 | 5-GAGCTATAGAGGCCTGGGGATTAC-3 |
MDR-1 | 5-GCCTGGCAGCTGGAAGACAAATAC-3 | 5-ATGGCCAAAATCACAAGGGTTAGC-3 |
GAPDH | 5-GACCCCTTCATTGACCTCAAC-3 | 5-CTTCTCCATGGTGGTGAAGA-3 |
2.5Flow cytometry
For isolation of
2.6Quantitative reverse transcription (qRT)-PCR
Total RNA was extracted from
2.7Plasmids
Retroviral-based plasmids containing a short hairpin RNA (shRNA) against human
2.8Sphere formation assays
Sphere-formation assays were carried out as previously published [10]. Briefly,
2.9Cell differentiation assays
Figure 1.
Expression of
![Expression of α2δ1 in primary LSCC tissues. (A) Representative images of immunofluorescence expression of α2δ1 in laryngeal tissue, paracancerous tissue and normal adjacent tissue in a patient with T3N0M0 laryngeal cancer are shown. α2δ1 (green) was stained with specific monoclonal antibody and nuclei (blue) were stained with DAPI. Arrows indicate α2δ1-positive cells. (B) Summary of α2δ1 expression in LSCC tissues, paracancerous tissues and normal adjacent tissues.](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g001.jpg)
2.10Matrigel assays
For detection of migration and invasion of
2.11Tumor xenograft assays
Four to 6 week-old female non-obese diabetic/severe combined immunodeficient (NOD/SCID) female mice were purchased from Huafu Kang Experimental Animal Co., Ltd (Beijing, China), and maintained in a SPF facility. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Animal Care and Use Committee at Peking University Cancer Hospital.
Cells were suspended in 50 mL in a 1:1 mixture of RPMI 1640 and Matrigel (BD Biosciences) and 10
2.12Statistical analysis
Data was analyzed using SPSS 17.0 software (IBM, Armonk, New York, NY, USA). Student’s
Figure 2.
Expression of stem cell and drug efflux and resistance genes in
![Expression of stem cell and drug efflux and resistance genes in α2δ1-versusα2δ1+ subpopulation of TU686 cells. qRT-PCR analysis of the expression of stem cell markers and drug-resistance-related genes in purified α2δ1+ and α2δ1- subpopulations of TU686 cells. Data are presented as fold difference over α2δ1- cells for each gene. Compared with α2δ1- cells, α2δ1+ cells consistently expressed 1 to 4.5 fold higher levels of BMI1, SOX2, EPCAM, OCT4, CNTTBKLF4, NANOG, ABCG2 and MDR1. *Error bars indicate SD.](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g002.jpg)
Figure 3.
Sphere-forming efficiency of
![Sphere-forming efficiency of α2δ1+ and α2δ1- cells. (A, B) The sphere-forming ability of α2δ1+ and α2δ1- cells was examined by sphere formation assays as detailed in Methods. (C) Histograms show the spheroid forming efficiency of FACS-sorted α2δ1+ and α2δ1-fractions from indicated sources. The ability of the spheres formed by α2δ1+ TU 212 cells (D) and TU 686 cells (E) to form secondary spheroids is also shown (α2δ1+/2nd passage). (F) The sphere forming ability of purified α2δ1+ TU212 cells was reduced when α2δ1+cells were incubated with indicated lentivirus for 4 hours, and were plated at 100 cells per well (n= 5). One hundred cells per well were plated(n= 5). Spheroids (⩾ 100 μm) were counted under a stereomicroscope. Statistical analysis was carried out using data on sphere formation in 100 cells (C, F). Data are represented as mean ± SD.](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g003.jpg)
3.Results
3.1α 2δ 1 is predominantly expressed in LSCC tissues
Immunofluorescence staining revealed abundant
3.2Stem cells-associated genes and drug efflux and resistance genes are significantly upregulated in α 2δ 1+ TU686 cells
We isolated
3.3Self-renewal property of α 2δ 1+ cells
Sphere formation assays further showed that
3.4Differentiation properties of α 2δ 1+ cells
After purified TU212
3.5Migratory and invasive potential of α 2δ 1+ cells
Our Matrigel assays showed that
3.6Chemoresistance of α 2δ 1+ cells
We treated TU686 cells with cisplatin (6
3.7Tumorigenesis of α 2δ 1+ cells
As few as 10
Figure 4.
Differentiation properties of
![Differentiation properties of α2δ1+ TU212 and TU686 cells. Flow cytometry shows the percentage of α2δ1+ cells in TU212 and TU686 cells, FACS-purified α2δ1+ cells and purified α2δ1+ cells cultured in 10% serum-containing medium for 1 week (cultured).](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g004.jpg)
Figure 5.
Migration and invasive potential of
![Migration and invasive potential of α2δ1+ cells. Sorted α2δ1+ and α2δ1- cells were assayed for their invasive ability on Matrigel using a Boyden chamber assay. The invasive and migratory potential of α2δ1+ cells (A, C) and α2δ1- cells (B, D) were detected. Statistical results of invasive cells were calculated (E). Data represent the mean ± SD of three independent experiments. *Student’s t-test.](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g005.jpg)
Figure 6.
Chemoresistance ofTU686 cells. The percentage of sorted
![Chemoresistance ofTU686 cells. The percentage of sorted α2δ1+ TU686 cells after exposure to cisplatin (6 μmol/L) or paclitaxel (0.2 μmol/L) for 72 hours.](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g006.jpg)
Figure 7.
Tumorigenesis of
![Tumorigenesis of α2δ1+vs.α2δ1-subpopulation. (A) TU212 cells were transplanted subcutaneously in NOD/SCID mice as indicated. (B, C) Representative photographs showing dissected tumors by FACS-purified α2δ1+ (red arrows) and α2δ1- (blue arrows) TU212 cells. (D) TU686 cells were inoculated subcutaneously in NOD/SCID mice as indicated. (E) Photographs showing dissected tumors formed by FACS-purified α2δ1+ and α2δ1- TU686 cells. (F, G) Serial inoculation of 102 and 103 TU686 cells into NOD/SCID mice from tumors formed by α2δ1+ TU686 cells. (H, I) Tumorigenicity of TU686 α2δ1+ cells FACS-purified from indicated sources after α2δ1 shRNA53/shRNA56 knockdown. TU686 α2δ1+ cells were incubated with indicated lentiviruses for 4 hours, and 102/103 cells per mouse were injected. The statistical results of tumorigenic formation in 5 mice were calculated. (J) The volume and weight of tumors by α2δ1+ cells, and α2δ1+ cells knockdown with shRNA53and shRNA56 were detected.](https://ip.ios.semcs.net:443/media/cbm/2019/24-1/cbm-24-1-cbm181947/cbm-24-cbm181947-g007.jpg)
4.Discussion
Immunofluorescence staining of 16 LSCC specimens showed that
Next, we showed that TU212 and TU686 cells also contain a
We next evaluated the differentiation and migration/invasion potential of the
Consistent with a previous study on
Previous studies have confirmed that
Upon in vivo tumor xenograft assays, 100 purified
5.Conclusion
In summary, the results from the current study indicated the presence of a
Acknowledgments
This research was supported by the Scientific Research Common Program of Beijing Municipal Commission of Education (#KM201510025028) and Research Foundation of Beijing Friendship Hospital, Capital Medical University (#yydszx2015-02, #yyqdkt 2014-23).
Conflict of interest
The authors declare that they do not have any potential conflict of interests in relation to the contents of this manuscript.
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