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Keywords:

  • differentiation;
  • FABP7;
  • glioblastoma;
  • stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Glioblastomas are the most aggressive brain tumors. Glioblastoma stem cells (GSCs) are thought to be responsible for the recurrence, chemoresistance, and poor prognosis of glioblastoma. Fatty acid binding protein 7 (FABP7), which is a cellular chaperone for a variety of omega-3 fatty acids, is a known marker for neural stem cells. In this study, using a newly developed anti-FABP7 antibody and patient-derived GSC lines, we evaluated the expression of FABP7 in GSCs. Using immunocytochemistry, Western blotting, and qPCR analyses, FABP7 was found to be highly enriched in GSCs and its localization was found in cytosol and nuclei. FABP7 expression was significantly downregulated in differentiated GSCs induced by the addition of serum. In the glioma surgical specimens, FABP7 was highly expressed in the majority of glioblastoma. Double immunostaining for FABP7 and Sox2 showed that FABP7+Sox2+ tumor cells were significantly increased in glioblastoma (grade IV) compared with diffuse astrocytoma (grade II) and anaplastic astrocytoma (grade III). Our data introduces FABP7 as a marker for GSCs and further highlights its possible significance for glioma diagnosis and treatment.

Glioblastomas are the most aggressive brain tumors and have limited therapeutic options. Prognosis remains poor with a median survival of approximately 15 months despite concomitant treatment with surgical intervention and radio-chemotherapy.[1] Improvement in the current treatment process requires a deeper molecular understanding of this disease. Progress in stem cell research has shown, as in various other solid tumors, the presence of cancer stem cells within glioblastomas.[2-4] Glioblastoma stem cells (GSCs) are quiescent, slowly cycling cells which reside in the hypoxic niches of the tumor mass which can escape from conventional anticancer therapy and can differentiate to tumor cells.[5] Conventionally, a subset of stem cell markers co-expressed by both GSCs and normal NS cells (CD133, CD44, nestin, Sox2, and Musashi) has been widely cited for use in the identification of GSCs.[6] However, identification of GSCs, which is essential for glioma research, has so far been a challenging issue.[7]

The fatty acid binding protein (FABP) family, which consists of ten isoforms in mammals, serves as an intracellular chaperone of long chain fatty acids. Among the FABP family, FABP7 and FABP5 are abundantly expressed in neural stem/progenitor cells and radial glia of the developing brain[8] and FABP7 is widely used as a neural stem cell marker. In the adult mouse brain, FABP7 and FABP5 are localized to astrocytes and oligodendrocyte precursor cells[9] with spatio-temporal heterogenity, suggesting their specific roles in glial cell biology. Although the precise mechanism by which both FABPs regulate neural cell proliferation/differentiation remains unknown, it has been shown that FABP7 is involved in the maintenance of neural stem cells, and positively regulates self-renewal of neural stem cells[10] and proliferation of astrocytes.[9] Recently, possible links between FABP7 and glioma pathology have been shown: the FABP7 promoter was found to be hypomethylated in samples of glioblastoma multiforme (GBM) with concomitant overexpression of FABP7 mRNA;[11] FABP7 is associated with shorter survival and invasiveness in GBM patients;[12, 13] FABP7 is dominantly expressed in glioma stem cells cultured by sphere method.[14]

Apart from other stem cell-like characteristics such as tumorigenicity and multipotency, GSCs also have the ability to grow as nonadherent 3-dimensional aggregates termed neurospheres when cultured in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in serum-free media.[5] GSC lines derived from freshly resected tumor specimens and cultured in serum-free medium have been shown to mirror the phenotype and genotype of primary tumors more closely than serum cultured cell lines.[15, 16] Such cell lines have been regarded as better models to study the glioma stem cell biology and the efficacy of various therapeutic agents.[17-20] To examine the value of FABP7 as a marker for glioblastoma stem cells, using a newly developed anti-FABP7 antibody and patient-derived GSC lines, we evaluated the expression of FABP7 in GSCs before and after differentiation, by immunohistochemistry, Western blotting, and quantitative PCR (qPCR).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Tumor samples and development of patient-derived GSC lines

