Non-small cell lung cancer (NSCLC) mainly included lung adenocarcinoma (LUAD, 40%), lung squamous cell carcinoma (LUSC, 30%), and large cell carcinoma (15%) (1). LUAD and LUSC had different characteristics in the following aspects, including mutation models, pathogenesis, molecular mechanisms, treatment plans, and personalized medical services (2). Cancer stem cells (CSCs) were a population of cells in tumors with self-renewal and infinite proliferation capability and could be the resource to generate heterogeneous tumor cells (3). CSCs played an important role in tumor survival, proliferation, epithelial-to-mesenchymal transition, invasiveness and migration capability, metastasis, recurrence, and therapy resistance (4). According to the theory of CSCs, CSCs had the ability to escape from antitumor therapies; thus, CSCs could not be killed by conventional chemotherapies, which only kill the bulk of differentiated and differentiating cancer cells (5). In this process, CSCs might express a variety of resistant molecules and mediate some resistance-related pathways, including polycomb group transcriptional repressor Bmi-1 pathway, Notch pathway, sonic hedgehog, Wnt pathway, phosphoinositide 3-kinase (PI3K)/AKT, tyrosine kinase receptors pathways [such as epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR)/MET, insulin-like growth factor receptor (IGFR), and platelet-derived growth factor (PDGF)], and transforming growth factor β (TGFβ)/SMAD pathway (6). CSCs were commonly identified with fluorescence-activated cell sorting plus specific antibodies binding to CSC-surface markers (ALDH1A1, CD24, CD34, CD44, CD133, and EPCAM) (7, 8). CSCs were found in multiple cancers, including lung cancer, breast cancer, colorectal cancer, liver cancer, prostate cancer, and blood tumor (9). Recent studies found that lung resident epithelia such as variant club cells, basal cells, club cells, and alveolar epithelial type-2 cells had facultative stem cell reparative activities (10). Additionally, the CSC theory introduced a new concept—mRNA expression-based stemness index (mRNAsi) based on machine learning algorithm (11). The mRNAsi was calculated with the transcriptomic expression profile of tumor samples from The Cancer Genome Atlas (TCGA) according to transcriptomic datasets of non-transformed pluripotent stem cells and their differentiated progeny (12). CSC theory proposed that tumor tissue contained its own cancer itself. Therefore, CSC theory might cast new lights on the nature of tumors and clinical differentiation therapy between LUAD and LUSC, respectively.

The development of tumor immunotherapy promoted the applications of immune checkpoint inhibitors in NSCLC, such as anti-PD-1/PD-L1 immune checkpoint therapy (13). Currently, eight kinds of PD-1/PD-L1 checkpoint inhibitors were approved worldwide, including six kinds of PD-1 checkpoint inhibitors [Merck Keytruda, Bristol-Myers Squibb Opdivo, Pfizer/Merck Bavencio, Sanofi/regenerative Libtayo, Jun Tolpalimab (Treplimab), and Cinnabi/Lilidabo Shu^® (Tyvyt, Sintelimab)] and two kinds of PD-L1 checkpoint inhibitors (Roche Tecentriq and AstraZeneca Imfinzi) (14). With the renaissance of anticancer immunotherapy and the occurrence of CSC escape from antitumor therapies, it might be essential to further explore the interplay between tumor immune microenvironment (TME) and CSCs to discover novel cancer approaches to treat drug resistance and immune escape of CSCs (15). Recent studies found that mRNAsi was closely related to TME and that higher mRNAsi was identified in many tumors with a lower-expressed PD-L1 and a reduced leukocyte fraction (11). Immunotherapy strategy largely depended on the infiltrated immune cells and upregulated PD-L1. However, those cancers with higher mRNAsi might be less susceptible to immune checkpoint blockade therapies, which suggested a potential mechanism of immune evasion (16). TME contained various cell types such as monocytes, natural killer (NK) cells, macrophages, eosinophils, dendritic and mast cells, and neutrophils (17). The relationships between these cell types and CSCs were assessed in tumors; for example, stem-cell markers (Nanog, Lgr5, CD44v6, and ALDH1A1) were highly associated with immune cell counts, which revealed that that cancer stemness and immune state should be considered as a whole (18). Additionally, some cytokine signals were also important for CSCs; for example, IL33 could mediate stem cell genes to stimulate cell sphere formation and increase tumor resistance to chemoradiotherapy, and IL33 activated OCT3/4, NOTCH3, and NANOG and enhanced transcription factor (TF) c-Jun that binds to the promoters of hub stem-cell-related genes (19). Moreover, CSCs had immunomodulatory capabilities and communicated with TME through producing immune system inhibitory factors. Thus, CSCs could interact with immune checkpoint molecules, such as Tim3, CD47, CTLA4, PDL-1, and LAG3, to protect cancer cells from immune clearance (20). Therefore, it was necessary to study the correlation between stemness (mRNAsi) and immunity (the fraction of immune cell populations, activation state of immune cells, or alterations of immune-related genes and pathways).

