Conspectus Borealis
Volume 4 | Issue 1 Article 4
8-31-2018 Immunohistochemical Analysis of Basigin and Axl Expression in Gliomas Brittney N. Moore Northern Michigan University, [email protected]
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Recommended Citation Moore, Brittney .N (2018) "Immunohistochemical Analysis of Basigin and Axl Expression in Gliomas," Conspectus Borealis: Vol. 4 : Iss. 1 , Article 4. Available at: https://commons.nmu.edu/conspectus_borealis/vol4/iss1/4
This Article is brought to you for free and open access by the Journals and Peer-Reviewed Series at NMU Commons. It has been accepted for inclusion in Conspectus Borealis by an authorized administrator of NMU Commons. For more information, please contact Kevin McDonough. Immunohistochemical Analysis of Basigin and Axl Expression in Gliomas
Abstract:
Glioblastoma (GBM) tumors are a type of brain cancer with few effective treatment options. The median survival time of patients who receive the standard care of surgical resection, radiation, and chemotherapy is little more than a year. Characterization of novel biomarkers is essential in developing improved therapies for GBM. Receptor tyrosine kinases (RTKs) are an attractive target in cancer therapy because they are often overexpressed in cancers and there are many inhibitors which reduce their signaling capacity. The protein called Axl is an RTK that is highly expressed in GBM. Axl is involved in epithelial to mesenchymal transition (EMT), which is a process that underlies cancer metastasis. In order for cancer to metastasize, however, the cells must be able to move through the extracellular matrix (ECM), and therefore ECM remodeling must occur. A key protein in stimulation of ECM degradation is Basigin, a transmembrane glycoprotein that stimulates matrix metalloproteinase (MMP) expression, the enzymes responsible for ECM remodeling. This action facilitates tumor invasion by degrading physical barriers to proliferation. Basigin has previously been demonstrated to be overexpressed in GBM by immunohistochemistry assay. Little work has been done to characterize Axl expression in lower grade brain tumors such as astrocytomas. As the expression of Axl and
Basigin individually correlate with a more aggressive tumor phenotype, it was hypothesized that the expression of Axl and Basigin are directly correlated in astrocytic tumors.
Background and Context:
Glioblastoma multiforme (GBM) is the most severe type of glioma with no cure. Median survival time of patients with treatment is 15 to 31 months (Louis et al., 2016). Treatment for glioblastoma includes surgical resection, radiation, and treatment with temozolamide, an alkylating chemotherapy agent. Glioblastoma is a high-grade astrocytoma, a cancer that develops from astrocytes in the brain. There are four grades of astrocytomas, including: pilocytic astrocytoma (grade 1), diffuse astrocytoma/low-grade astrocytoma (grade 2), anaplastic astrocytoma (grade 3), and GBM (grade 4) (Louis et al., 2016). Tumor diagnosis typically begins with a histological diagnosis, at which time tumors are characterized as astrocytoma, glioblastoma, or another brain tumor type, such as oligodendroglioma or oligoastrocytoma.
Following diagnosis, immunohistochemistry is used to test for IDH1 mutation status, which is followed by DNA sequencing to confirm diagnosis. If sequencing is not available, the tumor will have a subclass of ‘NOS’ (not otherwise specified). Therefore, all astrocytomas and GBM tumors are classified first by the type of tumor, then by the IDH mutation status (Louis et al.,
2016). Thus far, it appears that GBM patients with an IDH mutation have a ‘better’ prognosis.
IDH1-mutant tumors develop from previous astrocytic lesions; therefore they are ‘secondary’ lesions, and typically develop in younger patients. Unfortunately, IDH mutant GBM only represents about 10% of GBM diagnoses. The IDH1 gene encodes for the enzyme isocitrate dehydrogenase 1 (IDH1), which functions in the citric acid cycle to produce the intermediate metabolite -ketoglutarate (-KG). The mutant IDH1 enzyme, however, converts - ketoglutarate to 2-hydroxyglutarate (2-HG) which accumulates in the cytoplasm of the cell and competitively inhibits enzymes such as DNA and Histone demethylases (Parker & Metallo,
2015). This mutation therefore may contribute to a better overall prognosis in GBM because it prevents demethylation of DNA, and therefore does not inhibit the function of temozolamide, which acts to methylate DNA. The majority of GBM tumors typically occur in older individuals and arise ‘de novo’, meaning that there was no previous lesion from which they developed. These patients have a very poor prognosis and a median survival time of 15 months with standard treatment (Louis et al., 2016).
