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Introduction

Esophageal cancer (EC), which is mainly classified into esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) based on histopathology, remains one of the major global healthcare challenges due to its poor prognosis (1,2). Cancer stem cells (CSCs), a small subset of cells within solid tumors, have the capacity to self-renew and differentiate into several types of cells that constitute the tumor (3,4). Esophageal CSCs (ECSCs) directly regulate cancer initiation, progression, metastasis, resistance to therapy and recurrence in both EAC and ESCC (5,6). Therapeutic strategies aimed at targeting CSCs may be among the most promising ones for the comprehensive treatment of tumors (7). Various trials have achieved satisfactory efficacy in the treatment of certain hematopoietic malignancies, e.g. the trials on chimeric antigen receptor T-cell therapy (8–10). However, studies on the treatment of solid tumors have not yielded satisfactory results. One of the reasons for this is the difficulty involved in identifying a relatively specific antigen in solid tumors (11).

In the tumor microenvironment, immune cells and tumor cells are in close contact and are mutually influenced (12), which may result in a change in phenotype. For instance, CD70, which is generally expressed on the surface of activated T-lymphocytes, B-lymphocytes and a portion of dendritic cells, is also ectopically expressed in certain solid tumors (13,14). CD14, a specific surface marker of monocytes, macrophages and neutrophils, in combination with lipopolysaccharide, induces pro-inflammatory responses to invading pathogens via the Toll-like receptor 4 signaling pathway (15,16). CD14 has also been indicated to be associated with tumor recurrence, growth, metastasis and resistance to treatment (17–19), which is in conformity with the characteristics of CSCs. It may thus be hypothesized that there is an inevitable connection between CD14 and CSCs.

In the present study, paraffin-embedded sections of human EC (HEC) and tissues adjacent to the tumor (AT) were examined to qualitatively determine the expression of CD14 in ECSCs using immunofluorescence double staining with CD14 and aldehyde dehydrogenase-1 (ALDH1), which is expressed in EAC and ESCC CSCs as an ECSC marker (20–22). CD14+ cells were then isolated and stemness properties were examined by the detection of proliferation, migration, invasion and tumorigenicity.

Materials and methods

Tissue samples

Paraffin-embedded sections of HEC (12 well-differentiated, 11 moderately differentiated and 9 poorly differentiated ESCC tissues; 9 well-differentiated, 9 moderately differentiated and 7 poorly differentiated EAC tissues) and 9 AT tissues were acquired from Mudanjiang Tumour Hospital (Mudanjiang, China) between January 2017 and May 2022, and all tissues of patients were pathologically verified by the Department of Pathology. The HEC tissue specimens were obtained from the surgical specimens of patients with EC undergoing surgery without pre-operative chemotherapy or radiotherapy at the Affiliated Hongqi Hospital of Mudanjiang Medical University (Mudanjiang, China) for cell culture. One patient was a 62-year-old woman with well differentiated ESCC, another patient was a 71-year-old man with moderately differentiated ESCC, and the third patient was a 74-year-old man with moderately differentiated EAC. The clinical and pathological data were collected (Table I) and written informed consent was obtained from all patients. The present study was approved by the Research Ethics Committee of Mudanjiang Medical University (approval no. 2022-MYGZR06).

Table I. Clinicopathological characteristics of the 60 patients with esophageal cancer.

Table I.

Clinicopathological characteristics of the 60 patients with esophageal cancer.

Characteristic	Value
Sex
Male	35 (58.3)
Female	25 (41.7)
Age, years
≥60	36 (60.0)
<60	24 (40.0)
Median (range)	60.3 (45–78)
Differentiation
Squamous cell carcinoma	34 (56.7)
Well	13 (21.7)
Moderate	12 (20.0)
Poor	9 (15.0)
Adenocarcinoma	26 (43.3)
Well	9 (15.0)
Moderate	10 (16.7)
Poor	7 (11.7)

[i] Values are expressed as n (%) unless otherwise specified.

