Evaluation of Pro- and Anti-Inflammatory Cytokines in Glioma Patients: Correlation with Tumor Grading

doi:10.21203/rs.3.rs-2981213/v1

Download PDF

Research Article

Evaluation of Pro- and Anti-Inflammatory Cytokines in Glioma Patients: Correlation with Tumor Grading

https://doi.org/10.21203/rs.3.rs-2981213/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objective and design:

In this case control observational study of glioma patients, using ELISA technique, we have examined the tissue cytokine profile of pro- and anti-inflammatory cytokines (IFN-γ, IL-6, IL-1β, IL-8, TNF-α and IL-10, IL-4), and angiogenic factors (VEGF, FGF-2), and compared with the tumor type and stage. We found that a significant upregulation of pro-inflammatory cytokines (IFN-γ, IL-6, IL-1β, IL-8, and TNF-α), and a significant down regulation of anti-inflammatory cytokines (IL-10, and IL-4) compared to control brain tissues. Similarly, when compared to control group, we found a significant increase in angiogenic cytokines (VEGF, FGF-2) in tumor tissues.

Methodology:

We have examined the tissue cytokine profile of pro- and anti-inflammatory cytokines (IFN-γ, IL-6, IL-1β, IL-8, TNF-α, and IL-10, IL-4), as well as angiogenic factors (VEGF, FGF-2), in order to evaluate tumoral aggressiveness by using ELISA technologies.

Result

Our results suggest that even though there is a significant alteration in the cytokines between the control and tumor samples, we found that the difference between various glioma grades and meningioma are not statistically significant. Based on our results we conclude that the cytokine levels alone may not be suitable enough for tumor staging.

Conclusion

These cytokines contribute to the development of pain associated with the disease as well as the growth and aggressiveness of tumours. With a focus on cytokines, our goal in this study is to investigate the microenvironment of gliomas. Pain is one of the main symptoms of glioblastoma, but it might be delayed or neglected. Cytokines might accelerate diagnosis and treatment.

Glioma

Meningioma

Cytokines

Angiogenic growth factors

Brain cancer

ELISA

Gliomas are the most common primary intracerebral tumors in adults, of which GBM (Glioblastoma Multiforme) is the most aggressive and terminal stage of brain cancer. The physical, psychological, and social status of patients and their families is significantly impacted by the disease, which has a death prognosis for the majority of glioma patients (Ostrom et al., 2014). Gliomas originate from glial cells of the central nervous system (CNS) and are the most common primary neoplasm of the brain, accounting for approximately 80% of primary malignant brain tumors (Ostrom et al., 2022). The median survival time for primary GBMs is approximately 14.6 months, and the average age at presentation is 62 years (Stupp et al., 2005). Although there has been advancement in medicine and surgery, the GBM's poor prognosis is well known, and survival rates are still dismally low.

Smoke, alcohol, and growth hormones are a few glioma risk factors that can activate signaling pathways linked to inflammation (such as NF-κB and STAT3 signaling). Chronic infections like the hepatitis B virus (HBV), cause inflammatory conditions, etc. can also lead to the GBM development. Chemo- and radiation therapies also disrupt the control of several genes involved in angiogenesis, invasion, proliferation, survival, and cancer spread (Zhu, Zhong, & Shen, 2011). Mutations, genomic instability, and epigenetic alterations also contribute to inflammation, which has a definite role in cancer development (Grivennikov, Greten, & Karin, 2010). Although headache is one of the most frequently reported symptoms in the case of glioblastoma, diagnosis is frequently made too late for a successful therapeutic strategy.

Till 2016, the classification of gliomas was previously based on histology findings supported by auxiliary tissue-based analyses (immunohistochemical, ultrastructural). The new categorization now includes molecular diagnostics in addition to histology and immunohistochemistry as a result of recent developments in molecular genetics (2021, 5th edition) (Louis et al., 2021). Virchow, who discovered "lymphoreticular infiltrates" in neoplastic tissues, for the first time observed a connection between inflammation and cancer. The same group showed that the presence of these lymphoreticular infiltrates at sites of chronic inflammation might indicate the origin of cancer. Recent years have seen an enormous amount of experimental evidence that supports Virchow's theory (Balkwill & Mantovani, 2001). According to Colotta et al., inflammation is "the seventh hallmark of cancer" in a synthetic formulation (Colotta, Allavena, Sica, Garlanda, & Mantovani, 2009). Inflammation and repair are analogous to the body's reaction to cancer in many ways; The tumor milieu, other than the native tumor cells, also harbors immune cells and cytokines, as a consequence of to host immune response. The combined action of pro- and anti-inflammatory cytokines together decide the glioma progression. Functional polymorphisms in cytokine genes are frequently connected to the susceptibility to and severity of cancer. According to Balkwill and Mantovani, some forms of inflammation may act as the "fuel that feeds the flames" in cases where genetic damage is the "match that lights the fire" of cancer (Balkwill & Mantovani, 2001). Additionally, inflammation can encourage angiogenesis, contribute to the growth of malignant cells, and aid in the spread of metastatic disease through M1 and M2 macrophage activation.

The treatment of GBM remains the most difficult clinical oncology task, despite several multinational initiatives (Mrugala, 2013). Numerous treatments have been researched over the past ten years with very little success. The primary difficulties in treating GBM are due to the disease's location and its complicated and varied biology (Kesari, 2011). The prognosis for individuals with GBM remains dismal despite improvements in surgical techniques, radiation, and adjuvant chemotherapy that have shown progressive improvements in survival and quality of life (Ohka, Natsume, & Wakabayashi, 2012). Glioma treatment is multimodal and includes chemo- and radiotherapy. Despite these efforts, high-grade glioma recurrence rates and outcomes have not significantly changed (Arribas Alpuente, Menéndez López, & Yayá Tur, 2011). A wide variety of cytokines, including glioblastoma multiforme, have altered expression in malignancies (Iwami, Natsume, & Wakabayashi, 2011). The alterations result from the interaction of tumor cells with non-tumor cells, such as macrophages, lymphocytes, or stromal cells, and they offer regulatory support for tumor growth, angiogenesis, invasion, and metastasis (Iwami et al., 2011). Taking into account the overall research effort in the field, in the present study, we have analyzed the expression of various pro-inflammatory, anti-inflammatory, and angiogenic cytokines, among various grades of glioma’s and also meningiomas, to enable us for further in-depth understanding of glioma tumor microenvironment.