Glioblastoma tumors are heterogeneous in nature with various subpopulations of tumor cells, including cancer stem cells (J. Chen et al., 2012). A population of quiescent cancer stem cells often remain within the patient following treatment, as the chemotherapeutic agents like temozolamide target growing and dividing cells. Over time, these cancer stem cells will grow and differentiate, leading to cancer recurrence, which is nearly inevitable with GBM
(Heddleston, Li, McLendon, Hjelmeland, & Rich, 2009). Another contributing factor to GBM recurrence is the tumor morphology. As GBM originates from astrocytes, the star-shaped supportive glial cells of the nervous system, these tumors tend to be spindly, diffuse, and penetrate into surrounding normal brain tissue. This makes it difficult to distinguish normal tissue from tumor tissue preventing complete surgical resection of the tumor.
The expression of specific biomarkers in GBM tumors vary significantly within the tumor. For example, FISH analysis on formalin fixed paraffin embedded tissues demonstrates that there is non-equal distribution of receptor tyrosine kinase expression in patient samples
(Szerlip et al., 2012). These subpopulations may arise from selective pressure generated in the microenvironment surrounding the tumor. It is plausible that variance in protein expression results from tumor niches or from cellular interactions (Szerlip et al., 2012). This concept is important in developing therapy for GBM as many anti-RTK therapies are monotherapies.
Szerlip et al. further elucidate the heterogeneity of the tumor microenvironment and the need for complex multi-targeted therapy rather than monotherapies.
Many cell-surface proteins have been the target of recent GBM research. One of these proteins, Basigin (CD147/EMMPRIN), is a transmembrane glycoprotein that belongs to the immunoglobin protein superfamily. The protein has four known isoforms, which are expressed at varying levels in human tissues. Basigin-2, which has two immunoglobin domains, is the principal isoform expressed in tumor cells. Basigin-1, expressed in retinal tissue, has three immunoglobin domains. Isoforms -3 and -4 were most recently discovered and little is known regarding their expression and function. However, Basigin-3, with one immunoglobin domain, has been demonstrated to inhibit cell proliferation and invasion by forming hetero-oligomers with Basigin-2 (Liao et al., 2011). In addition, Basigin-4, also with one immunoglobin domain, has been shown to be upregulated in cervical cancer (Wu, Zhou, & Zheng, 2011).
Basigin-2 is known to interact with various membrane proteins implicated in cancer cell survival and metastasis, such as CD98, Monocarboxylate Transporters (MCTs), MMPs, and
Integrins. These proteins are implicated in various cellular functions, including amino acid transport, lactate transport, extracellular matrix degradation, and cytoskeletal rearrangement, respectively. Basigin expression also upregulates MMP-1, MMP-2, and MMP-9 expression, inducing degradation of the extracellular matrix (ECM) surrounding tumors (Zhong et al., 2008).
Degradation of the ECM facilitates cancer invasion and metastasis by providing an environment in which micro-metastases can grow.
Immunohistochemical analysis has shown upregulated CD98 and Basigin in non-small cell lung cancer to contribute to poor overall survival. Furthermore, co-immunoprecipitation demonstrated that these proteins formed a complex at the plasma membrane (Fei et al., 2014).
CD98 is a transmembrane heterodimer that interacts with Integrins to induce cell motility, proliferation, and invasion. Additionally, data showed that the CD147-CD98 complex affected cell proliferation via the PI3K/Akt pathway (Fei et al., 2014). This is significant because it demonstrates that complexes formed with CD147 affect cellular pathways, which contribute to a cancerous phenotype. It is important to continue to investigate proteins that complex with CD147 to evaluate whether they are therapeutic targets in GBM. Recently, Basigin has also been implicated in immune modulation by regulating T-cell signaling (Supper et al., 2016).