Reagents

The following main reagents were utilized: Collagenase Ӏ (Coolaber); D-MEM/F12 powder (Gibco; Thermo Fisher Scientific, Inc.); basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF) (PeproTech, Inc.); ALDH1 (cat. no. 60171-1-Ig) and CD14 polyclonal antibodies (cat. no. CL647-65056; ProteinTech Group, Inc.); the EasySep™ Human CD14 Positive Selection Kit II (EasySep™; Stemcell Technologies, Inc.); the Cell Counting Kit-8 (CCK-8; Shanghai Yeasen Biotechnology Co., Ltd.); and the EdU Assay/EdU Staining Proliferation kit (Abcam).

Immunofluorescence staining

The paraffin-embedded sections (5 µm thickness) were used to determine the co-expression of CD14 and ALDH1 in ECSCs. Following conventional deparaffinization and rehydration, antigen retrieval was performed using 0.01 mol/l sodium citrate buffer at 100°C for 15 min. The sections were then permeabilized with 0.3% Triton X-100 for 15 min and blocked with 3% methanol-H2O2 solution for 15 min at room temperature. To label the ECSCs, the sections were incubated with ALDH antibody (1:100 dilution) at 37°C for 100 min and stained with the secondary antibody IgG Texas Red (cat. no. ab6800; 1:100 dilution; Abcam, Inc.) at 37°C for 40 min. For the analysis of CD14, the sections were incubated again with CD14 antibody (1:100 dilution) at 37°C for 100 min and stained with another secondary antibody IgG FITC (cat. no. Abs20004-500 ul; 1:100 dilution; Absin Inc.) at 37°C for 40 min. For the cellular count, the sections were counterstained with DAPI (1:100 dilution) at 37°C for 20 min. A total of 7 images per section were captured at a magnification of ×200 (n=3). Quantified results are presented as a percentage of positive staining (red and green) out of the total number of cells, as visualized using DAPI nuclear staining (blue).

Primary culture of human EC

When obtained from surgical specimens, the HEC tissue was transferred to the laboratory as soon as possible. After being washed with PBS three times, the tissue was cut into small sections with a maximum size of 4 mm. The sections were then cultured in serum-free medium (DMEM/F12 containing 10 ng/ml bFGF, 20 ng/ml EGF and 20 ng/ml LIF) under a constant temperature of 37°C in a humidified atmosphere containing 5% CO2. The culture solution was replaced every other day for ~2 weeks until the cells stopped migrating out from the tissue specimens.

Purification of CD14+ cells

CD14+ cells were isolated using the EasySep™ Human CD14 Positive Selection Kit II according to the protocol provided by the manufacturer, as follows: The cells were washed, detached using 0.25% trypsin (Solarbio, Inc.; without EDTA), centrifuged at 800 × g for 4 min at room temperature and suspended with 1 ml PBS in a 5-ml polystyrene round-bottom tube. The cells were incubated with 50 µl EasySep™ Human CD14 Positive Selection Cocktail II for 40 min at 37°C; they were then further incubated with 25 µl EasySep™ RapidSpheres™ for 10 min at 37°C. After 1.5 ml PBS was added to the tube, the Stemcell 18,000 EasySep™ Magnet was used to hold the tube for 5 min at room temperature. The solution which then contained CD14− cells in the tube was poured into another tube, and the CD14+ cells remained.

CCK-8 assay

Cell proliferation was detected using a CCK-8 assay kit according to the manufacturer's protocol. The CD14+ and control CD14− cells were seeded in 96-well plates in 100 µl serum-free medium at a density of 2×103 cells/well and cultured for 24, 48 or 72 h. At the designated time-points, 10 µl CCK-8 reagent was added separately to a well of each corresponding group and the optical density values were measured at 450 nm after the cells were continually cultured for 4 h.

