† These authors contributed equally.
Academic Editor: Graham Pawelec
Background: The expression levels of the programmed cell death ligand 1
(PD-L1), known as an immune-inhibitory molecule, are closely associated with
cancer stem cell (CSCs) immune escape. Recently, PD-L1 has also been reported to
be able to regulate the self-renewal of cancer stem cells. However, The
expression and intrinsic role of PD-L1 in endometrial cancer stem-like cell
(ECSC) maintenance and its underlying mechanism of action remain unclear.
Methods: Using flow cytometry and western blot assays, we have
demonstrated that PD-L1 expression is higher in ECSCs derived from endometrial
cancer than in nonstem-like cancer cells. Using mouse xenograft assays for ECSC
tumorigenicity. Using gene reporter assay for uncovering the regulation mechanism
of PD-L1 in the hypoxia. Results: We revealed the high expression levels
of PD-L1 in ECSCs and its correlation with self-renewal. We further found that
PD-L1 knockdown reduced expression of several pluripotency-related genes
(aldehyde dehydrogenase 1 (ALDH1), CD133, OCT4, SOX2, NANOG), impaired ECSC
proliferation and undifferentiated colonies and decreased the number of CD133
positive ECSCs and the number of stem-like spheres. Furthermore, we found that
PD-L1 knockdown inhibited ECSC tumorigenicity and the PD-L1 induced self-renewal
capability of ECSCs was dependent upon hypoxia HIF-1
Endometrial carcinoma (EC) is one of the three common malignant tumors of female reproductive system and the most common gynecological genital tract swelling. In recent years, the incidence and mortality of endometrial cancer are gradually increasing, and the trend is getting younger [1, 2, 3]. Studies have shown that cancer stem-like cells are a small population of tumor cells with self-renewal and pluripotency capabilities , and endometrial carcinoma stem-like cell (ECSC) homeostasis plays an important role in the occurrence, invasion, drug resistance and metastasis of endometrial carcinomas [5, 6].
Cancer immunotherapy based on immune checkpoint molecules such as PD-1/PD-L1 can achieve better outcomes for advanced cancers including those with high PD-L1 expression. PD-1/PD-L1 represents a pair of immunocostimulatory factors [7, 8] involved in the immunomodulatory processes of the body, such as autoimmunity, transplantation immunity and tumor immunity. Blockade of the PD-1/PD-L1 signaling pathway with targeted antibodies can enhance their endogenous anti-tumor immune effect .
PD-L1 is not only highly expressed in tumors and involved in their immune escape , but also has been found to be highly expressed in some tumor stem cells where it can regulate their self-renewal [11, 12]. It has been reported that PD-L1 is highly expressed in breast cancer stem cells, where the PI3K/Akt signaling pathway is activated to promote the expression of stemness factors OCT4 and NANOG, and thus maintain the characteristics of breast cancer stem cells. There is also a strong correlation between PD-L1 expression and stemness in breast cancer patients . Furthermore, tumor stem cells with higher PD-L1 can promote their immune escape . Therefore, it is of great value to study the role of PD-L/PD-L1 signal transduction mechanisms in tumor stem cells, and to elucidate the molecular mechanism between PD-L1 and tumor stem cell self-renewal, with an aim of inhibiting this signaling pathway for tumor immunotherapy. Ultimately, this may represent a novel approach for the development of tumor therapies. However, the relationship between PD-L1 and ECSC maintenance and its underlying mechanism of action remains elusive. Therefore, our study aims to determine the relationship between the expression of PD-L1 and the accumulation of stemness-related genes, and thus dissect the potential mechanism involved in the maintenance of stemness.