Glioma tissue samples for Western blotting and histopathological examination were derived from patients who underwent tumor resection after glial tumor diagnosis at the Yamaguchi University Medical Center, treated between 1996 and 2012. Diagnosis was verified by a neuropathologist, and tumors were classified and graded according to WHO criteria. For Western blotting, 20 surgically-removed glioma tissues (16 glioblastoma, 2 oligodendroglioma, 1 anaplastic astrocytoma, and 1 anaplastic ganglioglioma) and normal cortex as a control were examined. For immunohistochemical analysis, 34 pathological specimens including 18 glioblastoma (grade IV), 8 anaplastic astrocytoma (grade III) and 8 diffuse astrocytoma (grade II) were examined. Pathological specimens used in the immunohistochemical study were derived from 23 male and 11 female patients aged from 17 to 80 years (mean 56 years). In detail, grade IV samples were from 11 male and 7 female patients aged from 48 to 80 years (mean 64 years); grade III samples were from 6 males and 2 females aged from 17 to 78 years (mean 54 years); grade II samples were from 6 males and 2 females aged from 28 to 72 years (mean 49 years).

The study protocol followed the principles outlined in the Declaration of Helsinki, and informed consent was obtained from all patients. This study was approved by our institutional review board.

The GSC line, G144, was kindly donated by Dr Peter Dirks (Arthur and Sonia Labatt Brain Tumor Research Center, Toronto, Canada).[21] GSC lines Y02, Y04, and Y10 were generated as described before,[22] using surgically resected tumors from three patients (70-year-old female, 63-year-old male, and 80-year-old male) diagnosed as GBM. Tumors were dissociated into single cells using trypsin (1.33 mg/mL), hyaluronidase (0.67 mg/mL), and kynurenic acid (0.17 mg/mL) for 50 min at 37°C. The dissociated cells were cultured in serum-free neurobasal medium supplemented with EGF (20 ng/mL) and FGF (20 ng/mL) to form neurospheres in suspension culture. Neurospheres were subsequently dissociated into single cells by placing in Accutase, and then transferred to fresh culture vessels coated with poly-L-ornithine for 15 min in 1% Laminin overnight at 37°C. GSC expansion was carried out using serum-free media supplemented with B27, hormone mix, EGF (20 ng/mL), and FGF (20 ng/mL). After reaching confluency, GSCs were dissociated using Accutase, and then passaged 1:5 to 1:8. Medium was replaced every 3–5 days. Each GSC line was differentiated into a non-GSC line in DMEM supplemented with 10% fetal calf serum (FCS), without EGF or FGF for 28 days. Neurobasal medium and B27 were purchased from Invitrogen (Carlsbad, CA, USA); FCS was purchased from HyClone Laboratories, Inc. (Logan, UT, USA). All other chemicals and reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).

Transplantation of GSCs into NOD-SCID mouse brain

All experimental procedures involving mice were approved by the Institute of Laboratory Animals of Yamaguchi University (No. 51-033). Adult male and female NOD-SCID mice weighing 20–25 g (4–12 weeks old) were used in the study. Surgical procedures were performed in a sterile fashion. Mice were anesthetized with an intraperitoneal injection of ketamine and xylazine, and then positioned in a rodent stereotaxic frame. 100 000 GSCs in 2 μL of cold PBS) were stereotactically injected into the right putamen (1 mm forward, 2 mm right lateral from the bregma, and 2.5 mm down from the dura) using a Hamilton syringe and a 30 G needle. The inoculation period was 2 min and the needle was left in place for another 5 min before withdrawal. Mice were returned to their cages and monitored for signs of illness. Mice were sacrificed when symptoms of brain tumor were observed or at approximately 22 weeks after transplantation. Brains were used for preparation of histological sections. All xenograft coronal sections were stained with hematoxylin-eosin. Primary antibody staining for anti-FABP7 (1:100) was carried out after deparaffinization, followed by incubation with biotined anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA, 1:200). The immunoreaction sites were visualized using the avidin-biotinylated peroxidase complex (ABC) system (Vector Laboratories) with diaminobenzidine as a substrate.

