Analysis of Lung Ground-glass Nodules From the Perspective of Inammation-to-cancer Transition Based on Il-6 Level and Clinical Features

Objective(cid:0)To explore the potential impact of interleukin-6 (IL-6) in promoting the development and progression of lung ground-glass nodules (GGN) based on multiple clinical features using multi-dimensional analysis (MDA). Methods: Sample collected from data taken from patients with lung GGN who received treatment in Shanghai Tenth People's Hospital, between September 2018 and September 2020. Sample data were grouped according to GGN diameter as well as IL-6 levels and screened according to the solitary GGN (d<0.8cm) after more than one year of follow-up observation. Differences in associated complications, peripheral superoxide dismutase (SOD), IgE and T cell subsets, tumor markers, and CT malignant signs were compared between the two sample groups.

difference in T helper (Th) and T suppressor (Ts) cell levels between the two groups (p<0.05). Levels of tumor markers such as NSE and CY21-1 were also found to be elevated in the group with elevated IL-6 levels than in the group with normal IL-6 levels (p<0.05). Finally, the group with elevated Il-6 levels was observed to have a higher proportion of patients with vascular convergence sign (VCS) and mixed ground-glass opacity (mGGO) as compared to the sample with normal IL-6 levels (p<0.05).
Conclusion: Chronic in ammation is an important factor in the malignant transformation of GGN. IL-6 plays a signi cant role in promoting the formation of early lung cancer through multiple pathways, including mediating imbalance of the human mitochondrial antioxidant system and reactive elevation of IgE, inhibiting the anti-tumor immune function of T cells, and promoting tumor tissue angiogenesis. The probability of malignant transformation is even higher in lung GGN accompanied with IL-6 elevation.

Research Background
Lung cancer is one of the malignant tumors with the highest morbidity and mortality rate worldwide [1]. With the increasing public awareness of health and wider application of low-dose spiral CT, the detection rate of lung nodules has been increased substantially [2]. The prevalence of lung ground-glass nodules (GGN) is high. Clinically, GGN presents diversely with a wide range of clinical manifestation and are prone to cancerations [3]. As a result, it is important to intensify clinical analysis of GGN and its associative factors responsible for lung cancer development, to prevent or improve the prognostic value of lung cancer positively.
It is generally believed that lung GGN formation is related to long-term in ammatory stimulation.
Interleukin-6 (IL-6) is a common proin ammatory factor [4] and is closely associated with the development and prognosis of lung cancer [5,6]. The goal of this study is to analyze in ammatory factors and clinical features of lung GGN and explore the role of in ammation in the biological behavior of lung GGN.

Subjects
Clinical data of patients with lung GGN who received treatment in Shanghai Tenth People's Hospital (Shanghai, China) between September 2018 and September 2020 were collected. A total of 145 patients with lung GGN were included in the study. The inclusion criteria were patients with lung GGN of a minimum diameter of 0.8. Patients must have been followed up for more than a year. Patients must have undergone a high-resolution CT (HRCT) scan, and excluded for lung nodules arising from other reasons such as malignant tumor metastasis or acute infective diseases.
The research protocol was approved by the ethics committee of the said hospital, and informed consent was signed by all participating subjects.

Data collection
Sample data taken from GGN patients included: (1) patient's general information, such as sex, age, smoking history, and family history of lung cancer; (2) clinical manifestations such as cough, chest pain, shortness of breath, fatigue, diarrhea, or constipation, and sleep disturbances; (3) past medical histories including hypertension, diabetes, chronic pulmonary disease, chronic liver disease, chronic kidney disease, cardiocerebrovascular disease and malignant tumors; and (4) investigations included chest CT and laboratory tests consisting of T cell subset analysis, in ammatory markers, humoral immunology, tumor markers, and adenosine triphosphate (ATP) level of peripheral mononuclear cells.

Grouping
Samples were classi ed into two groups: patients with GGN≥5mm in diameter and those with GGN<5mm in diameter. Comparison based on levels of in ammatory mediators were made between the two groups. Results showed signi cant differences in IL-6 levels. Consequently, patients were further classi ed into those with elevated levels of IL-6, and those with normal levels of IL-6, with 5.3pg/ml considered as the higher limit of normal.

Statistical methods
All statistical data were analyzed by using SPSS 22.0. Measurement of data was expressed as mean and a standard deviation (SD), and veri ed by t-test. Sample data was expressed as a percentage and veri ed by the chi-square test. P values <0.05 were considered statistically signi cant.