Chemicals and ELISA kits

The following human ELISA kits: IL-1β (Interleukin 1 Beta), IL-4 (Interleukin 4), IL-6 (Interleukin 6), IL-8 (Interleukin 8), IL-10 (Interleukin 10), VEGF (Vascular Endothelial Cell Growth Factor), TNF-α (Tumor Necrosis Factor Alpha), IFN-γ (Interferon-gamma), and FGF-2 (Heparin-binding growth factor 2) were supplied by Fine Test, Fine Biotech Co., Ltd., China. All other necessary reagents were obtained from Sigma Aldrich. ProLiant New Zealand Ltd. provided the bovine serum albumin (BSA). The BCA protein assay kits were obtained from G-Biosciences, St. Louis, USA. Management details like surgery performed and details of surgery were documented in the form of a case sheet, which mainly follows standard protocol and is not affected by the study. Anaesthetic agents were obtained from Baxter Pharmaceuticals Ltd. In the induction phase of anaesthesia, a combination of thiopentone sodium, fentanyl, and atracurium was used. Further, in the maintenance phase of anaesthesia, a combination of O2, air, sevoflurane or isoflurane, fentanyl, and atracurium infusion was used.

Design of the study site and duration

After receiving approval from the institutional ethics committees (EC/NIMS/2977/2022), this prospective, observational study of glioma patients was carried out in the Department of Neurosurgery of NIMS (Nizams Institute of Medical Science), Hyderabad, India from March 2022 to March 2023. Further extended research work was also carried out at the ICMR-National Institute of Nutrition, Food Safety Division, Hyderabad, following receipt of ethical clearance (EC/NIN/6/I/2023). The hospital operates a 24-hour emergency room that treats a wide range of critical illnesses.

Selection of meningioma and glioma-related cytokines in human subjects

To assess the cytokine levels of pro-inflammatory cytokines IFN-γ, IL-1β, IL-6, IL-8, and TNF-α; anti-inflammatory cytokines such as IL-4, and IL-10; and pro-angiogenesis cytokines VEGF, and FGF-2 in the control (the corticectomy tissue of capsuloganglionic bleeds from control patients) and patient brain tissues (cancerous tissue samples from glioma, and meningioma patients).

Selection of subjects

This prospective, observational study was conducted in the Department of Neurosurgery of NIMS and ICMR-NIN, Hyderabad, after clearance from the institutional ethics committee (no EC/NIMS/2977/2022) and (no EC/NIN/6/I/2023) to conduct the present study. Collection of cancerous tissue samples from glioma and meningioma patients and the corticectomy tissue of capsuloganglionic bleeds from control patients. All the methods were performed as per the standard ethical guidelines and regulations made by the Indian Council of Medical Research's Institutional Ethics Committee for Human Subjects. (https://ethics.ncdirindia.org/asset/pdf/ICMR_National_Ethical_Guidelines.pdf). Informed consent was obtained from all the subjects and guardians before the collection of the tissue samples

Method of patient selection and experimental groups

A total of 52 study participants took part in the investigation: the control group (n = 14), the glioma group (n = 32), and the meningioma group (n = 6). Based on the following inclusion criteria as patients aged ≥ 18 years with gliomas of any grade; patients aged ≥ 18 years with meningiomas of any grade; and patient with capsuloganglionic bleed undergoing surgical intervention (control group). The exclusion criteria of this study as patients with other malignancies; patients with inflammatory or degenerative diseases and chronic medical illnesses; patients on immunotherapy; post-transplant patients. We estimated the cytokine marker in cancerous tissue samples after each patient's surgery. The expression levels of pro-inflammatory cytokines markers IFN-γ, TNF-α, IL-1β, IL-6, and IL-8; anti-inflammatory cytokines such as IL-4 and IL-10; and pro-angiogenesis cytokines (VEGF and FGF-2) in the control and patient tissues were the main areas of focus in this study. The World Health Organization's (WHO) glioma grading system was used to divide participants into subgroups for this study Table S1. Participants in this study were divided into five groups Table 1.

Table 1

**The total number of study participants was divided into five groups according to WHO glioma grades.**
Total Study Participants (A + B + C + D + E)	Control (A)	Glioma Grade 2 (B)	Glioma Grade 3 (C)	Glioma Grade 4 (D)	Meningioma (E)
N = 52	N = 14	N = 6	N = 11	N = 15	N = 6

Sample collection

Routine blood investigations were done to exclude co-existing chronic systemic illnesses. The tissue samples were obtained from gliomas, meningiomas, and corticectomy tissue from capsuloganglionic bleeds. Epidemiological data were collected. Tumour tissue and normal brain tissue from the control group and patients were collected in DMEM (Dulbecco’s modified Eagle’s medium) and stored at -20°C. Tissue samples were subjected to homogenization (Sinha et al., 2022) and then centrifugation of the sample; the supernatant was collected and stored at -80°C for estimation of cytokines. To do additional analysis, such as ELISA techniques for the assessment of cytokine levels of pro-inflammatory, anti-inflammatory, and pro-angiogenesis cytokines or chemokines in the control and patient tissues.

Tissue homogenization and sample preparation for estimation of cytokines in control, glioma, and meningioma patients

Each glioma, meningioma tissue, and control tissue were weighed, sliced, and homogenized into a 20% homogenate using a protease inhibitor cocktail pill (Roche Indianapolis, IN) in cooled phosphate-buffered saline (PBS, pH 7.4). The amount of total protein in the supernatant was determined using a BCA protein assay kit (G-Biosciences, St. Louis, USA) and bovine serum albumin (BSA) as a reference standard. Concentrations were represented as pg/mg of protein in tissue homogenate. The glioma, meningioma tissue, and control tissue homogenate were centrifuged for 20 minutes at 5000 RPM and 4°C, and the supernatant was used to measure the levels of the following human ELISA kits: IL-1β (Interleukin 1 Beta), IL-4 (Interleukin 4), IL-6 (Interleukin 6), IL-8 (Interleukin 8), IL-10 (Interleukin 10), VEGF (Vascular Endothelial Cell Growth Factor), TNF-α (Tumor Necrosis Factor Alpha), IFN-γ (Interferon-gamma), FGF-2 (Heparin-binding growth factor 2), according to manufacturer instructions. All cytokine concentrations, including those for human IL-1β, IL-4, IL-6, IL-8, IL-10, VEGF, TNF-α, IFN-γ, and FGF-2 were expressed as pg/mg of protein in brain tissue samples. These cytokines were analyzed according to the instructions of the manufacturers. The assay principle is a competitive ELISA with colorimetric detection, performed using an ELISA multimode microplate reader (Synergy H1 hybrid reader, Biotek) at 450 nm (Sangaraju, Alavala, Nalban, Jerald, & Sistla, 2021; Sangaraju et al., 2019).