Preparation of anti-FABP antibodies

The full-length cDNA of the human FABP7 gene (GenBank accession no. 24 759), which encodes a protein of 132 amino acid residues, was amplified by PCR from a human brain cDNA library. The amplified gene was subsequently inserted into the pGEX-6p-2 expression vector, and confirmed by sequencing. The recombinant protein was expressed in Escherichia coli BL21 (DE3) cells after transformation of the recombinant plasmid. Conditions for recombinant protein induction were optimized under different concentrations of isopropyl-beta-D-thiogalactopyranoside (IPTG) and different induction time points. The induced culture pellet was harvested, and the supernatant was saved. After the extraction procedure, the recombinant protein was analyzed by SDS-PAGE. Generation of the anti-human FABP5 antibody was described previously.[23]

Antibodies against recombinant FABP7 were raised in male New Zealand white rabbits. The rabbits were injected subcutaneously with purified recombinant FABP7 protein dissolved in 0.2 M NaCl and emulsified in Freund's complete adjuvant to enhance the response to the immunogen. Six booster injections were given with recombinant protein each in incomplete Freund's adjuvant at 2-week intervals. Ten days after the final injection, blood was collected and kept overnight at room temperature (RT) to allow clotting. The crude antiserum was collected by centrifugation and the antibody was isolated by affinity purification. The titer and the specificity of the antibody were checked by Western blot analysis.

Immunostaining

Double immunostaining of FABP7 and Sox2 was performed following the protocol as described previously with slight modifications.[24] Briefly, tumor samples were fixed with 10% formalin in 0.1 M phosphate buffer (pH 7.4) and were then dehydrated and embedded in paraffin. Paraffin sections were deparaffinized, washed thoroughly with water, and treated with microwave in HISTOFINE (pH 9) (Nichirei, Tokyo, Japan) for 40 min. After washing with TPBS for 5 min, sections were blocked in Protein Block Serum-Free Ready-To-Use (Dako Japan, Tokyo, Japan) for 10 min and incubated with rabbit anti-FABP7 antibody (1:300) for 60 min, followed by incubation with alkaline phosphatase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, 1:50) for 30 min. The chromogenic reaction was performed by using a Vulcan Fast Red Chromogen Kit2 (BIOCARE MEDICAL, Concord, CA, USA) according to the manufacturer's manual. After using denaturing Solution (BIOCARE MEDICAL) to denature the alkaline phosphatase activity, sections were incubated with goat anti-Sox2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1:100) and subsequently with alkaline phosphatase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, 1:50). Then the chromogenic reaction was performed by using PermaBlue/AP (Diagnostic BioSystems, Pleasanton, CA, USA) according to the manufacturer's manual. The sections were air dried and cover slipped. For counting the FABP7+ and Sox2+ tumor cells, high magnification images of the area where tumor cells were highly accumulated were obtained through x40 objective lens. More than 200 tumor cells were randomly selected from the images obtained. The staining intensity was scored as absent, weak, moderate or strong, and the percentage of tumor cells showing cytosolic or nuclear staining with a moderate to strong intensity was determined.

Cells were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 30 min. After blocking in 5% skim milk or goat serum for 30 min, the cells were incubated with combinations of the following primary antibodies overnight at 4°C: rabbit anti-FABP7 (1:50); mouse anti-nestin (Millipore, 1:200); or goat anti-Sox2 (1:500). The cells were incubated with suitable combinations of the following secondary antibodies for 30 min at RT: Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG, or Alexa Fluor 568 donkey anti-goat IgG (Invitrogen, 1:1000 for all). Cells were cover-slipped using Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA).

Western blotting

Cells or tissues were lysed using buffer containing 100 mM Tris-Cl (pH 6.8), 4% SDS, 20% glycerol, 200 mM β-mercaptoethanol. Protein quantitation was performed using the BCA Protein assay (Pierce, Rockfold, IL, USA) method with a Viento multi-spectrophotometer at 562 nm. Equal amounts of protein (15–30 μg) were separated by SDS-PAGE, transferred to Immobilon PVDF membranes (Millipore), and blocked with either 5% skimmed milk or rabbit serum in TBST (10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20). Membranes were incubated with primary antibodies diluted in blocking buffer overnight at 4°C. Primary antibodies used were anti-FABP7 (1:1000), anti-nestin (Millipore, 1:5000, MAB5326), anti-Sox2 (1:1000), anti-FABP5 (1:1000), and anti-β-actin (Santa Cruz Biotechnology, 1:5000, sc-47778). Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Immunoreactive protein bands were visualized using ECL Western blotting detection reagents (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, England).