Results
Baseline data, clinical manifestations, complications, and laboratory ndings.
Out of the 145 included patients, 95 had GGN ≥ 5mm, and 50 had GGN < 5mm. The study found no signi cant difference in the baseline data between the two groups. There was also no signi cant difference in sex, age, IL-4, IL-8, and TNF-α levels between the two groups. However, the study found signi cant differences in IL-6 levels between the two groups (p < 0.05) ( Table 1). While IL-6 was found to be elevated in 53 patients, levels were normal in 92 patients. Details of the symptoms, complications, and laboratory ndings are shown in Table 2. The proportion of GGN patients with vascular convergence sign (VCS) and mixed ground-glass opacity (mGGO) in the group with elevated levels of IL-6 was signi cantly higher than the group with normal IL-6 levels (p < 0.05) ( Table 3).

Discussion
The release of reactive oxygen species (ROS) from the mitochondria of in ammatory cells under chronic in ammatory conditions could directly cause a genotoxic effect and damage to tissues, thereby initiating and promoting tumor development, invasion, and migration [7]. As a response, the expression of SOD released from mitochondria to the peripheral blood is increased [8,9]. Increased SOD expression was found to be related to the upregulation of matrix metalloprotein (MMP), hence blocking SOD could partly reverse epithelial-mesenchymal transition (EMT) and migration of A549 cells [10 ]. Other studies found that the content of SOD in tissues of adenocarcinoma of the lung was signi cantly higher than that in adjacent normal tissues [11]. We found that SOD levels in the IL-6 elevated level group were signi cantly higher than those in the normal IL-6 level group. In addition, GGN in the group with elevated levels of IL-6 exhibited more malignant signs on imaging, suggesting that lung GGN with SOD elevation may have a more malignant transformation potential [12].
IgE can provide a protective effect on the mucosal epithelium exposed to carcinogenic environments. It was found that exposure of the mouse skin to benzopyrene could produce a local IgE response, which induced the basophilic granulocyte-mediated anti-tumor effect to clear cancerous cells following DNA damage by cancerogenic substances. When the IgE response was blocked, the cancerous tissue induced by the carcinogenic substances grew even faster [13,14]. It was found in our study that the IgE level in the GGN patients of the IL-6 elevated group was increased signi cantly. IgE could promote the transformation of the activated macrophages to the proin ammatory phenotype [15], and enhance the anti-tumor proin ammatory signal and promote the killing effect of human mononuclear cells on tumor cells [16]. Therefore, a reactive elevation of IgE may be a tumor protective mechanism in early canceration of lung GGN. However, this postulate could on the other hand imply that GGN lesions with an elevation of IL-6 and IgE could be associated with an increased risk of malignancy.
We found from the sample of GGN patients with elevated levels of IL-6 that, while the levels of Ts cells were increased, that of Th cells was decreased signi cantly. IL-6 has a role in promoting tumor formation [17,18]. Myeloid-derived suppressor cells (MDSC) are a group of cells generated in the process of tumor progression, that function to inhibit T cells and natural killer (NK) cells, promoting tumor progression. IL-6 is an important regulatory factor aggregated and activated by MDSC, resulting in the inhibition of adaptive immunity [19]. Studies demonstrated that CD40 of upregulated MDSC could induce T cell tolerance in tumor-bearing mice. In addition, MDSC could stimulate M2-like macrophages to produce large amounts of IL-1 to promote tumor progression and induce Treg cell-mediated immune suppression. Under the stimulation of tumor-derived transforming growth factor TGF-β, MDSC upregulates the expression of peripheral ATP hydrolase-1 and nucleotidase and catalyzes the degradation of ATP into adenosine [20], which has an immunosuppressive effect on T and NK cells, thus reducing their killing effect on tumor cells. Other studies [21] also found that IL-6 could inhibit T cell activation in tumors, suggesting that IL-6 may be an important factor in inhibiting anti-tumor immunity of T cells.
Chronic in ammation and decreased immune function are common phenomena of immunosenescence [22]. Most GGN patients in our study were between 50 and 60 years of age, which is a period of quick immunosenescence. Therefore, complications in GGN patients with IL-6 elevation should not be ignored. Clinicians should rst assess whether complications of the patients would produce a negative impact on their lung GGN, such as chronic intestinal diseases. Recent studies have demonstrated a close relationship between the gut and lung [23]. For instance, patients with chronic pulmonary diseases do not only present microecological changes in the respiratory tract but their gut microbiota as well. Gut microbiota metabolites such as short-chain fatty acids (SCFAs) have systemic anti-in ammatory effects. Destruction of intestinal microecology will lead to GI dysfunction and imbalance between anti-and pro-in ammatory cells of the human body [24]. Gut microbiota also undergoes abnormal changes in patients with anxiety. In addition, metabolites from intestinal ora have important impacts on human in ammatory responses [25]. Both IL-6 and TNF-α mediators of pro-in ammation in the in ammatory mechanism of the gut-lung axis as well as gut-brain axis [24,26]. Coronary stenting is quite common in lung GGN patients with elevated levels of IL-6, which may re ect that in ammatory mediators could be implicated in aggravating coronary arterial damage. Higher levels of IL-6 and TNF-α participate in coronary arterial damage, as well as in the development and progression of tumors [27,28]. Besides, IL-6 also mediates the systemic in ammatory response of cancer patients [29,30]. For this reason, lung GGN is to some extent a systemic disease accompanied by other complications. Regulating gut microbiota and controlling in ammation are important critical ways to prevent GGN canceration [31,32].
It was found in our study that tumor markers were elevated in GGN patients with elevated levels of IL-6. IL-6 promoted {had a role in promoting} the transformation of lung tumor stem cells to foci of early canceration. In addition, combined use of the anti-IL-6 antibody and cisplatin could destroy lung cancerlike organs, while use of cisplatin alone did not produce such an effect. A study detected the presence of positive expression of IL-6mRNA in lung cancer cells [33]. Tumor cell-derived IL-6 promotes tumor cell proliferation by promoting glycolytic metabolism of tumor cells and activating the MEK/Erk1/2 hypoxiainducible factor 1α (HIF-1α) pathway [34,35]. IL-6 can also confer stem cell-like properties on cancer cells by activating IL-6/STAT3 signaling [36,37]. Elevation of NSE and CY21-1 in GGN patients of IL-6 elevated group suggests that in ammation has a promoting effect on tumor development and progression [38][39][40][41][42]. IL-6 promotes angiogenesis of early cancer tissues. It was found that the vascular endothelial growth factor receptor (VEGFR) level in non-small cell lung cancer (NSCLC) patients with elevated levels of IL-6 were even higher than that in those with normal IL-6 levels, and IL-6 and VEGFR were independent prognostic factors [43]. In ammation and angiogenesis are two important features in tumorigenesis. Tumor microvascular density (MVD) was found to correlate positively with the expression of IL-6 and VEGFR in human lung adenocarcinoma tissues [44]. IL-6 and VEGFR were found to play a key role in tumor angiogenesis [45]; when IL-6 formation was inhibited, tumor in ltration and metastasis were decreased [46]. Lung GGN with VCS may most probably represent lung cancer in its early stage. This could be evident on CT imaging as gathering or displacement of a single or multiple pulmonary microvessels to the GGN lesion due to traction, disrupting the GGN lesion, or penetrating through it [47]. It is reported in the literature that the canceration rate of mixed GGN (mGGN) is more than 50% [48,49]. Such canceration rate is even higher in GGN patients with IL-6 elevation and VCS, and in mGGN patients.