This study aimed to identify prognostic immune-related gene model related to stem cell characteristics based on mRNAsi in LUAD and LUSC, respectively. The immune cell types were profiled with CIBERSORT in lung cancers, which provided insights into the relationships between mRNAsi and infiltrating immune system cells and between mRNAsi and immune-related genes. Moreover, the constructed network between TFs and stemness-related differently expressed immune-related genes (DEIRGs) revealed the potential mechanisms of stemness-related DEIRGs in LUAD and LUSC, respectively. The overall experimental flow chart was showed for this study ( Figure 1 ), and this method would also be easy to transit for other cancer studies.

With in-depth study of CSC theory, it was found that CSCs had (i) the obvious characteristics such as self-renewal and unlimited proliferation; (ii) pathological behaviors to participate in radio-resistance, metastasis, relapse, and drug resistance; and (iii) extensive clinical application in diagnosis and treatment, in lung cancers (22, 23). One study separated CSCs with single-cell assay of human LUAD cancer cells (A549), with CD44+/CD24−/phenotype characteristics, special stemness-related gene expression, and Hoechst 33342 dye efflux assay, and the secondary protein structure of CSCs changed when compared to non-stem cancer cells (24). It clearly demonstrated that CSCs might be prognostic biomarkers in cancer therapy (24). Multidrug resistance and epithelial–mesenchymal transition (EMT) were closely associated with the existence of CSCs, which offered promise in developing reliable CSC markers of NSCLC-associated multidrug resistance and EMT. Some studies began to focus on specific stemness-related pathways such as Wnt/β-catenin pathway and revealed the mechanism of malignant phenotype (25). A CSC-targeted therapy with good biocompatibility and targeting capability was developed to disrupt the growth and survival of CSCs, which might provide an effective approach to prevent the relapse caused by CSCs (26). A study on CSC might provide valuable clues to solve the poor prognosis of lung cancer. Some lung cancer CSCs might be retained, but not eliminated, under the traditional therapies. The interaction between TME and CSCs might provide new clues to treat CSCs in a cancer (27). Especially in TME, the relationship between lung CSCs and tumor-infiltrating lymphocytes was very marginally considered. High ALDH expression was a key biomarker for CSCs. Immunohistochemistry revealed statistically positive correlations between ALDH+ and CD8+ and between ALDH+ and CD3+ cells populations. CSCs existed in different lymphocyte distribution, which might contribute to precision medicine management of different kinds of lymphocytes (28). CSCs also showed obvious properties to shape TME and help themselves to escape recognition from the immune system and become immunosuppressive (29). In turn, tumor-infiltrating immune cells also affected the status of tumor cells. For example, inflammatory factors [TGF-β combined with tumor necrosis factor alpha (TNF-α)] could induce tumor cells to turn CSCs with high expression of CD133, CD44, Bmi1, and Oct4 (30). CD4+ CD25+ Tregs were well-known to reduce antitumor immunity, and Tregs increased side population of mouse breast cancer cells, promoted sphere formation, and upregulated stemness-related genes. It revealed a certain interaction between Tregs and CSCs, which suggested that targeting the crosstalk between Tregs and CSCs was a promising strategy (31). Thus, better understanding of individualized immune signature and tumor immunology of CSCs might be one of the most effective treatments under the background of immunotherapy strategy (32).

This study analyzed the associations between cancer stemness and TME in lung cancer, which identified DEIRGs between high- and low-mRNAsi subtypes in LUAD and LUSC, respectively. The literature reported that some DEIRGs played an important role in stemness processes. Interleukin-27 (IL27) encoded one of subunits of a heterodimeric cytokine complex (33). The IL27–IL12B complex drove rapid expansion of naive CD4(+) T cells and trigger interferon gamma (IFNG) production of naive CD4(+) T cells. IL-27 downregulated stemness-related genes, such as SONIC HEDGEHOG in LUAD cells, and NOTCH1, SOX2, KLF4, Nestin SNAI2/SLUG, OCT4A, SNAI1/SNAIL, and ZEB1 in LUSC cells (33). Additionally, clinical data showed that even immunocompromised or advanced NSCLC patients could benefit from the treatment of IL-27 based on its ability to downregulate stemness-related genes (33). Bone morphogenetic protein 7 (BMP7) encoded a member of TGF-beta family of proteins. The function of BMP7 was to activate a dormant state in cancer cells by p38 MAPK signaling and p21 expression to reduce stemness (34).