Specifically, Basigin was shown to inhibit IL-2 production by inhibiting NFAT and NFB transcription via association with PMCA-4 (Supper et al., 2016). These data link CD147 to T- cell suppression in later stage activation, which would indicate that Basigin is involved in regulating the normal immune response.
Interestingly, Basigin is expressed in cells which are participating in invasion, such as the embryo during the implantation phase (L. Chen, Belton, & Nowak, 2009). For example, in uterine tissue, Basigin has been shown to be expressed at high levels during embryo implantation
(L. Chen et al., 2009). Basigin is vital to MMP dependent degradation of uterine fibroblasts, which is necessary for embryo implantation. This invasion cascade is similar to the cascade necessary for metastasis, and therefore Basigin expression in tumors results in higher rates of metastasis and invasion (Fu et al., 2011).
In addition to the effects described above, Basigin also induces angiogenesis and lymphangiogenesis in the tumor microenvironment by upregulating vascular endothelial growth factor (VEGF) (Jia, Wang, Qu, Miao, & Zhang, 2008). Stimulating lymph and blood vessel growth aids in tumor growth by providing a route for nutrients to supply the tumor.
Furthermore, Basigin, among other proteins, serves as a cell surface receptor for soluble
Basigin released in microvesicles and exosomes (Belton, Chen, Mesquita, & Nowak, 2008). This provides a mechanism for Basigin to stimulate surrounding cells in a paracrine fashion in addition to signaling in an autocrine fashion, thereby promoting MMP expression and extra- cellular matrix degradation. Expression of proteins like Basigin at the invading edge of the tumor would result in stromal degradation and may contribute to invasion.
Axl is a receptor tyrosine kinase also implicated in cancer metastasis. In addition, Axl has been implicated in development of chemotherapy resistance, and the immune response to tumors.
Axl has been demonstrated to affect intracellular pathways such as the Akt, NF-B, MEK-ERK, and metalloproteinase intracellular cascades (Gay, Balaji, & Byers, 2017). Axl is believed to promote epithelial-mesenchymal transition (EMT) in tumor cells (Antony & Huang, 2017). EMT is essential for tumor metastasis as epithelial cells must transition to mesenchymal cells to migrate to other tissues, and the majority of cancers are epithelial in origin. Mesenchymal cells lack the specialized contacts that forces epithelial tissue to be tightly regulated, and therefore they gain the ability to invade vasculature and travel to distant tissues to create metastases in the body.
Axl’s immunosuppressive activity is also likely to influence to ability of micro- metastases to develop into a proliferative metastasis by suppressing immune response, specifically natural killer cell response in the secondary lymphoid tissues (Gay et al., 2017).
Gas6 is the natural ligand for Axl, and binding with Gas6 causes homo-dimerization of Axl.
However, Axl can homo-dimerize without Gas6 when expression levels are high, causing activation within the cell (Antony & Huang, 2017).
Vouri and colleagues demonstrated that Axl and the epidermal growth factor receptor
(EGFR) form heterodimers at the plasma membrane and induce intracellular effects (Vouri et al.,
2016). Specifically, epidermal growth factor (EGF) stimulation causes activation of Axl, but
Gas6 stimulation did not cause activation of EGFR. Stimulation with EGF activated downstream signaling event such as Akt phosphorylation and ERK phosphorylation, and inhibition of EGF downregulated these effects. Furthermore, Vouri et al. demonstrated that the Axl-EGFR heterodimer is found constitutively, regardless of EGF stimulation. Axl has additionally been shown to be a strong predictor of chemotherapy resistance in triple negative breast cancer cells via interaction and signal amplification of EGFR (Meyer, Miller, Gertler, & Lauffenburger,
2013).
Additionally, Basigin, EGFR, and CD44 have been shown to form a complex in lipid rafts at the plasma membrane. CD44 is a transmembrane glycoprotein which is involved in cell adhesion, migration, and invasiveness. CD44 binds hyaluronan and various growth factors in the tumor microenvironment and is important in maintaining stemness of cancer cells (Morath,
Hartmann, & Orian-Rousseau, 2016). Furthermore, CD44 has been demonstrated to be overexpressed in invasive and metastatic tissues (Grass, Tolliver, Bratoeva, & Toole, 2013).