Highlights and limitations
To the best of our knowledge, this is the most comprehensive clinical study on factors affecting the biological behavior of lung GG by summarizing the occurrence and progression of lung GGN from the perspective of how IL-6 mediates in ammation-induced canceration. We also found that lung GGN patients with IL-6 levels elevated were more likely to be in icted with anxiety and GI dysfunction. By inhibiting the anti-tumor immune function, tumor markers became even higher and CT imaging revealed more malignant signs in such patients. These ndings may provide clinicians with more clues to identifying high-risk GGN at an earlier stage and preventing them from cancerous transformation.
The main limitation of the study is that the level of evidence is relatively low due to the lack of surgicalpathologic results and postoperative follow-up observations. Future research will seek to observe changes in GGN after treatment by inhibiting the in ammatory factors. This will help gain knowledge about the impact, of which inhibiting in ammatory mediators will have on cancer prevention.

Conclusion
Chronic in ammation is an important factor in promoting lung GGN and its progression to cancer. IL-6 promotes early cancer formation through multiple pathways such as by mediating imbalance of the human mitochondrial antioxidant system and reactive elevation of IgE, inhibiting the anti-tumor immune function of T cells, and promoting tumor tissue angiogenesis. The canceration rate of lung GGN with IL-6 elevation is higher than that of lung GGN with normal IL-6.

Declarations
Ethics approval and consent to participate