C–C motif chemokine ligand 1 (CCL1) belonged to chemokine CC family and was secreted by activated T cells and displayed chemotactic activity for monocytes. CD4+ CD25+ Tregs could reduce antitumor immunity and had a crosstalk with CSCs. The activated NF-κB-CCL1 signaling could recruit CD4+ CD25+ Tregs by reducing the binding of H3K27Me3 on promoter regions of p65 and Ccl1, and recruited CD4+ CD25+ Tregs increased the expressions of stemness-related genes (31). Heparin-binding growth factor 8 (FGF8) encoded by this gene was a member of the fibroblast growth factor (FGF) family. FGF family members played crucial roles in mitogenic and cell survival activities. FGF8 was also reported to dramatically enhance stemness (35). Activated macrophages (M2) secreted chemokines, such as chemokine ligand 17 (CCL17), whose expression was significantly associated with clinical pathological characteristics of hepatocellular carcinoma and with poorer overall survival rates. The stemness could be examined by flow cytometry, sphere formation, and Western blot. When cancer cells treated with CCL17, stemness-related markers were highly expressed, and EMT process was enhanced (36). The cytokine colony-stimulating factor 2 (CSF2) controlled production, differentiation, and function of granulocytes and macrophages. Myeloid-derived suppressor cells (MDSCs) were one kind of immune cells leading to tumor immune escape, which was verified with cell colony formation, tumor sphere formation, and CSC biomarkers (NANOG and c-MYC). MDSCs could induce CSCs and promote tumor immune evasion in different kinds of cancers through CSF2/p-STAT3 signaling pathway. When CSF2 was deleted in tumor cells, cell stemness could be markedly reversed through downregulating CSF2 expression (37). Further studies on the interactions between immune system and CSCs and targeting on the key point among them might be a promising strategy in cancer therapy.

This study found that DEIRGs between high- and low-mRNAsi subtypes in LUAD and LUSC were enriched in some crucial signaling pathways. Some enriched signaling pathways were reported to play an important role in stemness processes. For example, Janus kinases/signal transducer and activator of transcription (JAK/STAT) signaling pathway took part in cellular processes such as proliferation, cell death, cell division, immunity, and tumor formation, and this pathway could help transfer communication information from extracellular chemical signals to cell nucleus to activate gene transcription process (38). A previous study on microRNA-related signaling pathways contributing to stemness features of CSCs found that JAK/STAT was one of the various targeted signaling pathways that contributed to dysfunctions of stemness-related microRNAs (39). This finding prompted one to further study whether DEIRGs between high- and low-mRNAsi subtypes enriched in JAK/STAT pathway (CNTFR, CRLF2, CSF2, IL12B, IL2, and IL22RA2 in LUAD) affect stemness in lung cancer. The chemokine family depended on seven-transmembrane-spanning G-protein-coupled receptors to generate activity. The diversity of chemokine–chemokine receptor interaction made the function of chemokines become more complicated and plastic. Chemokines and their receptors did not only mediate leukocyte infiltration into TME but also multiple hallmark processes of a cancer. However, chemokines and their receptors were not fully understood yet (40). A study found chemokines to be implicated in contributing to tumor heterogeneity through maintenance or promotion of stem-like phenotype. The CXCR4 (chemokine)/CXCL12 (chemokine receptor) interaction helped to promote tumor-initiating cells in lung carcinomas, which was associated with resistance to chemotherapy (41). DEIRGs between high- and low-mRNAsi subtypes enriched in cytokine–cytokine receptor interaction pathway (BMP7, CCL17, CCR9, CNTFR, CRLF2, CSF2, IL12B, and IL2 in LUAD, and CCL16, CCL1, IL2, CCNA2, IL26, CCL23, IL27, and DOLKL in LUSC) could provide potential and novel cytokines and cytokine receptors for CSC study. Antigen processing and presentation was a vital immune process to trigger T-cell immune response. The recognition of fragmented antigens in tumor cells by immune cells depended on major histocompatibility complex (MHC) and related receptors on immune cell surfaces (42). Studies showed that immunogenicity of CSCs was lower than non-stemness cancer cells, and CSCs were good at concealing their specific antigens to escape the immune effect of immune cells (43). Therefore, it was necessary to recognize and increase the specific antigen on the surface of tumor stem cells to increase immune. Renin–angiotensin system (RAS) was commonly known to regulate systemic vascular resistance, body fluid homeostasis, and blood pressure. However, RAS was also involved in maintenance and differentiation of CSCs, which showed that identification of RAS components in CSCs could provide a novel way for therapeutic targeting with RAS modulators in common clinical use (44). DEIRGs between high- and low-mRNAsi subtypes enriched in RAS-related pathways (CMA1 and CTSG in LUAD) were consistent with previous studies. Recently, a great improvement has been achieved from immune checkpoint therapy; for example, immune checkpoint therapy can kill CSCs (45). PD-L1 was one of definite checkpoints for tumor cells to escape from immune surveillance because PD-L1 binds PD-1 receptor to block T cell function. A study found that PD-L1 was highly expressed in CD133+CD44+ colorectal CSCs, and the altered PD-L1 could upregulate the expressions of stemness-related genes to increase CSC self-renewal ability to form tumor spheres. Thus, anti-PD-L1 monoclonal antibodies might crucially contribute to kill CSCs. DEIRGs between high- and low-mRNAsi subtypes enriched in antigen processing and presentation pathway (KLRC4, KIR2DL1, KLRC3, KLRC1, KIR2DS4, and KIR2DL3 in LUSC) could offer much more possible mechanisms of immunological process of CSCs. Further study on the interaction between immune-related pathways and CSCs would make potential mechanism of immunotherapy more clearer in cancer therapy.