RNA preparation and Real Time PCR

Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions, treated with DNase (Promega Corporation, Madison, WI, USA), and re-extracted using phenol/chloroform and LiCl precipitation. RNA concentration was determined using the Quant-iT RNA BR Assay kit in a Qubit fluorometer (Invitrogen). First-strand cDNA synthesis was performed with Transcriptor High Fidelity reverse transcriptase kit (Roche Diagnostics GmbH, Mannheim, Germany) using oligo d(T) primers. qPCR was performed using the Taqman Universal PCR Master Mix kit (Applied Biosystems, Carlsbad, CA, USA) and Taqman Gene Expression Assays for FABP7 (Hs00361426_m1) and GAPDH (Hs99999905_m1). Reactions were performed in triplicate using 96-well optical plates on a StepOnePlus Real-Time PCR System (Applied Biosystems).

Statistical analysis

All numerical data are shown as mean ± SEM. Comparison between two experimental groups was made using the unpaired Student t-test for qPCR. Immunohistochemical analyses were analyzed using two-way ANOVA, followed by one-way ANOVA for each group and Dunnett's tests. P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Sensitivity and specificity of anti-FABP7 antibody

Gel electrophoresis followed by Coomassie staining confirmed the expected molecular weight (MW) of human recombinant FABP7 (15 kDa, Fig. 1a). Using Western blotting, the sensitivity and specificity of the anti-FABP7 antibody was confirmed by specific immunoreaction to recombinant FABP7 as well as protein extracts from human brain cortex and glioma with the bands showing the expected MW (Fig. 1b).

figure

Figure 1. Characterization of an anti-human FABP7 antibody. (a) SDS-PAGE followed by Coomassie staining shows the expected molecular weight (MW) of the recombinant FABP7 (15 kDa). (b) Western blotting showing the sensitivity and specificity of the anti-FABP7 antibody by positive immunoreaction with recombinant human FABP7, tissue lysate from normal human brain cortex, and tissue lysate from GME. The bands show the expected MW (15 kDa).

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Characteristics of GSC lines

We cultured GSCs from glioma surgical specimens and established three cell lines, Y02, Y04, and Y10. For validation, the G144 cell line was also used alongside the newly established cell lines.[21]

The characteristics of GSCs including neurosphere formation, multipotency and tumorigenicity were confirmed in Y02, Y04, and Y10.[22] Western blotting and immunocytochemistry showed that all cell lines in their undifferentiated state in the presence of EGF and FGF strongly expressed the neural stem cell markers, nestin and Sox2 (Fig. 2), and GFAP, one of the differentiated glial cell markers, was absent in these GSCs (data not shown). Expression levels of neural stem cell markers were down-regulated in non-GSCs (Fig. 2, Fig. S1).

figure

Figure 2. Expression of FABP7 in GSC lines before and after differentiation and in the NOD-SCID mouse brain after GSC transplantation. (A) Western blotting and (B) qPCR results showing the strong expression of FABP7 together with other neural stem cell markers, Sox2 and nestin, in 4 GSC lines in the presence of EGF and FGF. Upon differentiation in the presence of FCS, FABP7 expression is down regulated similar with that of Sox2 and nestin. (C) Expression of FABP7 in GSC lines before and after differentiation. (a–f) Phase contrast micrographs showing the morphology of GSC lines (G144, Y10, and Y04) in the presence of EGF and FGF (a,c,e) or after differentiation in the presence of FCS (b,d,f). (a′–f′,a″–f″) Immunofluorescence micrographs showing the co-expression of FABP7 and Sox2 (a′–f′) and co-expression of FABP7 and nestin (a″–f″) in GSC lines G144, Y10, and Y04, respectively. (D) Localization of FABP7 in the mouse brain transplanted with Y10 GSCs. An inset shows high magnification image of the area enclosed by rectangle. Bars in C = 50 μm, Bars in D = 1 mm, *P < 0.05. ■, EGF+FGF; □, FCS.