Statistical Analysis

The mean and SEM were used to express the data. One-way ANOVA was used to examine more than two group differences (control, glioma grade-2, 3, 4, meningioma), followed by the Tukey HSD post hoc test comparison analysis to establish the statistical significance, and a T-test was used between two groups (control verses glioma grade-2, 3, and 4 (glioma patients); glioma verses meningioma patients); p values < 0.05 were regarded as statistically significant and p values > 0.05 as non-significant (ns). Data analysis was employed by utilising the software as statistical analysis of IBM SPSS Statistics version 23.0 and Prism version 8.3.

Sociodemographic, case-specific, and epidemiological information on glioma and meningioma cases

We evaluated the demographics of our sample of men (N = 34) and women (N = 18); and patients (N = 38) verses control (N = 14). Geriatric patients were more likely to be male (N = 28 patients and control; 53.84%) than female (N = 18 patients and control; 34.61%), while non-geriatric patients were more likely to be male (N = 06 patients and control; 11.53%). Glioma grade 2 was found in males (N = 5; 9.61%) and females (N = 1; 1.92%) in the experimental grouping on glioma grades; glioma grade 3 was found in males (N = 6; 11.53%) and females (N = 5; 9.61%); glioma grade 4 was found in males (N = 9; 17.53%) and females (N = 6; 11.53%); meningioma grade 1 in males (N = 1; 1.92%) and females (N = 3; 5.76%); Overall, males were more affected by gliomas compared to females. Gliomas have a disproportionately negative impact on males. Our research shows that females, not males, are disproportionately impacted by meningioma. According to our findings, gliomas and meningiomas are equally as likely to strike persons of any age. There is no statistically significant (p > 0.05) difference in age or sex between geriatric and non-geriatric subjects. Further there is a significant change (p < 0.05) difference in sex verses WHO glioma grading subjects. As per WHO classification and experimental grouping showed that males were more likely to be diagnosed with glioma than females, while the reverse was applicable to meningiomas.

Table II. Displays the Sociodemographic as age wise and epidemiological information on WHO grading b
SEX		Male		Female		Pearson Chi-Square
Based on Socio-demographic and experimental grouping analysis		No. of persons (N)	Percentage (%)	No. of persons (N)	Percentage (%)	(p-value)
Age	Non-Geriatric	28	53.84	18	34.61	0.058^ns
Age	Geriatric	6	11.53	0	0	0.058^ns
According to WHO grading for glioma, Meningioma, and Control	Glioma Grade 1	0	0	0	0	0.024^γ
	Glioma Grade 2	5	9.61	1	1.92
	Glioma Grade 3	6	11.53	5	9.61
	Glioma Grade 4	9	17.30	6	11.53
	Meningioma 1	1	1.92	3	5.76
	Meningioma 2	0	0	2	3.84
	Control	13	25	1	1.92

Table 2. Displays the Sociodemographic as age wise and epidemiological information on WHO grading on glioma (n=32), meningioma (n=6) cases and control (n=14) subjects between males and females.^γ p< 0.05, ^β p < 0.01, and ^α p< 0.001, and ns; nonsignificant, by using Pearson Chi-Square analysis.

Determination and investigation of the role of various pro-inflammatory cytokines in glioma, meningioma, and control group

Based on ELISA analysis, the pro-inflammatory cytokine levels in the tissue samples of glioma and meningioma patients and controls were estimated. We found a significant increase in the average levels of pro-inflammatory cytokines such as IFN-γ (p < 0.001), IL-6 (p < 0.001), IL-1β (p < 0.001), IL-8 (p < 0.001), TNF-α (p < 0.001) in glioma and meningioma patients when compared to those pro-inflammatory cytokines in controls (Fig. 1A–E). Additionally, there was a non-significant change (p > 0.05) in the pro-inflammatory cytokines such as IFN-γ, IL-6, IL-1β, IL-8, TNF-α in the glioma patients when compared to those pro-inflammatory cytokines in meningioma patients as well as among the glioma grade patients (Fig. 1A-E). Results of pro-inflammatory cytokines among all experimental groups were expressed as mean ± SEM in Table S2. The data were expressed as pg/mg of protein.

Determination and investigation of the role of various anti-inflammatory cytokines in glioma, meningioma, and control group

Based on ELISA analysis, the anti-inflammatory levels in the tissue samples of glioma and meningioma patients and controls were estimated. We found a significant decrease in the average levels of anti-inflammatory cytokines such as IL-10 (p < 0.001), and IL-4 (p < 0.001) glioma and meningioma patients when compared to those anti-inflammatory cytokines in the control group (Fig. 2A and Fig. 2B). Additionally, there was a non-significant change (p > 0.05) in the anti-inflammatory cytokines such as IL-10 and IL-4 in the glioma patients when compared to those anti-inflammatory cytokines in meningioma patients as well as among the glioma grade patients (Fig. 2A and Fig. 2B). The results of anti-inflammatory cytokines among all experimental groups were expressed as mean ± SEM in Table S3. The data were expressed as pg/mg of protein.

Determination and investigation of the role of various angiogenic cytokines in glioma, meningioma, and control group

Based on ELISA analysis, the angiogenic cytokine levels in the tissue samples of glioma and meningioma patients and controls were estimated. We found a significant increase in the average levels of angiogenic cytokines such as VEGF (p < 0.001) and FGF-2 (p < 0.001) in the glioma and meningioma patients when compared to those angiogenic cytokines in controls (Fig. 3a and Fig. 3b). Additionally, there was a non-significant change (p > 0.05) in the angiogenic cytokines such as VEGF and FGF-2 in the glioma patients when compared to those angiogenic cytokines in meningioma patients as well as among the glioma grade patients (Fig. 3a and Fig. 3b). The results of angiogenic cytokines among all experimental groups were expressed as mean ± SEM in Table S4. The data were expressed as pg/mg of protein.