The interaction between CSCs and immune cells and alteration of tumor-infiltrating immune cells around CSCs were observed in various cancers (27). This study found that the distribution of immune cells between high- and low-mRNAsi subtypes were significantly different, including B cells memory, dendritic cells resting, macrophages M0, mast cells resting, monocytes, plasma cells, T cells CD4 memory resting in LUAD, and macrophages M1, plasma cells, T cells CD4 memory resting, T cells CD4 memory activated, and T cells CD8 in LUSC. Some of these immune cells were reported to play an important role in stemness processes. For example, cellular colocalization of cathepsins B and D on CSCs and cathepsin G on mast cells suggested that the altered distribution of mast cells was observed in TME CSCs (46). The efficiency of dendritic cell (DC)-based treatment targeting CSCs was tested in breast cancer treatment. After DCs pulsed with breast CSC total RNA, total lymphocytes were cocultured with CSC total RNA-pulsed DCs and populations of lymphocytes and were analyzed with flow cytometry. The results showed that cocultured productions contained cytotoxic CD8 T lymphocytes, CD4 T lymphocytes, NK, and NKT cells (47). Tumor-associated macrophages, one population of key immune cells in TME, were closely associated with the progression of NSCLC. In terms of molecular mechanisms, macrophage-derived IL-10 activated JAK1/STAT1/NF-κB/Notch1 signaling to enhance properties of stemness in NSCLC. In turn, when IL-10/JAK1 signaling was blocked, CSC-related genes might be reversed (48). Monocytes, as the largest type of leukocyte, differentiated into dendritic cells and macrophages. Even though they belonged to innate immune system, monocytes also involved in the process of adaptive immunity. Tumor-associated monocytes created a CSC niche through juxtacrine secretion pathway to enhance CSC activities of carcinoma cells (49). Hepatocarcinoma cells HepG2 were cocultured with mouse splenic B cells (MSBCs). Those HepG2 cells were verified to obtain stem-cell-like characteristics, such as high tumorigenic capacity, self-renewal, extensive proliferation, overexpression of CSC-related genes and proteins, drug resistance, and highly activated Notch and SHH signaling pathways (50). CD8+ T-cells-mediated antitumor immunity was one of developed strategies to target CSCs. One previous study isolated CSCs from human lung cancer cell line H460 with special marker ALDEFLUOR, and CSC lysate-pulsed dendritic cells was used to stimulate CD8+ T cells as a treatment strategy. The results indicated that ALDH-high-CD8+T cells might directly target against ALDH-high CSCs to mediate antitumor immunity (51). This study found that the distribution of immune cells was significantly different between high- and low-risk score groups in LUAD and LUSC, respectively. Most of these results were coincident with the distribution of immune cells between high- and low-mRNAsi subtypes. Moreover, immune cells might recognize, attack, and eliminate cancer cells, and they might also have the ability to induce a small population of cancer cells to acquire stem cell-like characteristics. Our results revealed that CSC-cell-based tumor-infiltrating immune cell immunotherapy might be clinically useful.