EdU assay

Cell proliferation was also detected using the EdU Assay/EdU Staining Proliferation kit. The cells were seeded in 24-well plates in 500 µl serum-free medium at a density of 5×103 cells/well and cultured for 24 h. The cells were then treated with EdU medium (10 µM final concentration) at 37°C for 21 h, and were then fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 10 min. Subsequently, the cells were thoroughly washed with PBS three times to remove the residual paraformaldehyde before being incubated with DAPI at 37°C for 20 min. Quantified results are presented as the percentage of positive staining (red) out of the total number of cells, as visualized using DAPI nuclear staining (blue).

Colony-formation assay

The CD14+ and control CD14− cells were seeded in 6-well plates at a density of 200 cells/well and maintained in 2.5 ml serum-free medium in an incubator at 37°C with 5% CO2 for 14 days. During this period, the medium was changed on the 11th day. Subsequently, the cells were washed with PBS three times, fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 0.2% crystal violet solution at room temperature for 10 min. Colonies (number of cells, >50) were counted under an optical microscope (Olympus Corporation).

Transwell assay

Cell invasion was detected using Nunc™ Polycarbonate Cell Culture Inserts in Multidishes (cat. no. 140644; 8 µm pore size, 6-well plates; Thermo Fisher Scientific Inc.). Matrigel® (Solarbio, Inc.) was diluted to a 1 mg/ml concentration using DMEM/F12 and added at 100 µl to the chamber, followed by incubation overnight at 37°C for gelling. A total of 1×104 cells in DMEM/F12 (without serum or nutrient factors) were placed in the upper chamber, while 2 ml serum-free medium was added to the lower chamber. Following incubation at 37°C with 5% CO2 for 24 h, the cells at the top side were wiped off and the cells at the lower side of the membrane were fixed with 4% paraformaldehyde at room temperature for 10 min and dyed using 0.1% crystal violet at room temperature for 10 min. Cells were visualized and counted using an optical microscope.

The Transwell migration assay was performed in the same manner but without Matrigel coating.

Wound-healing assay

Cell migration was also detected using a wound-healing assay. The cells were seeded in 6-well plates and cultured in serum-free medium until reaching ~98% confluency. A 100 ul pipette (Thermo Fisher Scientific Inc.) tip was used to create a straight scratch on the monolayer of cells. Following culture at 37°C with 5% CO2 for 24 h, images of the wounded areas were obtained using an optical microscope (model TH4-200; Olympus Corp.).

Transplantation assay

The cell tumorigenicity was assessed in vivo using nu/nu nude mice (male; age, 4–5 weeks; median body weight, 20 g) obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed with three or four animals per cage under specific pathogen-free conditions at a controlled temperature of 24±2°C with 50±10% relative humidity, a 12-h light/dark cycle, ventilation (15 times/h) and free access to food and water. Animal health and behavior were monitored every day. The experiment would be terminated in advance when the weight of the mouse was reduced by 20–25% of the body weight, the tumor weight exceeded 10% of the body weight of the mouse, the tumor was ulcerated or damaged or the animal had poor appetite (50% less than normal; food intake <2g/d) for 3 days. The animals were divided into 2 groups (CD14+ and CD14−) with 7 animals in each group. After being resuspended with PBS, ~5×106 cells were subcutaneously injected in a single flank on the dorsal surface of the mice. The tumors were measured using calipers every other day and once the length of a tumor reached a maximum size of 10 mm, all of the mice were euthanized by excess CO2 in the euthanasia chamber for ~5 min at 45 days after injection. When the animals were euthanized, the EZ SmartBox Prodigy (E-Z Systems, Inc.) was used during the process in compliance with the American Veterinary Medical Association guidelines. The controlled displacement rate of CO2 was 30% volume of the euthanasia chamber per minute. Death was verified by the mice being motionless with absence of breathing and dilated pupils for 5 min. The xenograft tumors were extracted and their weight was determined with an electronic balance (SARTORIUS Corp.; model TH4-200). The present study was approved by the Institutional Animal Care and Use Committee of Mudanjiang Medical University (approval no. 20220228-26).

