Discussion
Lung cancer is the most widespread cancer globally, and NSCLC is the primary subtype of lung cancer. Most patients are resistant to immunotherapy, which may be related to their TIME. With the rapid development of scRNA-seq in cancer medicine, it is now possible to study highly heterogeneous tumors, including NSCLC, which will bring epochal shifts in the understanding of TIME and the exploration of novel cellular biomarkers [
25,
26].
This study comprehensively analyzes TIME in NSCLC by integrating two large scRNA-seq datasets. After quality control and dimensionality reduction clustering, ten cell types were initially annotated, and further subdivisions resulted in 28 major cell types. Annotation of cellular abundance in patients in the TCGA cohort based on the overall expression of mRNAs characteristic of 28 cells found that most cells differed significantly between tumors and normal tissues. Neutrophils were more abundant in tumor tissue, consistent with previous studies [
27]. Tumor-immune cell interactions lead to metabolic competition within the tumor ecosystem, which limits the effective supply of nutrition, and thereby hinders immune cell function. It has been reported that IL-18 may positively regulate autophagy to promote myocardial cell mitochondrial function and the steady state maintenance of gap junctional turnover [
28]. Close binding between mitochondria and gap junctions regulates the ionic permeability of gap junctions and influences metabolic reprogramming [
29,
30]. Multiple reliable ligand-receptor pairs were collected using cellular communication analysis in the research, characterizing the complex regulatory network in the NSCLC tumor microenvironment. The immune checkpoint
TNFSF14 in Neutrophils, NK Cells, and Monocytes and
BTLA in Treg Cells, Granulosa Cells, and B Cells interact with
LTBR and
TNFRSF14 in Cancer Cells to mediate cytotoxicity and promote tumor killing [
31‐
34]. In addition, the high expression of
CD24 in Cancer Cells affects the expression of
SIGLEC10 in Dendritic Cells, Macrophages, GMP Cells, B Cells, and Mast Cells, which in turn affects immune disorders and leads to tumor immune escape responses [
35]. The highly expressed cytokine
CCL5 in CD8 + T Cells, NK Cells, NKT Cells, CD4 + T Memory Cells, Plasma Cells, and Langerhans Cells work together with
SDC1 and
SDC4 in Cancer Cells to affect the occurrence, development, and mediation of cancer survival of cancer cells [
36]. Cytokines
CXCL1,
CXCL2, and
CXCL8 in Cancer Cells interact with
CXCR1 and
CXCR2 in Neutrophils, chemotactic the activity of Neutrophils, and promote the generation of tumor immune microenvironment [
37]. The growth factor
HBEGF, which is highly expressed in Monocytes, Neutrophils, Dendritic Cells, GMP Cells, Macrophages, and various Epithelial Cells, interacts with
CD9 expressed in Cancer Cells to help mediate tumorigenesis and proliferation [
38]. We also found that Cancer Cells interact with
ITGB1, highly expressed in multiple cells such as Fibroblasts, Endothelial Cells, and Basal Cells, through angiogenesis signal molecules (
VEGFA) to stimulate tumor growth and metastasis [
39]. This discovery provides a new research idea for tumor immunotherapy. Further mining the high heterogeneity of Neutrophils, we identified Neutrophil states with four distinct differentiation fates through developmental trajectory analysis. Using GSEA to functionally characterize signatures of differentiation, we found that this pattern of differentiation is intrinsically linked to intratumoral immune and metabolic biology as well. NDRGs in different differentiation states showed a highly mutated state, with a mutation rate greater than 91%, indicating that NDRGs play a crucial role in the occurrence and development of tumors. Based on the above findings, we established a prognostic risk model consisting of six NDRGs,
MS4A7, CXCR2, CSRNP1, RETN, CD177, and
LUCAT1, according to the rate of four reference genes (
ACTB, GAPDH, TFRC, TUBB). Overall, the model was suitable for various detection data and can effectively predict the prognosis and immunotherapy response of NSCLC patients, providing a theoretical basis for formulating individualized treatment for patients.