There was no substantial variation in the angiogenic cytokine such as VEGF and FGF-2 milieu based on glioma grade. These angiogenic cytokines were also examined in glioma tissue to those in meningioma tissue, and no significant difference was found. See the Fig. 3.

Because of their variety and complex pathophysiology, gliomas are the least understood of all cancers. Multiple epigenetic and genetic alterations affecting tumour suppressor genes such as PTEN, p53, MGMT, Rb, and others are typical features of diverse brain neoplasms. Recent research has established a relationship between inflammatory and tumour development, citing the inflammatory milieu as yet another distinguishing hallmark of all brain neoplasms. Tumour growth involves numerous molecular and cellular pathways, as well as a wide spectrum of components. Cytokines are crucial components of physiological inflammatory responses and help to maintain homeostasis. Cytokines significantly boost the proliferation and invasiveness of gliomas. Some of the pro-inflammatory cytokines such as IFN-γ, IL-6, IL-1, IL-8, and TNF-α all play key roles in inflammation by modulating T and B cell development and activation. According to research, many solid tumours release inflammatory cytokines that can regulate cancer cell proliferation and invasion. Some of the anti-inflammatory cytokines include IL-4 and IL-10, which help suppress tumour growth and invasion via suppressing the activation of pro-inflammatory cytokines. In their study of murine gliomas, Qian et al. (2018) found elevated IFN-γ and demonstrated its direct correlation with PD-L1 receptors (Qian et al., 2018). In their study on human glioma tissue, Zhang et al. (2022) concluded that IFN-γ-associated genes are independent prognostic factors for predicting glioma patients' overall survival (Zhang et al., 2022). In the present study on human glioma tissue, IFN-γ expression was significantly higher in gliomas than in the control group, which is consistent with previous research. Nonetheless, a comparable increase of IFN-γ was also observed in meningiomas. See Table 3. Shan et al., (2015) hypothesised that the poor prognosis of glioma is due to IL-6-induced tumour growth and invasion in their study on human glioma tissue, serum, and CSF(Shan et al., 2015). Chen et al., (2016) discovered that the cytomembrane MMP14 was activated by IL-6 produced by astrocytes, promoting glioma cell motility and invasion via MMP2 (Chen et al., 2016). West et al. (2018) demonstrated the role of IL-6/STAT3 signalling in glioblastoma in their study comparing prior papers (West et al., 2018). Ansari et al., (2023) discovered high amounts of IL-6 and TNF-α in the peripheral blood of high-grade gliomas in their investigation on human serum, indicating the disease's invasiveness (Ansari et al., 2023). In the current investigation on human glioma tissue, there was a significantly increase in IL-6 expression with a when compared to the control group, which was consistent with earlier studies (West et al., 2018) (Ansari et al., 2023). Nonetheless, a comparable increase of IL-6 was also observed in meningiomas. See Table 3. In their investigation of the glioma cell lineage culture model, Lu et al. (2007) revealed biological responses to IL-1β and TGF-β in close proximity to the tumour (Lu, Tian, Han, Vogelbaum, & Stark, 2007). Tarassishin et al. (2014), discovered an abnormal expression of IL-1β in gliomas in their work on the glioma cell lineage culture model (Tarassishin, Casper, & Lee, 2014). Fathima et al. (2014) found evidence that IL-1β enhances glioma cell motility, invasion, and proliferation in their investigation of glioma cell lineage culture (Fathima Hurmath, Ramaswamy, & Nandakumar, 2014). IL-1β was elevated in the current investigation on human glioma tissue and demonstrated a significant difference when compared to the control group, which was consistent with earlier results. Nonetheless, a comparable increase of IL-1β was also observed in meningiomas. See Table 3. Hands et al. (2013) used a bioplex immunoassay to estimate cytokine levels in human serum and discovered higher expression of IL-8 in glioma patients' serum (Hands et al., 2013). This investigation on human glioma tissue found considerably higher levels of IL-8 expression as compared to the control group, which was consistent with earlier findings (Hands et al., 2013). Nonetheless, a comparable increase of IL-8 was also observed in meningiomas. See Table 3. Maruno et al. (1997) discovered endogenous TNF-α in cells of numerous origins in glioma tumours, including tumour vasculature, in their investigation on human glioblastoma tissue (Maruno, Kovach, Kelly, & Yanagihara, 1997). Peng et al. (2014) demonstrated the effects of TNF-α on glioma cell viability, proliferation, and apoptosis in their investigation on glioma cell lineage culture (Peng & Ying, 2014). In their study, Wang et al. (2022) created a TNF-α family-based signature to predict the prognosis of a glioma patient (Wang, Lin, Zhu, & Signaling, 2022). Ansari et al. (2023) discovered high amounts of IL-6 and TNF-α in the peripheral blood of high-grade gliomas in their investigation on human serum, indicating the disease's invasiveness (Ansari et al., 2023). This investigation on human glioma tissue found a considerable increase in TNF-α expression when compared to the control group, which was consistent with earlier research. Nonetheless, a comparable increase of TNF-α was also observed in meningiomas. See Table 3.