Statistical analysis

Statistical analysis was performed using SPSS 14.0 software (SPSS, Inc.) and Origin 2021b SR1 v9.8.5.204 (Origin Software, Inc.) statistical software. The data are expressed as the mean ± standard deviation. One-way ANOVA followed by Dunnett's post-hoc test was used to compare the means among multiple groups, whereas statistical comparison of only two groups was performed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of CD14 in ALDH1-labeled ECSCs

Immuno-fluorescence staining was performed to examine whether CD14 is expressed in ECSC tissues. The tissue donors (n=60) had a median age of 60.3 years (range, 45–78 years) and 58.3% were male. Squamous cell carcinoma was the pathology of 56.7% of cases, while the remaining ones were adenocarcinoma (Table I). The ECSC marker ALDH1 was first used to label the ECSCs, and CD14 was then detected to determine its expression in the same EC tissues. In all types of EC tissues, it was observed that the ALDH1-labeled ECSCs had small cell bodies and exhibited more nuclear division, mainly located in the areas of relatively loose tissues and were rich in blood vessels around cancer nests. In addition, the tissues significantly expressed CD14 relative to the AT tissues (Fig. 1).

Figure 1. CD14 is expressed in the ALDH1-labeled esophageal CSCs. (A) Results of immunofluorescence assay of CD14 and ALDH1 expression in human esophageal carcinoma (A2-A7) and adjacent tissues A1 (scale bars, 100 µm). (B) The number of cells with coexpression of ALDH1 and CD14 in esophageal CSCs. *P<0.05 vs. A1. ALDH1, aldehyde dehydrogenase-1; CSCs, cancer stem cells.

Morphological characterization of primary cells

Primary cells were isolated and cultured from HEC tissues by the method of explanted tissue culture. During the cultivation for ~2 weeks, multiple shapes or types of cells were observed and selection bias was not found. As indicated in Fig. 2A, after 4-day culture, viable and adherent cells, which infiltrated from fragments and were uneven in shape, were observed to surround fragments. Subsequently, the adherent cells surrounding fragments developed into multiple shapes and predominantly tended to form parallel spindle-shaped morphology (Fig. 2B). When the fragments were removed from the plate after 12-day culture, the cells under fragments, which were uneven in size and shape, grew side by side with an untypical cobblestone-like morphology (Fig. 2C) and certain cells formed amorphous cell clusters due to overproliferation (Fig. 2D). Following immunomagnetic separation and culture, the CD14+ cells exhibited a spindle-like cell shape, with a smaller cell body relative to the CD14− cells, and the CD14− cells exhibited a predominantly polyhedral or irregular spindle-shaped morphology (Fig. 3A).

Figure 2. Morphology of esophageal cancer cells during primary culture. (A) After culture for 4 days, the viable and adherent cells infiltrated from fragments (the lower shadow) were uneven in shape. (B) Subsequently, the adherent cells developed into multiple shapes and predominantly tended to adopt a parallel spindle-shaped morphology. (C) After 12 days of culture, the cells under the fragments, which were removed from the plate, grew side by side with an untypical cobblestone-like morphology. (D) Certain cells formed amorphous cell clusters (scale bars, 100 µm).

Figure 3. CD14+ cells of esophageal carcinoma possess proliferative abilities. (A) The CD14+ cells exhibited a spindle-like cell shape with a smaller body size relative to the CD14− cells; the CD14− cells exhibited a predominantly polyhedral or irregularly spindle-like shape. (B) The results of the Cell Counting Kit-8 assay revealed that the cellular growth of the CD14+ cells was faster than that of the CD14− cells. (C) The EdU assay revealed that the proliferative ability of the CD14+ cells was higher than that of the CD14− cells. (D) The colony-formation assay further confirmed that the proliferative capacity of the CD14+ cells was higher than that of the CD14− cells (scale bars, 100 µm). *P<0.05 vs. CD14−. OD, optical density.