In this study, we found that PD-L1 expression increased with the ECSC separation process from day 1 to day 8, and the elevated PD-L1 can promote the growth and proliferation of ECSCs, while PD-L1 inhibition prevented this effect. Further analysis of the HIF signaling pathway in the hypoxic microenvironment showed that it regulated PD-L1 by interfering with its promoter region. Thus, we speculate that detecting the expression and mechanism of action of PD-L1 in ECSCs may be critical for the future application of immunotherapy, and the molecular mechanism for the self-renewal in ECSC, therefore, represent a new reference point and basis for further research and clinical application.
The endometrial cancer cell line Ishikawa (ISK) cells were cultured in the
medium of DMEM/F12 (11320082, Gibco, Carlsbad, CA, USA), supplemented with 1
percent penicillin/ streptomycin (15140122, Gibco, Carlsbad, CA, USA), and 10
percent FBS (SV30087, HyClone). ECSC cells were cultured in DMEM/F12 media
supplemented with 10 ng/mL Human Fibroblast Growth Factor Protein (FGF) (AA
10-155, Sigma-Aldrich, St. Louis, MO, USA), 20 ng/mL Human Epidermal Growth
Factor Protein (EGF) (HY-P7109, MedChemExpress, Monmouth Junction, NJ, USA),
0.4% Bovine Serum Albumin (BSA) (HZB0148, Sigma-Aldrich, St. Louis, MO, USA) 20
ng/mL Insulin (HY-P73243, MedChemExpress, Monmouth Junction, NJ, USA) and 1%
penicillin/ streptomycin. For hypoxia exposure, cells were placed in a modular
incubator chamber (Billups-Rothenberg, San
Diego, CA, USA), which was reported before . In testing conditions, ECSC
cells were cultured in defined media before supplemented chemicals as indicated
Analysis of CD133 and ALDH1 population of the ECSCs isolated from Ishikawa cells
by flow cytometry. The ECSCs spheres were harvested after 3 days after formed and
dissociated into single cells, and ALDH1, CD133 antibody was added, then
analysed by flow cytometry. We used cell lysis (C3702, Beyotime, Shanghai, China)
buffer of Red blood cell to remove red blood cells in tumor sample. Cells were
suspended with 2 percent FBS in PBS solution, blocked with 5 percent BSA in PBS
before CD133 and ALDH1 antibody labelling. Cells were stained with antibodies on
ice for 20 min before washing. The CD133 (1:100, CST, Mouse mAb #60577), with
the second antibody anti-mouse IgG (H+L) (Alexa Fluor® 555
Conjugate), and the ALDH1A1 (1:100, CST, Rabbit mAb #36671), with the second
antibody anti-rabbit IgG (H+L) (Alexa Fluor® 488 Conjugate)
(1:100, CST, #4412). Cells were analysed on the FACS Calibur cell analyser (BD
Biosciences, San Jose, CA, USA), or sorted on the FACS cell sorter with
We used TRIzol (15596026, LIFE TECHNOLOGIES, Carlsbad, CA, USA) to extracted the total RNA according to the manufacturer’s instructions. cDNA synthesis was performed using the High Capacity RNA-to-cDNA Kit (4388950, LIFE TECHNOLOGIES, Carlsbad, CA, USA) and SYBR green master mix (Q111-03, Vazyme, Piscataway, NJ, USA). Primers used in this study are shown in Table 1. All results were performed in triplicate, and three independent experiments.
|PD-L1 F||CTACTGGCATTTGCTGAACG||qPCR for human PD-L1|
|PD-L2 F||AAAGAGGGAAGTGAACAGTGCT||qPCR for human PD-L2|
|ALDH1 F||GCTCCATCATCTATCACCCGT||qPCR for human ALDH1|
|CD133 F||CAGAAGGCATATGAATCCAAAA||qPCR for human CD133|
|SOX2 F||GACAGTTACGCGCACATGAA||qPCR for human SOX2|
|OCT4 F||GGTATTCAGCCAAACGACCA||qPCR for human OCT4|
|NANOG F||TTTGTGGGCCTGAAGAAAACT||qPCR for human NANOG|
The modified RIPA lysis buffer (50 mM Tris
SiRNA targeting PD-L1 were purchased from Ribo Bio. SiRNA sequences were
designed as follows: PD-L1, Sense: 5
For luciferase assay, HEK 293T cells were plated into a 24-well plate and
transfected with reporter plasmid plus a Renilla luciferase plasmid as internal
reference using Lipofectamine 2000 reagent in 21% O
Cells were trypsinizedinto single-cell suspensions, then seeded in six-well plates (3736, Corning) with 5000 cells per well. The cells were photographed 3 days later.