Table 3

**In this study, the results of IFN-γ, IL-6, IL-1β, IL-8, and TNF-α (pro-inflammatory cytokines) expression compare to earlier investigations.**
CYTOKINE	STUDY	MODEL	INFERENCE
IFN-γ	Qian et al (2018) (Qian et al., 2018)	Murine glioma	IFN-γ was INCREASED and showed its direct co-relation with PD-L1 receptors
	Zhang et al (2023) (Zhang et al., 2022)	Human glioma tissue	IFN-γ related gene signature (INCREASED)
	In the present study (2023)	Human glioma and meningioma tissue	INCREASED IFN-γ expression in both glioma and meningioma.
IL-6	Shan et al (2015)(Shan et al., 2015)	Human tissue, serum, and CSF	IL-6 was INCREASED
	Chen et al (2016)(Chen et al., 2016)	Cell lines	IL-6 was INCREASED
	West et al (2017)(West et al., 2018)	Comparison of previous articles (Human tissue)	Role of IL-6 - STAT3 signaling in glioblastoma. (INCREASED)
	Ansari et a l(2020)(Ansari et al., 2023)	Human serum	INCREASED expression of IL-6
	In the present study (2023)	Human glioma and meningioma tissue	INCREASED expression of IL-6 in gliomas and meningiomas.
IL-1β	Lu et al ( 2007) (Lu et al., 2007)	Cell culture	Dose-dependent cross-talk between TGF and IL-1 (increased)
	Tarassishin et al (2014) (Tarassishin et al., 2014)	Cell culture	Aberrant expression of IL-1β in gliomas (increased)
	Fathima et al 2014 (Fathima Hurmath et al., 2014)	Cell culture	IL-1β promotes the proliferation of glioma cells (increased)
	In the present study (2023)	Human glioma and meningioma tissue	IL-1β was increased in glioma and meningioma tissue.
IL-8	Hands et al (2012) (Hands et al., 2013)	Human serum	INCREASED IL-8 in serum of glioma patients.
IL-8	In the present study (2023)	Human gliomas and meningioma tissue	INCREASED IL-8 expression in gliomas and meningioma.
TNF-α	Maruno et al (1997) (Maruno et al., 1997)	Human glioma tissue	TNF-α INCREASED (distribution of endogenous TNFα in gliomas)
	Peng et al (2014) (Peng & Ying, 2014)	Cell lineage	Effects of TNF-α on cell viability, proliferation, and Apoptosis of glioma cells
	Wang et al (2022) (Wang et al., 2022)	Data sets of human gliomas	Comprehensive analysis of TNF family in diffuse gliomas (INCREASED)
	Ansari et al (2020) (Ansari et al., 2023)	Human serum	INCREASED expression of TNF-α
	In the present study (2023)	Human glioma and meningioma tissue	TNF-α was INCREASED in gliomas and meningioma tissue.

In the current study revealed about the pro-inflammatory cytokine levels of IFN-γ, IL-6, IL-1β, IL-8, and TNF-α were found to be dramatically elevated in gliomas as compared to the control group and showed a significant difference, which was consistent with previous studies. There was no difference in the cytokine milieu concerning the grade of gliomas, which was inconsistent with previous studies. These pro-inflammatory cytokines of glioma tissue were also compared with pro-inflammatory cytokines of meningioma tissue, and no significant difference was observed. See Table 3.

Huettner et al. (1995, 1997) discovered elevated mRNA expression of IL-10 in human glioma tissue and proposed that IL-10 may promote to the advancement of astrocytes by dampening the patient's immune system (Huettner, Czub, Kerkau, Roggendorf, & Tonn, 1997; Huettner, Paulus, & Roggendorf, 1995). Zhang et al. (2019) report in their cell lineage culture investigation that IL-10 enhances glioma growth by upregulating KPNA2 (Zhang et al., 2019). Ansari et al. (2023) found reduced IL-10 in human serum, indicating a suppressive effect of pro-inflammatory cytokines on IL-10 (Ansari et al., 2023). IL-10 expression was significantly lower in this current investigation on human glioma tissue compared to the control group, which was consistent with previous findings (Ansari et al., 2023). Nonetheless, a comparable decrease of IL-10 was also observed in meningiomas. See Table 4. Joshi et al. (2001) discovered that human brain tumors in situ overexpress IL-4R when compared to normal brain tissue in their work on glioma cell lines(Joshi et al., 2001). This present study on human glioma tissue found a significant drop in IL-4 expression when compared to the control group, which contradicted previous research (Joshi et al., 2001). According to Ansari et al. (2023), decreased IL-4 expression may be related to the suppressive action of pro-inflammatory cytokines (Ansari et al., 2023). Nonetheless, a comparable decrease of IL-4 was also observed in meningiomas. See Table 4.

Table 4

**In this study, the results of IL-10 and IL-4 (anti-inflammatory cytokines) expression compare to earlier investigations.**
CYTOKINE	STUDY	MODEL	INFERENCE
IL-10	Huettner et al (1995) (Huettner et al., 1995)	Human glioma tissue	INCREASED mRNA of IL-10
	Huettner et al (1997) (Huettner et al., 1997)	Human (in vivo/ in vitro)	INCREASED IL-10 expression
	Zhang et al (2019) (Zhang et al., 2019)	Cell culture	IL-10 promotes glioma progression via the upregulation of KPNA2.
	Ansari et al (2020) (Ansari et al., 2023)	Human serum	DECREASED IL-10 expression
	In this study (2023)	Human glioma and meningioma tissue	DECREASED IL-10 expression in gliomas and meningiomas.
IL-4	Joshi et al (2001) (Joshi et al., 2001)	Cell lines	INCREASED expression of IL 4 receptors
IL-4	In the present study (2023)	Human glioma and meningioma tissue	DECREASED expression of IL 4 cytokines in gliomas and meningiomas.

This current study pattern revealed about the anti-inflammatory cytokine levels of IL-10, IL-4 are consistent with the findings of Ansari et al., who hypothesised that higher pro-inflammatory cytokines such as IFN-γ, IL-6, IL-1β, IL-8, TNF-α may account for lower levels of IL-10 and IL-4 in glioma tissue when compared to the control group. Anti-inflammatory cytokine levels were comparable regardless of glioma grade. There was also no noticeable change in anti-inflammatory cytokine levels between glioma and meningioma tissue. See Table 3 and Table 4.

Gliomas are distinguished by their widespread invasiveness, tumour necrosis, and angiogenesis. VEGF, FGF-2, TNF-α, IL-1β, IL-6, and other inflammatory cytokines are the threads that connect angiogenesis and cancer (Coppola et al., 2014; Folkman, 2007; Lakka & Rao, 2008; Thomas & Omuro, 2014). When comparing the current study to prior studies, Mentlein et al. (2004), in their study on cell lineage culture, demonstrated the importance of VEGF receptor expression in glioma cells (Mentlein, Forstreuter, Mehdorn, & Held-Feindt, 2004). Folkins et al. (2009) discovered that glioma cancer stem-like cells increase tumour angiogenesis via VEGF in a mouse model (Folkins et al., 2009). Exogenous VEGF increases glioblastoma stem cell multiplication, according to Xu et al. (2013) in their cell culture study (Xu, Wu, & Zhu, 2013). This study on human tissue found significantly higher levels of VEGF expression when compared to the control group, which is consistent with earlier research. Nonetheless, a comparable increase of VEGF was also observed in meningiomas. See Table 5.