High expression of Basigin has also been shown to correlate with increased concentration of
CD44 and EGFR in lipid raft complexes (Grass et al., 2013).
It is currently unknown if Axl interacts with Basigin at the plasma membrane. However, as Axl and EGFR are known to form hetero-dimers at the plasma membrane, and Basigin, CD44, and EGFR interact in lipid rafts, it could be hypothesized that Basigin and Axl may interact within the plasma membrane as well. Furthermore, it is possible that Basigin mediates Axl distribution in lipid rafts at the plasma membrane, and may facilitate Axl activation via EGFR activation. Both Basigin and Axl are known to activate the MEK-ERK and PI3K-Akt pathways
(Fei et al., 2014; Gay et al., 2017). In addition, Basigin induced MMP9 expression and subsequent ECM degradation could coincide with Axl induced EMT and facilitate metastasis.
Understanding the interaction of Axl and Basigin will be beneficial to develop understanding of the metastasis cascade, and develop our understanding of prognosis and the efficacy of future therapeutic targets toward these complexes.
Immunohistochemical analyses have been previously used to characterize Basigin as a prognostic marker in glioblastoma (Yang et al., 2013). As Axl expression is likely correlated with metastasis, Axl expression could be a useful prognostic marker in glioblastoma. Current biomarkers in glioblastoma include Epidermal Growth Factor Receptor (EGFR), Telomerase
Reverse Transcriptase (TERT), Phosphatase and Tensin Homolog (PTEN), ATP-dependent
Helicase ATRX (ATRX), O-6-Methylguanine Methyltransferase (MGMT), IDH, and Tumor
Protein 53 (TP53) (Louis et al., 2016; Sasmita, Wong, & Ling, 2017). As mentioned previously,
EGFR is often amplified in GBM and contributes to chemotherapy resistance. Additionally,
PTEN mutations may be correlated with susceptibility to tumor progression and increased severity of tumors, as these mutations are not found in low grade gliomas (Arif et al., 2015).
TERT promoter mutations have a strong correlation with worse overall prognosis. These mutations turn on the TERT promotor, which results in telomere lengthening. This promotes survival of the rapidly dividing cancer cells by protecting the cancer genome from degradation during division (Bell et al., 2016; Louis et al., 2016). Methylation, and therefore silencing, of the
MGMT promoter results in a favorable prognosis. MGMT is a DNA repair enzyme, which removes methyl groups from DNA. As temozolamide functions by adding methyl groups to
DNA, active MGMT contributes to temozolamide resistance (Hegi et al., 2005). TP53 and
ATRX are both commonly lost in the progression of cancer. TP53 is a tumor suppressor gene and ATRX is involved in maintaining genomic stability through the regulation of histone proteins (Nandakumar, Mansouri, & Das, 2017; Olivier, Hollstein, & Hainaut, 2010). Therefore, these two mutations are common in the slower progressing IDH-mutant GBM, while TERT, EGFR, and PTEN mutations are more often found in IDH-wildtype rapidly-progressing GBM
(Louis et al., 2016).
Despite discovery of all these biomarkers in GBM, prognosis remains devastatingly poor.
In addition, development of novel therapies takes time and monotherapies have thus far proven to be minimally successful. Therefore, establishing new useful biomarkers is important in cancer therapy as effective biomarkers help to refine diagnosis, prognosis, and treatment of individual tumors. It is necessary to continue to explore potential prognostic markers in GBM and to elucidate the basic biology of these molecules to characterize their function and utilize them as a focus for multi-targeted therapies. Immunohistochemical analysis of Axl expression is an important first step in evaluating expression levels of Axl. Axl expression has been demonstrated to be correlated with a more invasive phenotype in GBM cancer cells and expression has been correlated with a shorter overall survival time in GBM (Onken et al., 2016, 2017). Given that
Basigin expression has also been correlated with a worse prognosis in GBM, it is hypothesized that Axl expression will be directly correlated with Basigin expression across tumor grades.