Proliferative abilities of CD14+ cells

Cell proliferation was evaluated using CCK-8, EdU and colony-formation assays. The results of the CCK-8 assay revealed that the cellular growth of CD14+ cells occurred more rapidly than that of the CD14− cells (Fig. 3B). In addition, the results of the EdU and colony formation assays further confirmed that the proliferative ability of the CD14+ cells was higher than that of the CD14− cells, which was consistent with the results of the CCK-8 assay (Fig. 3C and D).

Metastatic and invasive abilities of CD14+ cells

The results of the Transwell assays revealed that the number of CD14+ cells with an invasive and migratory ability was significantly higher than that of the CD14− cells (Fig. 4A and B). Furthermore, the wound-healing assay demonstrated that the migratory ability of the CD14+ cells was higher than that of the CD14− cells (Fig. 4C).

Figure 4. CD14+ cells of esophageal carcinoma exhibit metastatic abilities. (A and B) The results of the Transwell assay revealed that CD14+ cells exhibited (A) invasive and (B) migratory abilities, and the numbers of invasive cells and migratory CD14+ cells were significantly higher than those of the CD14− cells (scale bars, 100 µm). (C) The wound-healing assays demonstrated that the migratory ability of the CD14+ cells was higher than that of the CD14− cells (scale bars, 250 µm). *P<0.05 vs. CD14−.

Tumorigenic ability of CD14+ cells in vivo

Tumor xenografts were established using nude mice to examine the tumorigenic ability of CD14+ cells. At 45 days following implantation, the tumors in the CD14+ cell group were distinctly heavier than those in the CD14− cell group (Fig. 5).

Figure 5. CD14+ cells of esophageal carcinoma possess tumorigenic abilities in vivo. At 45 days following implantation into nude mice, the tumors in the CD14+ cell group were distinctly larger than those in the CD14− cell group. *P<0.05 vs. CD14−.

Discussion

The understanding of CSC morphology in tumor tissues may contribute to the observation, identification and localization of CSCs and may also avoid interference by false-positive staining in staining experiments related to CSCs. However, there is frequently no clear morphological distinction between tumorigenic and non-tumorigenic cancer cells (23). According to the data presented in the current study, the size of the labeled ECSCs was similar to that of the neutrophils, based on the size of the false-positive erythrocytes, and was evidently smaller than that of the cells of tumor lobes, particularly in moderately and poorly differentiated tissues; it was also possible to observe the nuclear division of the ECSCs, and the karyoplasmic ratio was relatively larger compared to the cells of tumor lobes. Of note, reports on CSC distribution in tumor tissues are controversial. It has been reported that CSCs are preferentially located in the core of the tumor lobes (a hypoxic environment) in vivo (24), and Ji et al (25) found diffuse CSCs in ESCCs. The present study demonstrated that the diffusive ECSCs were mainly located in areas that were relatively loose and rich in blood vessels, around the cancer nests. Regarding the quantification of CSCs, the statistical proportion of CSCs varies markedly in tumor tissues, even among primary cell lines, due to differences in sampling site, sample size, tumor grade, patient age and detection methods in different studies (26–29). The main aim of the present study was to qualitatively detect the expression of CD14 in ECSCs.

The three major characteristics of CSCs are an unlimited proliferative ability, self-renewal ability and an ability for strong tumorigenesis; these abilities were thus examined to identify and verify the stemness of isolated cells (30). CCK-8 and EdU assays are two common methods used to investigate the proliferation and/or self-renewal ability of cancer cells (31). In addition, two main methods have been applied to identify tumorigenesis characteristics in published studies. One approach is the colony formation assay, which is also used to detect the proliferation of cancer cells, which is considered the most appropriate in vitro (32); the other approach is the xenograft assay, an in vivo method involving the implantation of cancer cells into immunodeficient mice (33). However, several issues are associated with the transplantation assay. A total of 105 CSCs, which may not be indicative of a rare tumor-initiating cell, are frequently used in transplantation experiments, which poses difficulties in inducing efficient tumorigenesis due to species barriers, host strains, developmental stages and even sex (34). On the other hand, the majority of tumor cells, even those not associated with stem cell markers, result in tumor initiation due to CSC plasticity by the host microenvironments (35–37). However, the majority of studies to date using CSCs still utilize the transplantation assay to prove the existence of CSCs for a particular tumor, as a better alternative is not yet available. In addition, the metastatic ability of CSCs is usually examined using Transwell and wound-healing assays in vitro (38). Using the aforementioned methods, the present study determined that CD14+ cells of EC possessed the characteristics of ECSCs.