Anderson et al. (2008) discovered FGFR (the FGF receptor) overexpression in glioblastoma cells in their investigation on human glioma tissue (Anderson, McFarland, & Gladson, 2008). FGF-2 was highly overexpressed in this present investigation on human glioma tissue as compared to the control group, which was consistent with earlier reports. Nonetheless, a comparable increase of FGF-2 was also observed in meningiomas. See Table 5

Table 5

**In this study, the results of VEGF and FGF-2 (anti-inflammatory cytokines) expression compare to earlier investigations.**
CYTOKINE	STUDY	MODEL	INFERENCE
VEGF	Mentlein et al (2004)(Mentlein et al., 2004)	Cell culture	Significance of VEGF receptor expression in gliomas cells
	Folkins et al (2009) (Folkins et al., 2009)	Mice models	Gliomas tumor stem-like cells promote tumor angiogenesis via VEGF
	Xu et al (2013) (Xu et al., 2013)	Cell culture	Demonstrated that exogenous VEGF stimulates glioblastoma stem cell proliferation
	In the present study (2023)	Human glioma and meningioma tissue	INCREASED VEGF expression in gliomas and meningiomas
FGF-2	Anderson et al (2009) (Anderson et al., 2008)	Human glioma tissue	Glioblastoma cells have overexpression of FGF receptor
FGF-2	In the present study (2023)	Human gliomas and meningioma tissue	INCREASED expression of FGF-2 in gliomas and meningiomas.

Overall, glioma tissue displayed an upregulation of pro-inflammatory and angiogenic cytokines and a downregulation of anti-inflammatory cytokines (IL-10 and IL-4), with no discernible variation between glioma grades. There was no discernible difference between the expression patterns of cytokines in glioma and meningioma tissue. This demonstrates that the production of these cytokines in gliomas and meningiomas is merely a host immunological response. Tumorigenesis, invasiveness, and the ability to metastasize are hallmarks of gliomas and are directly attributable to epigenetic and genetic alterations. Immunotherapy against gliomas has been tried in clinical trials for decades, and while it has a good safety profile, it has not been proved to be effective in reducing the growth of gliomas. Only the anti-VEGF medication bevacizumab is currently licensed for recurrent glioblastoma. The immune checkpoint inhibitor nivolumab is now being tested in humans. Redirecting immunotherapy away from cytokines is necessary. Molecular studies in cancer biology are desperately needed so that the genetic alterations can be targeted. For instance, the tyrosine kinase inhibitor "imatinib" has proven to be an effective targeted therapy in CML for the single mutation (BCR/ABL fusion). Tamoxifen was created as a result of research into the HER2-Neu receptor in breast cancer. However, tumour heterogeneity and the fact that these genomic and epigenomic changes differ from patient to patient have made the development of targeted therapy for gliomas more challenging. As an adjunct to the current standard of care, it may be beneficial to evaluate the efficacy of newer medications targeting individual molecular subtypes of gliomas in upcoming clinical trials.

We found that pro- and anti-inflammatory cytokines were significantly up- and down-regulated in the brain tumour compared to the control brain. To a similar extent, we found that pro-angiogenic cytokines were significantly upregulated in the brain tumour compared to the control brain. Although the expression of these cytokines was dramatically changed in brain tumours, this change did not occur consistently across tumour grades. Neither the glioma nor the meningioma samples displayed any discernible variation in expression. Brain tumours alter their cytokine environment in a way that is more similar to a systemic host immune response. Additional analysis of protein expression, signalling, and protein interaction networks in conjunction with a clinical panel for pain scoring may help explain the relationship between inflammatory mediators, targets for tumoral progression, signalling pathways, and cancer pain therapy. Our small sample size suggests that while cytokine expression does distinguish between control and tumour patients, it may not be useful for identifying glioma sub-stages.

Our findings revealed that there was a statistically significant up and down-regulation in pro- and anti-inflammatory cytokines respectively in the brain tumor samples, compared to control brain samples. Similarly, we also observed a statistically significant upregulation in pro-angiogenic cytokines in the brain tumor supernatants, compared to control brain supernatants. Even though, brain tumors showed a significant alteration in these cytokine’s expression, it was not significantly altered amongst the tumor grades. Further, there was also no significant change in expression between the glioma and meningioma samples. The change in the cytokine milieu in brain neoplasms is more like a generalized host immune response. Based on our limited sample analysis, we conclude that while the cytokine expression differentiates between normal and tumor patients, the cytokine expression analysis may not be positively used for distinguishing the glioma sub-stages

Limitations

The small sample size for each tumour grade grouping is less for comparison between the tumor groups. There were no serum samples in this study. We analyzed commonly abrogated cytokines, and chemokines in this analysis, however, any unbiased analysis of all the human cytokines perhaps gives the best data in this kind of setting. However, due to funding limitations, we could not able to perform such an analysis.

Our research shows a strong relationship between cytokines and angiogenic factors and brain tumor development. Our study showed that a panel of inflammatory cytokines and angiogenic factors is more pertinent than a single molecule among all putative biomarkers for glioblastoma staging and prognosis. Because of their roles in inflammation and pain, cytokines are reliable study candidates that could be used for diagnosis and treatment of glioblastoma. Potentially useful for assessing tumoral development is ELISA technology. Less intrusive procedures, molecular marker screening, and validation of potential treatment targets could be advantages of ELISA technology. Glioblastoma patients commonly experience pain, which is one of the key signs that prompt an examination and diagnosis, but the diagnosis is sometimes delayed or made too late. Cytokines estimation could accelerate diagnosis and treatment.

ANOVA, analysis of variance

APCs, antigen-presenting cells

ARG1, arginase 1

CCL-2, chemokine (C-C motif) ligand 2

CNS, central nervous system

COX-2, cyclooxygenase-2

CSF-1, colony-stimulating factor 1

FGF, fibroblast growth factor

FGFR, fibroblast growth factor receptor

GBM, glioblastoma multiforme

GM-CSF, granulocyte macrophage colony stimulating factor

HGF, hepatocyte growth factor

IDO, Indolamine 2,3 dioxygenase

IFN-g, Interferon-gamma

IL-10, Inteleukin-10

IL-12, Inteleukin-12

IL-1β, Interleukin-1 beta

IL-4, Inteleukin-4

IL-4R, Interleukin-4 receptor

IL-5, Inteleukin-5

IL-6, Interleukin-6

IL-8, Interleukin-8

KPNA2, karyopherin alpha 2

MDSC, myeloid-derived suppressor cells

MHC, major histocompatibility complex

MT1-MMP, membrane type 1 matrix metalloprotease

NF-ҡB, nuclear factor kappa B

NK cells, natural killer cells

PD-1, programmed death-1

PDGF, platelet-derived growth factor

PD-L1, programmed death ligand 1

PTGS2, prostaglandin-endoperoxide synthase 2

STAT3, signal transducer and activator of transcription 3

TAMs, tumour associated macrophages

TGF-β,Transforming growth factor-beta

Th1 and Th2, T helper 1 and 2 cells

TIE, tumour immune escape

TIS, tumour immune surveillance

TNF-α, tumour necrosis factor-alpha

Tregs, regulatory T cells

VEGF, vascular endothelial growth factor.