Followed with the Tongji University Caring and Laboratory Animals using Guide,
approved by the Ethics Review Committee and Animal Care. We randomly assigned
each group blind. A tatal of 18 female BALB/c mice used each time, there were 3
groups for both NTC and ECSC cells, and six mice were randomly assigned to each
group with three times repeat. A total of 1
We studied the expression of PD-L1/L2 in ECSCs from ECs by firstly obtaining
ECSCs from an EC cell line and tumor samples using our previously described
method . We also enriched ECSCs from endometial cancer cell lines, the
commonly reported stemness marker, CD133, was used to characterize the
endometrial cancer stem cell (CSC). In this study, ECSCs isolated from ISK were
investigated from day 1 to day 8, and we found that the expression of CD133
increased by FACS data (Fig. 1A). To further confirm the high expression of the
PD-L1 in ECSC
PD-L1 is crucial for pluripotency in ECSCs. (A) FACS showing
the percentage of CD133 positive cells in ECSC
PD-L1 has been reported to have a regulatory effect on tumor stem cells, and therefore, this study investigated whether inhibition of PD-L1 signaling could influence ECSC phenotypes. We focused on PD-L1 signaling and its effects on ECSCs. PD-L1 RNA interference was used to elucidate the effect of PD-L1 on the state of the ECSCs and to further study the molecular mechanism of PD-L1 and its effect on the stem-like state of ECSCs. siRNAs were designed to specifically target PD-L1, and quantitative RT-PCR and western blots were used to confirm the effectiveness of the siRNAs (Fig. 2A and 2B). We found that PD-L1 was significantly knocked down after 3 days in ECSC, and these cells also lost their compact colonial morphology. However, the expression of a scrambled siRNA maintained ECSC-like morphology (Fig. 2C), whereas when PD-L1 was knocked down the ECSC cells lost their colonial morphology as well as a reduced expression of pluripotency-related genes (CD133, ALDH1, OCT4, SOX2 and NANOG) (Fig. 2D). Furthermore, cell proliferation and the presence of undifferentiation colonies were consistently decreased by knocking down PD-L1 (Fig. 2E & 2F). FACS analysis revealed that the scrambled siRNA maintained high levels of pluripotency-related markers, including ALDH1 and CD133, while the PD-L1 knockdown in ECSCs did not prevent the loss of these markers (Fig. 2G). The CD133 positivity was significantly reduced, and the percentage of CD133-positive ECSCs was highly correlated with results from PD-L1 knockdown in ECSC cells (Fig. 2H), along with a decrease in the number of spherical cells (Fig. 2I). Therefore, RNA interference of PD-L1 demonstrated that this protein is required for the stem-like state of ECSCs.
The effect of PD-L1 on ECSC stemness. Validation of the siRNAs
targeted to PD-L1 by qRT-PCR (A) and western blot (B). ALDH1 and OCT-4 protein
levels detected in ECSC
Here, we found that PD-L1 promoted a stem-like state in ECSCs and to further
reveal a role for PD-L1 in the modification of ECSCs, we identified the
tumorigenicity of the stem cells using a mouse xenograft assay (Fig. 3A). By
injecting mice with cells (1
PD-L1 induces an endometrial cancer stem-like state.
