The Relationship Among Integrin Alpha 7, CD133 and Nestin as Well as Their Correlation With Clinicopathological Features and Prognosis in Astrocytoma Patients

Changtai Li (  cai48527922@163.com ) Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region https://orcid.org/0000-0002-9372-9452 Jiao Liu Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region Wei Yang Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region Chongyi Chen Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region Bo Wu Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region


Introduction
Astrocytoma, as the most frequent central nervous system (CNS) tumor, has a high incidence in males compared to females with a ratio of 1.4:1 [1][2][3]. Regarding its management, different options are applied according to the world health organization (WHO) classi cation of astrocytoma [1]. In detail, surgery following radiotherapy is commonly applied in low grade astrocytoma patients (WHO grade I and grade II), while surgery in combination with adjuvant chemotherapy and/or radiotherapy is used to treat high grade astrocytoma patients (WHO grade III and WHO grade IV) since most astrocytomas in this classi cation are in diffuse form and may cause intracranial metastasis [4]. However, even after appropriate management, some astrocytoma patients (such as anaplastic astrocytoma patients) experience multiple, rapid recurrence which further decreases their long-term survival pro le [2,5,6]. Therefore, it is necessary and urgent to discover novel biomarkers to improve prognosis in astrocytoma patients.
Integrins are large, membrane-spanning, heterodimeric cell-surface proteins that contribute to cell proliferation, differentiation, adhesion and migration by interacting with extracellular matrix (ECM) and cytoskeleton [7,8]. Among which, integrin alpha-7 (ITGA7) is a key mediator in rectal cancer, breast cancer and astrocytoma pathogenesis [9][10][11][12]. In addition, in the clinical eld, ITGA7 high expression correlates with poor prognosis in clear renal cell carcinoma patients, rectal cancer patients and breast cancer patients [10,13,14]. Furthermore, ITGA7 not only acts as a potential cancer stem cell (CSC) marker, but also regulates multiple other CSC marker expressions (including CD44, CD90, CD133, SOX2, OCT3/4 and NANOG), meanwhile, these CSC markers (such as CD133 and Nestin) correlate with shorter overall survival (OS) in astrocytoma patients [9,11,[15][16][17]. Given the fact that ITGA7 regulates CSC marker expressions in several cancers, besides CD133 and Nestin are well-characterised CSC markers in astrocytoma, we hypothesized that ITGA7 assessment might have a potentially clinical value in astrocytoma patients [18][19][20][21]. However, no relevant research has been conducted yet. Herein, we performed this study and aimed to explore the relationship among ITGA7, CD133 and Nestin, as well as to explore their correlation with clinicopathological features and prognosis in astrocytoma patients.

Patients and specimens
The retrospective study was carried out with the approval from the Institutional Review Board of Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region (Hospital. C.T). Totally, 124 patients with primary astrocytoma who underwent surgical resection in the hospital between January 2016 and June 2020 were included in this study. By reviewing their clinical data, only patients who met the following criteria were screened out for study analysis: (i) histologically-con rmed astrocytoma in accord with World Health Organization (WHO) classi cation criteria [22]; (ii) age ≥ 18 years; (iii) directly treated by surgical resection without neoadjuvant therapy; (iv) formalin xed and para n embedded tissue (FFEPT) of astrocytoma was well preserved and available for immunohistochemical (IHC) assessment; (v) major clinical features, treatment information, and survival data were integral and available; (vi) no history of other malignant diseases; (vii) without history of exposure to radiotherapy or chemotherapy before diagnosis of astrocytoma. The written informed consents were obtained from the patients or their relatives. A total of 124 astrocytoma FFEPT specimens were acquired form the hospital's sample library, which were used for determination of ITGA7, CD133 and Nestin expression by IHC assay.

Data compilation
The major clinical feature data and treatment information, including patients' age, gender, WHO grade (2016 WHO Classi cation [22]), isocitrate dehydrogenase (IDH) mutation, preoperative Karnofsky Performance Scale (KPS) score and adjuvant therapy, were collected from the medical documents. In addition, follow-up records of patients were reviewed to abstract the survival information for estimation of overall survival (OS).

IHC assay
The FFEPT specimens were sliced into sections at a thickness of 4 µm, which were then depara nized with xylene, rehydrated with ethanol, and treated with hydrogen peroxide, followed by blocking with normal goat serum. Afterwards, the sections were respectively incubated overnight with primary antibodies (Invitrogen, Carlsbad, California, USA), including ITGA7 Polyclonal Antibody (1:25 dilutions), CD133 Polyclonal Antibody (1:500 dilutions) and Nestin Polyclonal Antibody (1:1000 dilutions). Following that, the sections were incubated with Goat anti-Rabbit IgG (H + L) Secondary Antibody (1:5000 dilutions, Invitrogen, Carlsbad, California, USA). Chromogenic reaction was realized using diaminobenzidine, followed by counterstaining with hematoxylin. At the end of IHC assay, the staining results of sections were viewed and photographed using a microscope. The intensity and density of the IHC staining were scored by two experts, and the detailed scoring procedures were performed in terms of the method described in the published literature [23,9,24]. The IHC score was the product of intensity score and density score, ranging from 0 to 12. The average IHC score of the two experts was used as the nal IHC score. In the analysis, the cut-off score of 3 was used to classify the high and low expression, Regarding their relationship, ITGA7 expression positively correlated with CD133 expression and Nestin expression (both P=0.001) in astrocytoma patients (Table 2). Moreover, CD133 expression positively associated with Nestin expression in astrocytoma patients (P=0.002).