In solid tumors, membrane-associated proteins are usually utilized for research into CSCs. The membrane-associated biomarkers associated with ECSCs include the following: CD34 (39), CD44 (40), CD90 (41), CD133 (42), CD271 (43), CD326 (44), LgR5 (45), integrinα7 (46) and podoplanin (47). However, these membrane-associated proteins are similarly found in normal tissue cells; thus, the availability of CSC-specific biomarkers is limited (48). As a type of stromal cells, telocytes express CD34 (49); CD44 and CD90 are the positive markers of mesenchymal stem cells (50) and CD44 is equally expressed in normal head and neck epithelium (51,52); CD133 has been used as a marker to identify prostate and neural stem cells, and is also expressed in differentiated epithelial cells in certain organs, such as the pancreas, liver, colon, gastric contents, as well as sweat, salivary and lacrimal glands (53); CD271 has been found to be expressed in human adipose-derived mesenchymal stem cells (54) and the glial cells of the central and peripheral nervous systems (55); CD326 is expressed in certain epithelial cells (56) and various stem and progenitor cells (57,58); LGR5 has been suggested to be a marker of adult stem cells of the intestine, stomach, skin and hair follicles (59,60); integrin α7 is a key adhesion receptor that is highly expressed in vascular smooth, skeletal and cardiac muscle (61,62); podoplanin expression has been detected in a variety of normal tissues, including glomerular podocytes, lymphatic endothelial cells, heart cells, type I alveolar cells and skeletal muscle (63). With regard to CD14, it is mainly expressed on myeloid-derived cells, such as monocytes, polymorphonuclear leucocytes and macrophages (15,16). In the present study, while CD14 was not found to be expressed in immunocytes in the tissues of esophageal cancer by immunofluorescence double staining, possibly due to incomplete detection, it remains to be further elucidated whether CD14 is a highly selective marker for ECSCs.

In conclusion, the present study demonstrated that the ALDH1-labeled ECSCs expressed the surface marker CD14; in vitro and in vivo experiments further confirmed that the primary CD14+ cells possessed the characteristics of CSCs. Regarding the distribution of CD14 in adult tissues, CD14 may be considered a novel surface marker of ECSCs; thus, this may provide a novel therapeutic target against ECSCs, which may be used in the treatment of EC. However, the lack of knockdown and CD14 expression rescue experiments was one limitation of the present study, and the lack of experiments on chemoresistance both in vivo and in vitro was another limitation. Further research is required to confirm the role of CD14 in the maintenance of ECSC characteristics.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Start-up Fund for Doctor Scientific Research of Mudanjiang Medical University (grant no. 2021-MYBSKY-024).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Authors' contributions

JuL, YoL and JD conceived and designed the study. YoL, WY, YuL, ZL, JiL and LZ performed the experiments. CW and JQ analyzed the data. YoL wrote the first draft of the manuscript. LuJ and YoL confirm the authenticity of all the raw data. All authors contributed to the article and approved the submitted version, and all authors have read and approved the final manuscript.

Ethics approval and consent to participate

The experiments involving human participants were reviewed and approved by the Research Ethics Committee of Mudanjiang Medical University (approval no. 2022-MYGZR06). The patients provided their written informed consent to participate in the study. The animal experiment was reviewed and approved by the Institutional Animal Care and Use Committee of Mudanjiang Medical University (approval no. 20220228-26).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References