Acknowledgments

The authors would like to thank The Indian Council of Medical Research for funding this study (ICMR-NIN) No. of File: 20-FT04, 21-FS01, and 21-FS03) and also the Director of the ICMR-National Institute of Nutrition for the support and motivation to work on the project. Nithin Kumar J is a Final Year Resident (M.Ch. Neurosurgery) in the Department of Neurosurgery NIMS, Hyderabad, India. Sangaraju Rajendra is a Research Associate supported by ICMR, New Delhi. All authors thank everyone who contributed technical assistance to our research as well as the patients.

Author contributions

The experiment's conceptualization and design, data analysis, and writing the original manuscript were all done by SKM. Surgery was carried out by NKJ, VM, RA, PSG, RK, VM P. The experiments were carried out by RS, SKM, SNS, BG, DA, SD, while the paper was drafted and edited the manuscript by NKJ, RS, SKM. Performers of the experiments and data analysis were NKJ, RS, VM, S N S, B G, SKM. Statistical analysis was carried out by NKJ, RS, VM, SKM. All authors were reviewed and accepted to publish the manuscript.

Data availability

All the data are available in the manuscript. On reasonable request, the corresponding author will provide raw data for research purposes.

Conflicts of interest

The authors declare no competing financial interest

Ethics statement

This prospective, observational study was conducted in the Department of Neurosurgery, NIMS, Hyderabad and ICMR-NIN, Hyderabad, after clearance from the institutional ethics committee EC/NIMS/2977/2022 and EC/NIN/6/I/2023.

Consent for publication

All listed authors have approved the manuscript before submission, including the names and order of authors