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FABP7 expression in GSC lines

Western blotting showed that all GSC lines expressed high levels of FABP7, although in varying amounts (Fig. 2a). Furthermore, it was noted that after differentiation in the presence of 10% FCS, protein levels of FABP7 were clearly reduced (Fig. 2a). We also performed quantitative RT-PCR (qPCR) to examine the relative expression of FABP7 in GSCs before and after differentiation. The mRNA level of FABP7 was drastically down regulated in non-GSCs consistent with the observation at protein level (Fig. 2b). In immunocytochemistry, FABP7 was localized in both cytosol and nuclei of GSCs, and all cell lines showed considerable co-expression of FABP7 with Sox2 and nestin, all of which showed a remarkable downregulation following differentiation (Fig. 2c). In addition, FABP7 immunoreactivity was found to be much higher in large sized Ki-67 positive cells with long processes (Fig. S2). When the expression of FABP7 was examined immunohistochemically in the mouse model transplanted with GSCs (Fig. 2d), it was strikingly similar to the distribution of nestin expression.[22]

We also checked the expression of another brain-expressed FABP, FABP5, in GSCs. Interestingly, based on our preliminary results, FABP5 showed relatively lower expression levels as compared to FABP7 in the GSC lines, whereas after differentiation FABP5 expression was substantially increased (Fig. S3). This may imply that in GSCs, FABP7 and FABP5 exhibit reciprocal expression patterns before and after differentiation.

FABP7 expression in glioma tumor samples

We also checked by Western blotting the expression of FABP7 in glioma samples that had been surgically removed. As a result, FABP7 was expressed, although at different levels, in 17/20 glioma samples, while Sox2 expression was detected in 15/20 samples and nestin expression was detected in 17/20 samples. A total of 14/20 samples expressed both nestin and FABP7, 13/20 expressed both Sox2 and FABP7, and 12/20 samples (65%) expressed all three markers (Fig. 3a). In double immunohistochemical examination using FABP7 and Sox2 antibodies on 34 glioma pathological specimen with different grades, FABP7 immunoreactivities were detected in both cytosol and nuclei in tumor cells with different intensity (Fig. 3b), while those of Sox2 were confined to the nuclei as previously reported.[25] The ratio of FABP7+ tumor cells and of Sox2+ tumor cells was significantly increased in grade IV (GBM) samples compared with grade II (diffuse astrocytoma) (Fig. 3b–d, P < 0.05). Population density of FABP7+Sox2+ cells among total tumor cells was significantly increased in GBM samples compared with grade II or grade III (anaplastic astrocytoma) samples (Fig. 3b,e, P < 0.05). As previously described, intensity of Sox2 expression was higher in GBM[25] and that of FABP7 expression was basically correlated with it, although FABP7 was hardly detected in a few glioma samples. In addition, FABP7+Sox2+ cells were frequently detected in the perivascular area and nuclear localization of FABP7 was more evident in the tumor cells located in invasive front, consistent with the findings by Mita et al.[26] (Fig. S4).

figure

Figure 3. Expression of FABP7 in human glioma samples. (a) Western blotting showing the expression of FABP7, nestin, and Sox2 in surgically resected glioma samples including GBM, oligodendroglioma, anaplastic ganglioglioma, and anaplastic astrocytoma. Note the difference in expression levels of FABP7, nestin and Sox2 in the different samples. Expression of β-actin was shown as an internal control. (b) Representative photomicrographs showing FABP7 and Sox2 immunoreactivity in different grades of human glioma. Note that Sox2 expression (blue) is confined to the nuclei, and that FABP7 (red) is in both cytosol and nuclei. (c,d,e) Population density of FABP7+ (c), Sox2+ (d), and FABP7+Sox2+ tumor cells (e) in different grades of human glioma. Bar = 40 μm, *P < 0.05, #P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

In this study, using a newly developed antibody and GSC lines, we show by Western blotting, qPCR, and immunocytochemistry, that FABP7 was highly enriched in GSCs and was downregulated in non-GSCs. These findings suggest that FABP7 can serve as a marker for identification of GSCs, along with other markers such as nestin and Sox2.