Methods:
Specimens were obtained from Upper Peninsula Health System (UPHS): Marquette.
Patient samples were obtained and fixated in 2005, 2006, and 2007 by UPHS Marquette.
Samples used in this experiment were previously evaluated by pathologists and graded as follows: pilocytic astrocytoma (grade 1), diffuse astrocytoma (grade 2), anaplastic astrocytoma
(grade 3), and GBM (grade 4). Two separate patient samples were used for each tumor grade.
Patient samples were de-identified by renaming samples to patients 1-8. Paraffinized tumor samples were sectioned to 5 µm widths (Leica RM2125 RTS Microtome), deparaffinized using xylene, and rehydrated with a graded series of histological grade alcohol. Immunohistochemistry assay was performed with an ImmPRESS Duet Double Staining HRP/AP Polymer Kit (anti- rabbit IgG-brown, anti-mouse IgG-red) (Vector Laboratories) according to the manufacturer’s instructions with minor modifications. Specifically, endogenous peroxidase and alkaline phosphatase activity of samples was deactivated with BLOXALL blocking solution for 20 minutes (Vector laboratories). Slides were incubated with 2.5% normal horse serum for 60 minutes, followed by incubation with polyclonal rabbit Axl antibody (1:200; Proteintech) and monoclonal mouse Anti-CD147 [MEM-M6/1] antibody (1:200; abcam) at 4°C overnight.
ImmPRESS Duet Detection Reagent was then incubated with slides for 10 minutes, followed by incubation with ImmPACT DAB EqV substrate for 2 minutes. ImmPACT Vector Red substrate was then incubated with slides for 20 minutes. Lastly, slides were counterstained with hematoxylin for 5 minutes. Negative controls were created by staining slides with secondary antibody, but no primary antibody, and also by using either only Basigin or Axl primary antibody with the Vector Laboratoies secondary antibody kit. Coverslips were mounted aqueously using Vectamount AQ solution (Vector Laboratories). Hematoxylin and Eosin staining were performed by standard methods. Coverslips were mounted with Vectamount
Permanent Mounting Media (Vector Laboratories). Sections were examined and imaged with an
Olympus BX41 microscope equipped with an Olympus DP72 camera using cellSens software.
Results:
Expression of Axl and Basigin was characterized by immunohistochemistry in two cases each for pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and GBM. All tumor samples positively immunostained for Basigin and Axl; however, expression level and stain development varied greatly between tumor types and patient samples (Figures 1-8). Hematoxylin and Eosin staining of patient samples demonstrate tumor progression
(Figures 9 and 10). As seen in progression from pilocytic astrocytoma (grade 1) to GBM (grade
4), there is an increase in cell number which is demonstrated by an increased number of cell nuclei (stained with hematoxylin). In addition, cell nuclei become more spindle shaped and anaplastic. Lastly, there is an appreciable increase in tissue necrosis and vascularity, common characteristics of GBM.
Figure 1. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in pilocytic astrocytoma (patient no. 1). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 2. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in pilocytic astrocytoma (patient no. 2). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 3. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in grade 2 astrocytoma (patient no. 3). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 4. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in grade 2 astrocytoma (patient no. 4). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 5. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in grade 3 astrocytoma (patient no. 5). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 6. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in grade 3 astrocytoma (patient no. 6). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 7. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in GBM (patient no. 7). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 8. Immunohistochemistry assay of Basigin (red) and Axl (brown) expression in GBM (patient no. 8). A, Axl expression; 10x. B, Axl expression, 40x. C, Basigin expression, 10x. D, Basigin expression, 40x. E, Dual-labeled Axl and Basigin expression, 10x. F, Dual-labeled Axl and Basigin expression, 40x. G, Negative control; 10x. H, Negative control; 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 9. Hematoxylin and Eosin staining of pilocytic astrocytoma (A-D) and grade 2 astrocytoma (E-G). A, pilocytic astrocytoma (patient no. 1); 10x. B, pilocytic astrocytoma (patient no. 1); 40x. C, pilocytic astrocytoma (patient no. 2); 10x. D, pilocytic astrocytoma (patient no. 2); 40x. E, grade 2 astrocytoma (patient no. 3); 10x. F, grade 2 astrocytoma (patient no. 3); 40x. G, grade 2 astrocytoma (patient no. 4); 10x. H, grade 2 astrocytoma (patient no. 4); 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Figure 10. Hematoxylin and Eosin staining of grade 3 astrocytoma (A-D) and GBM (E-G). A, grade 3 astrocytoma (patient no. 5); 10x. B, grade 3 astrocytoma (patient no. 5); 40x. C, grade 3 astrocytoma (patient no. 6); 10x. D, grade 3 astrocytoma (patient no. 6); 40x. E, GBM (patient no. 7); 10x. F, GBM (patient no. 7); 40x. G, GBM (patient no. 8); 10x. H, GBM (patient no. 8); 40x. Scale bars (A, C, E, and G) represent 100 µm. Scale bars (B, D, F, and H) represent 20 µm.