Anderson, J. C., McFarland, B. C., & Gladson, C. L. (2008). New molecular targets in angiogenic vessels of glioblastoma tumours. Expert Rev Mol Med, 10, e23. doi:10.1017/s1462399408000768
Ansari, T., Dutta, G., Srivastava, A. K., Jagetia, A., Singh, D., Singh, H., . . . Kumar, A. (2023). Serum cytokines in astrocytic brain tumors: a prospective study. Br J Neurosurg, 37(1), 35-40. doi:10.1080/02688697.2020.1859461
Arribas Alpuente, L., Menéndez López, A., & Yayá Tur, R. (2011). Glioblastoma: changing expectations? Clinical and Translational Oncology, 13, 240-248.
Balkwill, F., & Mantovani, A. (2001). Inflammation and cancer: back to Virchow? The lancet, 357(9255), 539-545.
Chen, W., Xia, T., Wang, D., Huang, B., Zhao, P., Wang, J., . . . Li, X. (2016). Human astrocytes secrete IL-6 to promote glioma migration and invasion through upregulation of cytomembrane MMP14. Oncotarget, 7(38), 62425-62438. doi:10.18632/oncotarget.11515
Colotta, F., Allavena, P., Sica, A., Garlanda, C., & Mantovani, A. (2009). Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis, 30(7), 1073-1081.
Coppola, D., Balducci, L., Chen, D.-T., Loboda, A., Nebozhyn, M., Staller, A., . . . Brem, S. J. J. o. G. O. (2014). Senescence-associated-gene signature identifies genes linked to age, prognosis, and progression of human gliomas. 5(4), 389-399.
Fathima Hurmath, K., Ramaswamy, P., & Nandakumar, D. N. (2014). IL-1β microenvironment promotes proliferation, migration, and invasion of human glioma cells. Cell Biol Int, 38(12), 1415-1422. doi:10.1002/cbin.10353
Folkins, C., Shaked, Y., Man, S., Tang, T., Lee, C. R., Zhu, Z., . . . Kerbel, R. S. (2009). Glioma tumor stem-like cells promote tumor angiogenesis and vasculogenesis via vascular endothelial growth factor and stromal-derived factor 1. Cancer Res, 69(18), 7243-7251. doi:10.1158/0008-5472.Can-09-0167
Folkman, J. J. N. r. D. d. (2007). Angiogenesis: an organizing principle for drug discovery? , 6(4), 273-286.
Grivennikov, S. I., Greten, F. R., & Karin, M. (2010). Immunity, inflammation, and cancer. Cell, 140(6), 883-899.
Hands, J. R., Abel, P., Ashton, K., Dawson, T., Davis, C., Lea, R. W., . . . Baker, M. J. (2013). Investigating the rapid diagnosis of gliomas from serum samples using infrared spectroscopy and cytokine and angiogenesis factors. Anal Bioanal Chem, 405(23), 7347-7355. doi:10.1007/s00216-013-7163-z
Huettner, C., Czub, S., Kerkau, S., Roggendorf, W., & Tonn, J. C. (1997). Interleukin 10 is expressed in human gliomas in vivo and increases glioma cell proliferation and motility in vitro. Anticancer Res, 17(5a), 3217-3224.
Huettner, C., Paulus, W., & Roggendorf, W. (1995). Messenger RNA expression of the immunosuppressive cytokine IL-10 in human gliomas. Am J Pathol, 146(2), 317-322.
Iwami, K., Natsume, A., & Wakabayashi, T. (2011). Cytokine networks in glioma. Neurosurgical review, 34, 253-264.
Joshi, B. H., Leland, P., Asher, A., Prayson, R. A., Varricchio, F., & Puri, R. K. (2001). In situ expression of interleukin-4 (IL-4) receptors in human brain tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary glioblastoma cell cultures. Cancer Res, 61(22), 8058-8061.
Kesari, S. (2011). Understanding glioblastoma tumor biology: the potential to improve current diagnosis and treatments. Paper presented at the Seminars in oncology.
Lakka, S. S., & Rao, J. S. J. E. r. o. n. (2008). Antiangiogenic therapy in brain tumors. 8(10), 1457-1473.
Louis, D. N., Perry, A., Wesseling, P., Brat, D. J., Cree, I. A., Figarella-Branger, D., . . . Reifenberger, G. (2021). The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol, 23(8), 1231-1251.
Lu, T., Tian, L., Han, Y., Vogelbaum, M., & Stark, G. R. (2007). Dose-dependent cross-talk between the transforming growth factor-beta and interleukin-1 signaling pathways. Proc Natl Acad Sci U S A, 104(11), 4365-4370. doi:10.1073/pnas.0700118104
Maruno, M., Kovach, J. S., Kelly, P. J., & Yanagihara, T. (1997). Distribution of endogenous tumour necrosis factor alpha in gliomas. J Clin Pathol, 50(7), 559-562. doi:10.1136/jcp.50.7.559
Mentlein, R., Forstreuter, F., Mehdorn, H. M., & Held-Feindt, J. (2004). Functional significance of vascular endothelial growth factor receptor expression on human glioma cells. J Neurooncol, 67(1-2), 9-18. doi:10.1023/b:neon.0000021737.89357.cc
Mrugala, M. M. (2013). Advances and challenges in the treatment of glioblastoma: a clinician’s perspective. Discovery medicine, 15(83), 221-230.
Ohka, F., Natsume, A., & Wakabayashi, T. (2012). Current trends in targeted therapies for glioblastoma multiforme. Neurology research international, 2012.
Ostrom, Q. T., Bauchet, L., Davis, F. G., Deltour, I., Fisher, J. L., Langer, C. E., . . . Walsh, K. M. (2014). The epidemiology of glioma in adults: a “state of the science” review. Neuro Oncol, 16(7), 896-913.
Ostrom, Q. T., Price, M., Neff, C., Cioffi, G., Waite, K. A., Kruchko, C., & Barnholtz-Sloan, J. S. (2022). CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2015–2019. Neuro Oncol, 24(Supplement_5), v1-v95.
Peng, Z., & Ying, L. (2014). Effects of TNFα on cell viability, proliferation and apoptosis of glioma cells U251. J buon, 19(3), 733-741.
Qian, J., Wang, C., Wang, B., Yang, J., Wang, Y., Luo, F., . . . Chu, Y. J. J. o. n. (2018). The IFN-γ/PD-L1 axis between T cells and tumor microenvironment: hints for glioma anti-PD-1/PD-L1 therapy. 15(1), 1-13.
Sangaraju, R., Alavala, S., Nalban, N., Jerald, M. K., & Sistla, R. J. I. I. (2021). Galangin ameliorates Imiquimod-Induced psoriasis-like skin inflammation in BALB/c mice via down regulating NF-κB and activation of Nrf2 signaling pathways. 96, 107754.
Sangaraju, R., Nalban, N., Alavala, S., Rajendran, V., Jerald, M. K., & Sistla, R. J. I. R. (2019). Protective effect of galangin against dextran sulfate sodium (DSS)-induced ulcerative colitis in Balb/c mice. 68, 691-704.
Shan, Y., He, X., Song, W., Han, D., Niu, J., & Wang, J. (2015). Role of IL-6 in the invasiveness and prognosis of glioma. Int J Clin Exp Med, 8(6), 9114-9120.
Sinha, S. N., Kumpati, R. K., Ramavath, P. N., Sangaraju, R., Gouda, B., & Chougule, P. J. S. R. (2022). Investigation of acute organophosphate poisoning in humans based on sociodemographic and role of neurotransmitters with survival study in South India. 12(1), 16513.
Stupp, R., Mason, W. P., Van Den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., . . . Bogdahn, U. (2005). Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New England journal of medicine, 352(10), 987-996.
Tarassishin, L., Casper, D., & Lee, S. C. (2014). Aberrant expression of interleukin-1β and inflammasome activation in human malignant gliomas. PLoS One, 9(7), e103432. doi:10.1371/journal.pone.0103432
Thomas, A. A., & Omuro, A. J. C. t. o. i. o. (2014). Current role of anti-angiogenic strategies for glioblastoma. 15, 551-566.
Wang, Q.-W., Lin, W.-W., Zhu, Y.-J. J. C. C., & Signaling. (2022). Comprehensive analysis of a TNF family based-signature in diffuse gliomas with regard to prognosis and immune significance. 20, 1-15.
West, A. J., Tsui, V., Stylli, S. S., Nguyen, H. P. T., Morokoff, A. P., Kaye, A. H., & Luwor, R. B. (2018). The role of interleukin-6-STAT3 signalling in glioblastoma. Oncol Lett, 16(4), 4095-4104. doi:10.3892/ol.2018.9227
Xu, C., Wu, X., & Zhu, J. (2013). VEGF promotes proliferation of human glioblastoma multiforme stem-like cells through VEGF receptor 2. ScientificWorldJournal, 2013, 417413. doi:10.1155/2013/417413
Zhang, Z., Huang, X., Li, J., Fan, H., Yang, F., Zhang, R., . . . Xin, T. (2019). Interleukin 10 promotes growth and invasion of glioma cells by up-regulating KPNA 2 in vitro. J Cancer Res Ther, 15(4), 927-932. doi:10.4103/jcrt.JCRT_284_19
Zhang, Z., Shen, X., Tan, Z., Mei, Y., Lu, T., Ji, Y., . . . Lv, Q. (2022). Interferon gamma-related gene signature based on anti-tumor immunity predicts glioma patient prognosis. Front Genet, 13, 1053263. doi:10.3389/fgene.2022.1053263
Zhu, Z., Zhong, S., & Shen, Z. (2011). Targeting the inflammatory pathways to enhance chemotherapy of cancer. Cancer biology & therapy, 12(2), 95-105.

No competing interests reported.

ManuscriptSuppl.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Evaluation of Pro- and Anti-Inflammatory Cytokines in Glioma Patients: Correlation with Tumor Grading

Status:

Version 1

Abstract

Objective and design:

Methodology:

Result

Conclusion

Figures

Introduction

Materials and Methods

Chemicals and ELISA kits

Design of the study site and duration

Selection of meningioma and glioma-related cytokines in human subjects

Selection of subjects

Method of patient selection and experimental groups

Sample collection

Tissue homogenization and sample preparation for estimation of cytokines in control, glioma, and meningioma patients

Statistical Analysis

Results

Sociodemographic, case-specific, and epidemiological information on glioma and meningioma cases

Discussion

Limitations

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1