Tumorigenicity of ECSC
We have found that PD-L1 promoted the stem-like state of ECSCs and to further
explore the underlying molecular mechanism responsible for the regulation of
PD-L1 expression during modification of ECSCs, we analyzed the PD-L1 promoter
sequence. We constructed a truncated PD-L1 promoter luciferase reporter, which
contained the sites –2685/+86 which are related to hypoxia (Fig. 4A). Hypoxia
has been reported to have a regulatory effect on ECSCs  and our luciferase
analysis showed that the HIF signal inducer, CoCl
The expression of PD-L1 is regulated by hypoxia. (A) A
full-length plasmid with truncated PD-L1 and luciferase reporter containing the
sites –2685/+86. (B) ECSC
Therefore, with the isolated ECSCs, we found that PD-L1 promoted a sustained
stem-like state. Mechanically, HIF-1
Mechanism of HIF-dependent expression of PD-L1 in ECSCs. In
ECSCs, the expression of PD-L1 is activated by HIF-1
PD-1/PD-L1 is a critical member of the immunoglobulin superfamily of costimulatory molecules and participates in many immunomodulatory processes. Under normal conditions, PD1 is an inhibitory receptor expressed on activated T cells. When bound to its ligand, PD-L1, it plays an important immune regulatory role, by inhibiting the activation and proliferation of T cells, regulate the expression and secretion of cytokines, and can participate in the immune escape mechanism. Currently, many studies have shown that PD-L1 is highly expressed in tumors, and is closely related to clinical pathology and prognosis and has become a biological indicator for tumor detection and future prognoses. When PD-L1 is expressed, tumor cells bind to the PD1 receptor on T cells which can lead to the formation of an immunosuppressive tumor microenvironment, transduce negative regulatory signals, and lead to the induction of apoptosis and immunosuppression of tumor antigen-specific T cells. These effects enable tumor cells to escape immune monitoring and destruction. However, PD-1/PD-L1 inhibitors can reverse this tumor immunosuppression microenvironment.
PD-1/PD-L1 is regulated by a multi-level network, and the regulation of the
expression of PD-L1 can be divided into five levels: genome level,
transcriptional level, miRNA level, post-transcriptional level and protein level
. In many tumors, soluble factors, oncogenic signals, miRNAs, genetic
variations and post-transcriptional modifications produced by immune cells are
involved in regulating the expression of PD-L1 such as INF-
The above preliminary data suggest that the increased expression of PD-L1 in
ECSCs effectively maintains their self-renewal, and HIFs bind to the PD-L1
promoter region to participate in the regulation of PD-L1 expression, thus
promoting self-renewal of cancer stem cells. Therefore, our research mainly
focused on to explore the mode of action, sites of interaction and specific
regulatory mechanisms of HIF1
In conclusion, we found that the increased PD-L1 expression in the ECSCs was regulated in the hypoxic microenvironment as HIFs bound to the PD-L1 promoter region, thus resulted the self-renewal of ECSCs. This work represent a new target of PD-L1 in cancer stem cells and may benefit for further cancer research and clinical therapy.
ECSCs: Endometrial cancer stem-like cells; HIFs: Hypoxia inducible factors; PD-L1/L2: Programmed cell death ligand 1/2; ALDH1: Aldehyde dehydrogenase 1.
GC, SY designed the study and write the draft. SY performed most of the experiments, with assistance from YG and XW conducted the scientific editing of language. HZ assisted with the data analyses. GC wrote the manuscript.
This study was approved by the Investigation Ethical Committee of Shanghai First Maternity and Infant Hospital, Tongji university school of Medicine, in accordance with the ethical standards or comparable ethical standards. The ethics approval code is KS21289. Written informed consent was obtained from each participant after detailed explanations regarding the study objectives and procedures were provided.
This study was supported by Guofang Chen from the National Natural Science Foundation of China (No.81802960), and the National Natural Science Foundation of China by Shasha Yin (No.81902874).
The authors declare no conflict of interest.