Studies have shown that
MS4A7 has a particular prognostic value in ovarian cancer [
40] and glioma [
41]. Our research found that
MS4A7 mainly mediates most immune-related pathways, such as immune receptor activity, but
MS4A7 is down-regulated in tumor tissues, and its low expression levels indicate a worse prognosis. It is speculated that the tumor produces immune tolerance by down-regulating the expression of
MS4A7, leading to a worse prognosis. As a LncRNA,
LUCAT1 is involved in the occurrence and development of lung cancer. Studies have found that
LUCAT1 can promote the metastasis of lung adenocarcinoma cells and glycolysis by regulating the miR-4316/VEGFA axis [
42]. It has been found in this research that the overexpression of
LUCAT1 increases the activity of most metabolic and carcinogenic pathways, and
LUCAT1 has a significant negative correlation with immune cells. It indicated that
LUCAT1 was an oncogene that promoted the occurrence and development of tumors by affecting metabolic pathways in the tumor microenvironment and inhibiting the immune response of immune cells, resulting in a lousy prognosis. Recent studies have shown that
CXCR2 can be used as a valuable independent prognostic marker in patients with cholangiocarcinoma, and its mediated immune response may have a tumor inhibition effect on cholangiocarcinoma cells [
43]. Our study confirmed that the down-regulation of
CXCR2 is associated with a poor prognosis.
CXCR2 has a significant positive correlation with most immune cells, and the activity of most immune response pathways, including acute inflammatory reactions, is increased, suggesting that
CXCR2 can inhibit cancer by inducing immune responses, and significant down-regulation of tumor tissues is also the main reason for worse prognosis. The tumor occurrence is usually related to the inflammatory reaction caused by excessive adipose tissue. It has been reported that the fat factor
RETN can activate obesity-related inflammatory responses through the combined action of the pro-inflammatory cytokine IL-1β [
44]. Studies have found that high expression of
RETN predicts a adverse prognosis because
RETN promotes an inflammatory response. Interestingly,
RETN is low expressed in the tumor. After the simulation of down-regulation, it was found that the activities of immune cell chemotactic related pathways were decreased, and a positive correlation between
RETN and immune cells. It indicated that
RETN helped improve the chemotaxis of immune cells, and tumors could ensure their survival by down-regulating
RETN. It has been reported that the
CSRNP1 gene can be used as a prognostic biomarker [
45,
46] for many cancers, indicating the essential prognostic value of
CSRNP1. When
CSRNP1 is simulated to be down-regulated, the activities of various biological modification-related pathways, including Spliceosome activity, are down-regulated. In addition,
CSRNP1 was positively correlated with most immune cells. It is speculated that
CSRNP1 is involved in the growth and development of immune cells, and the tumor produces immune resistance by down-regulating
CSRNP1. However, because
CSRNP1 is also involved in the growth and modification of tumor cells, its high level of expression will lead to a dreadful prognosis and high-risk score. As a Neutrophil surface glycoprotein,
CD177 triggers Neutrophil degranulation and superoxide production. Recently reported,
CD177 can regulate
PDPN and thus affect the physiological changes of cancer-related fibroblasts, which seems to be a new therapeutic target [
47].
CD177 was up-regulated in tumor tissue and correlated with Neutrophil content in this study. When
CD177 is overexpressed, many immune response pathways and biological regulatory pathways are significantly up-regulated, such as the IL-17 signaling pathway and the protein oxidation pathway. Therefore, low expression of
CD177 indicates a decrease in the content of immunocytes, especially Neutrophils, and a corresponding decrease in antitumor activity, resulting in a worse prognosis. Understanding the function of prognostic genes and their causes of dreadful prognosis will help to propose targeted therapy options.