In the present study, we observed that the expression of FABP7, nestin, and Sox2, though reduced, was not completely negated in non-GSCs. This could indicate that GSCs cultured in differentiation medium undergo asymmetric division or proliferative differentiation.[27] It is possible that the residual stem cell marker expression is due to remnant GSCs in the differentiation medium. In support of this view, it is worth mentioning a distinguishing feature of NS cells versus GSC cultures, where serum differentiation of the former is permanent, glioma lines established as serum cultures can be converted to neurospheres in serum-free media using neurosphere recovery assays.[20]

Our data suggest that FABP7 and its ligands may have regulatory roles in the maintenance and differentiation of GSCs. Notably, FABP7 has been reported as a regulator of stemness in neuroepithelial cells.[10] Interestingly, our present results showed that another FABP, FABP5, is weakly expressed in GSCs and upregulated in non-GSCs (Fig. S2), suggesting that reciprocal expression of FABP7 and FABP5 may reflect the differential status of cellular lipid metabolism in the hierarchy of GSC differentiation. FABP5 and FABP7 vary biochemically in their ligand binding affinity with FABP5 binding to saturated fatty acids and FABP7 to omega-3 polyunsaturated fatty acids (PUFAs) with high affinity.[28] Therefore, it is possible that GSCs residing in the niches of the tumor mass can maintain their stemness under the specific condition of cellular lipid metabolism, which is associated with omega-3 PUFAs. The hypothetical regulatory roles of FABP7 and their ligands in GSCs are currently being studied in our lab. In addition, expression of FABP7 in GSCs may partly explain the reported association of FABP7 with invasiveness of glioblastoma,[12, 29] as GSCs are considered as the main cause of invasiveness in glioblastoma.[30, 31]

CD133, cell surface stem cell marker, and stem cell transcription factors such as Sox2, Oct4, and Nanog play pivotal roles in maintaining the proliferation, survaival, and multi-lineage differentiation potential of GSCs.[32, 33] Among these proteins, Sox2 expression level in GSCs was shown to be associated with their self-renewal activity and tumorigenicity in mouse transplantation model.[25] Furthermore, it was also shown that expression level and distribution of Sox2 in human glioma are closely associated with their malignancy and patients' prognosis.[34] In this study, the ratio of FABP7+Sox2+ tumor cells was significantly increased in GBM and the localization pattern of FABP7 was well correlated with that of Sox2: FABP7+Sox2+ tumor cells were frequently detected in the perivascular area and the tumor invasive front, which is a well documented localization pattern of stem cell marker-positive tumor cells in human glioma.[35, 36] Therefore FABP7 can be a useful marker for identification of GSCs combined with other stem cell markers such as Sox2 in pathological examination of human glioma.

Taken together, our present data introduces FABP7 as a marker and candidate regulator for GSCs. Further studies on the function of FABP7 in GBM may shed light on the mechanism by which FABP7 controls the progression of GBM and we propose its significance as a therapeutic and diagnostic target for GBM.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

We would like to thank Mr Masakatsu Tamechika for his fine technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 24390047 to Y.O.) from The Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and from the Research Project of STRESS, Yamaguchi University.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
pin12109-sup-0001-fs1.tif473K

Figure S1 Characteristics of the GSC lines. (a–c) Phase contrast micrographs of GSC lines G144 (a), Y10 (b) and Y04 (c). (d–f) Immunofluorescence micrographs showing the co-expression of neural stem cell markers, nestin and Sox2, in GSC lines G144 (d), Y10 (e), Y04 (f). Bars = 50 μm.

pin12109-sup-0002-fs2.tif173K

Figure S2 Localization of FABP7 and Ki-67 in Y10 GSCs. Note that FABP7 immunoreactivity was found to be much higher in large sized Ki-67 positive cells with long processes. Bar = 40 μm.

pin12109-sup-0003-fs3.tif58K

Figure S3 Expression of FABP5 in GSCs before and after differentiation. Western blotting results showing the weak expression of FABP5 in four GSC lines enriched in the presence of EGF and FGF. Upon differentiation of GSCs in the presence of FCS, FABP5 expression is significantly upregulated. Expression of β-actin was shown as an internal control.

pin12109-sup-0004-fs4.tif1444K

Figure S4 Localization on FABP7 and Sox2 in GBM. Representative photomicrographs showing strong immunoreactivity of FABP7 and Sox2 in perivascular area (a and b) and invasive front (c and d). b and d are high magnification images of the areas enclosed by rectangles in a and c, respectively. Bars in a and c = 100 μm; Bars in b and d = 50 μm.

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