TFs were involved in regulating “turn on and off” genes by binding to a specific DNA sequence. Groups of TFs had diverse effects throughout the life of the cell and organism, such as cell migration, division, growth, death, and organization during embryonic development (52). To explore the underlying correlation between DEIRGs and TFs, this study screened TF-DEIRG regulatory networks. A total of 26 TFs in LUAD and 29 TFs in LUSC were identified as potential upstream regulatory mechanisms of DEIRGs. Some of these identified TFs in LUAD and LUSC were closely associated with stemness in lung cancer. For example, forkhead box M1 (FoxM1) was involved in cell proliferation and regulated the expression of cell cycle genes (cyclin B1 and cyclin D1). A study found that the upregulation of TF FoxM1 mediated the acquisition of cancer stem-like cell characteristics in NSSLC H460 cells with analysis of stemness markers (CD133, ALDH1, and CD44) (53). Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) was one of the members of polycomb-group (PcG) family, which played roles in maintaining the transcriptional repressive state of related genes during cell division (54). EZH2 altered stem-like phenotypes and progression of lung cancer via regulating the malignant gene modifier (histone methyltransferase) (55). Nuclear receptor NR5A2 was a regulator of stemness of pluripotent stem cells and embryonic stem cells, which was also reported to promote CSC properties and tumorigenesis in NSSLC by regulating Nanog (56). Hypoxia-inducible factor-2 alpha (HIF-2α) was involved in the induction of genes regulated by oxygen, which was induced by decreased oxygen levels. Hypoxia was considered to be one of the most important factors in TME. Most malignant tumors produced a hypoxic microenvironment that was conducive to their own development, which suppressed immune response (57). Lung cancer stem-like cells [CD133(+)] A549 were divided from cultured cells with serum-free culture conditions by fluorescence-activated cell sorting. Tissue sections from 50 NSSLC cases were used to estimate the correlation between HIF-2α levels and lung cancer stem-like cells [CD133(+)] with immunohistochemical analysis. HIF-2α levels were significantly higher in CD133(+) cells compared to CD133(−) cells, and HIF-2α caused radioresistance of CSCs (58). For this study, the construction of TF-DEIRG regulatory networks provided some molecular mechanisms of CSC processes in lung cancer.

Most CSCs are dormant and have strong drug resistance and insensitivity to radiotherapy. The traditional chemotherapy drugs and radiotherapy might not kill them effectively, which results in tumor metastasis and recurrence. Due to the development and application of immunotherapeutic drugs such as targeting PD-1 and PD-L1, immunotherapy has become one of the main treatment of lung cancer. As an effective therapeutic method, cellular immune intervention can target tumor-specific antigen and/or tumor-associated antigen and specifically remove tumor cells or CSCs. This study analyzed three levels of associations between mRNAsi and immunity, including (i) the associations of immune-related scores and mRNAsi, (ii) the associations of immune cells and mRNAsi, and (iii) differently expressed immune-related genes (DEIRGs) between high mRNAsi and low mRNAsi groups. Those provided potential immune molecular markers and mechanisms for immunotherapy in CSCs. In summary, this study focused on the alterations of immune-related genes and immune cells through the acquisition of stemness characteristics based on mRNAsi in LUAD and LUSC. Usually, it takes two to tango when ones explore the interaction between stemness and immunity. Two different prognostic models in LUAD and LUSC could be useful to further investigate their clinical application in lung cancers.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. All original data were extracted from public database.

NL designed the project, analyzed data, prepared figures and tables, and wrote the manuscript. YL and PZ participated in the data analysis. XZ conceived the concept, supervised results, critically revised/wrote manuscript, and was responsible for its financial supports and the corresponding works. All authors contributed to the article and approved the submitted version.

This work was supported by the Shandong First Medical University Talent Introduction Funds (to XZ), the Hunan Provincial Hundred Talent Plan (to XZ), the Shandong Provincial Natural Science Foundation (ZR202103020356 to XZ), the National Natural Science Foundation of China (82172866), and the Academic Promotion Program of Shandong First Medical University (2019ZL002).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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