Today, reorientation of drug function is a novel strategy for disease treatment. As disease mechanisms continue to deepen and treatment plans continue to be refined, a variety of drugs for treating disease including Valproic acid [
48] for the treatment of epilepsy have been applied to the treatment of cancer. Therefore, based on this strategy, we conducted targeted drug screening of prognostic genes with a view to proposing a therapeutic approach that modulates poor prognosis. As a small molecule compound that can efficiently bind to and upregulate
MS4A7 expression, more than 95% of estradiol in the bloodstream binds to sex hormone-binding globulin (SHBG) and alumina, which is commonly used to treat diseases related to estrogen reduction. However, excessive intake of estrogen can result in side effects such as nausea, vomiting, and vein thrombosis. Furthermore, estradiol functions as an immunomodulator in immune and inflammatory processes [
49]. Abrine also showed an exceptional performance in increasing
CXCR2 expression. Abrine was shown to be a competitive inhibitor of indoleamine-2,3-dioxygenase (IDO) in in vitro experiments, which could effectively reduce tryptophan degradation activity and enhance the efficacy of immunotherapies. Abrine is currently being used in conjunction with a series of chemotherapeutic drugs such as cisplatin, doxorubicin and paclitaxel, and has been shown to have excellent synergistic effects [
50]. Zhang et al. has shown that Abrine can regulate hepatocellular carcinoma cell growth and apoptosis via the KAT5/PD-L1 axis [
51]. The natural product ionomycin used in this study had high affinity to
RETN. The natural product of Ionomycin, which is found in Streptomyces polymerases, is also a calcium transporter that can increase the intracellular calcium level, which is linked to the activation of the endonuclease in lymphocytes and the reduction in the ratio of Bcl-2 to Bax, ultimately mediating apoptosis [
52,
53]. Beclomethasone was one of the compounds with up-regulation of
CSRNP1 that exhibited high affinity docking binding energy. Beclomethasone is a Corticosteroid with anti-inflammatory and immunomodulating properties for chronic obstructive pulmonary disease and COVID-19. It has been reported that Beclomethasone inhibits normal physiologic neutrophil migration and neutrophil chemotaxis upon detection of trauma induced inflammation [
54,
55]. In the cohort screened for drugs that promoted increased
CD177 mRNA expression, XL147 was found to have high affinity for the
CD177 mRNA. XL147 is a potent inhibitor of oral bioavailability and a member of the class I PI3K family of lipid kinases. In a variety of clinical cancer models, XL147 treatment has been found to significantly inhibit PI3K pathway signaling in tumors and lead to significant inhibition of tumor growth or tumor shrinkage [
56,
57]. Based on the data from the five targeted drugs targeting the five aforementioned prognostic genes, our study has proposed a novel targeted therapy scheme consisting of a combination of multiple drugs, which will help improve the poor prognosis brought by the five prognostic genes and improve patient survival rate. Among various targeted therapeutics, there has also been increased interest in novel biomaterials, including nanomaterials [
58] and hydrogel materials for hyaluronic acid [
59]. As a novel antioxidant with low toxicity and high efficacy, the nano-antioxidant is superior to the traditional antioxidant in improving superoxide dismutase and catalase activities in organisms, and has a lower biological toxicity [
60]. Hyaluronic acid-constructed hydrogel materials are brand new drug delivery vehicles, which can effectively reduce cytotoxicity, deliver drugs safely and efficiently to the site of action, and allow drugs to play the largest role. Of the five gene-targeted drugs chosen in this study, the primary goal is to regulate mRNA expression of prognostic genes. However, further research is needed on how to deliver drugs to drug targets. The drug delivery scaffold built with novel biomaterials may be an excellent choice.
The novelty of this study lies in the integration of large scale scRNA-seq to analyze the NSCLC regulatory network and further resolve the complex interactions within the TIME. We also employ a novel strategy of combining the Elastic Net Regression algorithm with housekeeping genes ratios for prognostic risk modeling. Further investigation discussing prognostic gene function and drug targeting research is also novel in this study. At the same time, there remain limitations to this study. First, although we had performed a batch correction for the two scRNA-seq data, the essential batch effect still exists. In that regard, future integration studies could begin with sequenced documents to ensure consistency and accuracy of data. Secondly, our results are still in the analytical and speculative stage and have not been experimentally validated, which is what future work will need. The combined therapeutic value of these five targeted drugs at the cellular and animal level will be the subject of future work. Furthermore, on the basis of our prognostic risk model, we hope to establish a shared network platform to aid in clinical diagnosis and prognostic therapy in NSCLC.