Discussion
ITGA7 is a cell adhesion molecule which is critical for cell migration, homeostasis and translocation [7,8].
Apart from its critical role in normal cell biology, ITGA7 is also able to induce cell malignant behaviours in several cancers [8,7,[9][10][11]. One study displays that ITGA7 regulates phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway to further promote glioma CSC proliferation and clonogenic survival [12]. Recently, ITGA7 has been identi ed as a novel biomarker in several cancer patients. For example, ITGA7 expression positively correlates with pathological grade and TNM stage in clear cell renal cell carcinoma patients, breast cancer patients and rectal cancer patients [10,14,13]. Furthermore, ITGA7 high expression is related to worse OS in rectal cancer patients and breast cancer patients [10,14].
However, limited study can be found in terms of the correlation of ITGA7 with prognosis in astrocytoma patients. Therefore, we performed this study and discovered that ITGA7 high expression correlated with advanced WHO grade and poor prognosis in astrocytoma patients. The possible reasons to explain these ndings were: (a) ITGA7 might promote astrocytoma cell proliferation, invasion and metastasis through the regulation of PI3K/Akt pathway, therefore led to deteriorating clinical features in astrocytoma patients [12,17]. (b) ITGA7 might regulate ERK signalling pathway to mediate cell differentiation, thereby led to increased WHO grade in astrocytoma patients [25]. (c) Regarding their survival pro le, ITGA7 high expression might lead to increased chance of developing chemotherapy resistance due to the clonogenic survival of CSC, therefore resulted in poor prognosis in astrocytoma patients [26].
Great attention has been paid in discovering the properties of CSC since they are responsible for cancer metastasis, recurrence and drug resistance [27,28]. CD133 and Nestin are two heavily investigated CSC markers, meanwhile they are commonly co-expressed in neural stem cells [29]. In the clinical eld, CD133 and Nestin correlated with advanced WHO grade, also their high expressions correlate with shorter OS in astrocytoma patients [15]. However, no relevant research reports their relationship with ITGA7 in astrocytoma patients. In the present study, we discovered that ITGA7 expression positively correlated with CD133 and Nestin expressions in astrocytoma patients. Besides, both CD133 and Nestin expressions correlated with advanced WHO stage and unfavourable survival pro le in astrocytoma patients. Furthermore, CD133 high expression could independently predict shorter OS in astrocytoma patients, which could be explained as follows: (a) Regarding the correlation of ITGA7 expression with CD133 and Nestin expressions, high ITGA7 expression suggested increased CSC cell proportion and density as discussed earlier, meanwhile CD133 and Nestin were glioma CSC markers, therefore, ITGA7 positively correlated with CD133 and Nestin in astrocytoma patients [29]. (b) In terms of the correlation of CD133 and Nestin expression with WHO grade, elevated CSC marker expressions (CD133 and Nestin in this case) suggested inhibition of glioma CSC apoptosis while promotion of CSC angiogenesis through Wnt/βcatenin and PI3K/Akt pathways, therefore led to clonogenic survival of glioma CSC and further led to differentiation of glioma CSC into astrocytoma cells, which resulted in elevated astrocytoma cell density and irregular nuclear morphology, therefore led to increased WHO grade in astrocytoma patients [27,28]. (c) In terms of the correlation of CD133 and Nestin with prognosis, elevated CSC marker expressions (CD133 and Nestin) indicated a higher proportion of CSC, which led to increased chance of developing drug resistance and distal metastasis, therefore resulted unfavourable survival pro le in astrocytoma patients [28,27].
There were several limitations in the current study. Firstly, the interacting mechanisms among ITGA7, CD133 and Nestin was not discovered in the present study, and further study was needed. Secondly, the sample size in our study was relatively small, therefore further study with larger sample size to validate the prognostic value of these biomarkers was needed. Thirdly, we detected ITGA7, CD133 and Nestin expressions from astrocytoma tissue samples harvested from patients, therefore these marker expressions from blood samples were not detected and further study could investigate this aspect. Finally, further experiments were warranted to determine the effect of ITGA7 on chemotherapy resistance in astrocytoma patients.
In conclusion, ITGA7, CD133 and Nestin are intercorrelated, also their high expressions correlate with deteriorating disease condition and poor prognosis in astrocytoma patients, suggesting their potentially clinical value for astrocytoma management.

Declarations
Ethics approval and consent to participate The retrospective study was carried out with the approval from the Institutional Review Board of Hospital of Chengdu O ce of People's Government of Tibetan Autonomous Region (Hospital. C.T). The written informed consents were obtained from the patients or their relatives.

Consent for publication
Not applicable.

Availability of data and material
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.