Discussion:
Due to the wide variety of expression patterns in Axl and Basigin expression between tissue types, a significant trend of expression along tumor progression is not appreciable in the data presented. Perhaps with further optimization and repeat trials, a more cohesive trend would be noted along tumor progression. A difference in expression that is appreciable between the samples is that Axl appears to be expressed more focally throughout the tissue while Basigin is expressed more diffusely throughout the sample. These findings are supported by previous findings by Yang et al. (2013) which demonstrate diffuse Basigin expression in GBM (Yang et al., 2013). In addition, Onken et al. (2017) elucidated two distinct phenotypes of phosphorylated
Axl (p-Axl) expression. Among 67 p-Axl positive GBM samples, 43 samples demonstrated focal p-Axl expression, while 14 samples demonstrated diffuse p-Axl expression (Onken et al., 2017).
These data further demonstrate the complexity of RTK expression in GBM.
The major shortcoming of this study is that the majority of the tumor samples have overdeveloped immunostaining. Due to limited volumes of kit reagents and tissue sections, these treatments could not be repeated and therefore some data is unevaluable. For this reason, the experimental methods should be repeated using different secondary antibodies and detection reagents. This would allow for more repetition with the tumor samples, allowing for increased optimization and data validation.
Furthermore, these data were unquantifiable due to limitations of software. With further optimization of staining, sections could be evaluated for expression by individuals blind to the study design; however, this method is subject to human error. Therefore, it would be ideal to have appropriate software to quantify expression data. With appropriate data evaluation, Axl expression could be effectively compared to Basigin expression. If there was a direct correlation between Axl and Basigin expression, this would indicate the potential for Axl as a prognostic marker in astrocytomas, as Basigin has previously been indicated as an independent prognostic marker (Yang et al., 2013).
Tissue sections which were dual-labeled with Axl and Basigin antibodies do demonstrate some overlap with chromogenic detection. This may provide evidence that expression levels are correlated between Basigin and Axl; however, further investigation is required to fully characterize their expression pattern among tumor types and individual patient samples. In addition, more clinical and pathological data is required for the patients to fully understand differences in expression levels of the tumors.
A visually significant difference can be noted between GBM (patient no. 7) and pilocytic astrocytoma (patient no. 1) (Figures 1 and 7). These data represent the best data collected in the study. Between these two samples, one can visualize an increased expression in both Axl and
Basigin in the GBM compared to the pilocytic astrocytoma. In addition, many of the characteristics associated with GBM are visible, such as tissue necrosis, increase in cell number, and anaplastic changes of cell nuclei (Figure 7). Characteristic vascular structures of GBM such as tubular and glomeruloid vessels are also visible in both GBM samples presented here (Figures
7, 8, and 10) (Onken et al., 2017).
In conclusion, further studies are required to evaluate any potential correlation between
Basigin and Axl expression in astrocytomas. This study represents a first step in characterizing
Basigin and Axl expression throughout astrocytic tumor grades, but shortcomings in data and study design limit the amount of usable data produced. It is important to continue exploring Axl expression and potential protein interactions in GBM as Axl facilitates EMT, chemo-resistance, and subsequent tumor metastasis. Therapies targeting Axl could potentially serve as important advances in GBM treatment. Literature Cited:
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