In vivo migration - MATERIAL AND METHODS - Glioma produced periostin attracts human neural stem

Ⅱ. MATERIAL AND METHODS

10. In vivo migration

Three micro-liter Hanks’ balanced salt solution (HBSS) or Dycycle green labeled p-NIH3T3 cells (1x10⁶ cells) had stereotaxically injected into the right striatum. The coordinates, anterior-posterior (AP) measured from bregma, lateral (ML) from the midline, and depth (DV) from dura, were as follow (in ㎜): AP +0.4; ML -2.3; DV -4.5. Five day after, each group was stereotaxically injected with DiI labeled 1x10⁶ NSCs into same hemisphere cortex that coordinates, anterior-posterior (AP) measured from bregma, lateral (ML) from the midline, and depth (DV) from dura, were as follow (in ㎜): AP +0.4; ML -2.3; DV -2.0. Cells and HBSS injected at a rate of 0.2ul/min using 26-gauge Hamilton syringe (Hamilton, Nevada, USA) attached to an automated microinjector (KD scientific INC, MA, USA). After transplantation, the needle was left in place for an additional 15min before being slowly retracted. 2-4weeks later, rats were sacrificed and collected rat brain fixed with ice-cold 4 % paraformaldehyde in 0.1M phosphate buffer. After 4hr, brains sink with 30 % sucrose in 0.1 M phosphate buffer at 4°C for overnight. Then brains frozen with OCT compound and sectioned at 30mm with cryostat. Slices were mounted with Vectashield® Hard Set™ mounting medium (Vector, CA, USA), and were observed with an Olympus IX71 confocal laser scanning microscope (Olympus, Tokyo, Japan).

14 11. Analysis of CD enzyme activity

F3-CD Cells (2X10⁵cells) were inoculated each well in 12-well plate and incubated for 48 hours with 1 mM 5-FC in 1ml medium. Incubated medium (50ml) was extracted with 500µl ethyl acetate:isopropanol:acetic acid (84:15:1 [v/v/v]), and the organic fraction was resuspended with 500µl H2O:methanol (4:1 [v/v]). HPLC carried out using a Kromasil 100-5C-18 column (Kromasil, Bohus, Sweden) and detected at UV 270nm. 5-FC and 5-FU were eluted isocratically at a flow rate of 1 ml/min with an isocratic mobile phase consisting of 40mM KH₂PO₄, adjusted to pH 7.0 with 10 % KOH. Retention times were 3.4 minutes for 5-FC, 3.9 minutes for 5-FU and 8.9 minutes for 5-bromouracil (Sigma), an internal standard.

Quantification of 5-FC and 5-FU was normalized with 5-bromouracil.

Fifty micrograms proteins of cell lysates were incubated in the presence of 1mM 5-FC at 37℃ for 8 hours for enzyme activity. The mixtures were extracted and analyzed by HPLC as described above.

12. In vitro bystander effect assay

To observe bystander effect of p-NIH3T3 cell by 5-FC treated F3-CD, Total 1x10⁴ p-NIH3T3 cells or mixed with F3.CD cells seeded in 96well plate (p-p-NIH3T3:F3.CD = 100:5~200). One day after, Culture medium was changed with 5-FC contained medium (100ug/ml, Sigma) and incubated for 3 day. Each cell washed with PBS and added MTT solution (0.5mg/ml, Sigma). 4 hours after, MTT were replaced with 200ul DMSO and

incubated for 1 hour. Each well supernatant collect and transfer to new 96 well plate and measured with microplate spectrophotometer (Bio Tek instrument Inc) at 550nm and 630nm as reference filter.

13. In vivo bystander efficacy

Dycycle green labeled p-NIH3T3 cells (0.5 - 2x10⁶cells) mixed with C6 rat glioma cells had stereotaxically injected into the rat right striatum. Five days after, each group was injected with DiI labeled 1x10⁶ NSCs into left hemisphere cortex. After transplantation, the needle was left in place for an additional 15min before being slowly retracted. Seven or ten days later, Animals were injected with 5-FC (500mg/kg/day) through intraperitoneal injection daily for 2 weeks. After that, animals sacrificed and collected rat brain fixed with ice-cold 4 % paraformaldehyde in 0.1M phosphate buffer. After 4hr, brains sink with 30 % sucrose in 0.1 M phosphate buffer at 4°C for overnight. Then brains frozen with OCT compound and sectioned at 30mm with cryostat. Slices were mounted with Gel mount (Biomeda corp, Foster city, CA). Tumor cells and size was measured using BMF stereo investigator program (MicroBrightField inc, Williston, USA).

14. Statistical analysis

Results are expressed as mean ± SD. Student’s t-tests were performed to determine statistical significance.

Ⅲ. RESULTS

1. Expression profiling of secreted genes from human brain tumor tissues

To search which genes were changed their expression pattern in human brain tumors, I performed microarray analysis using 3 different brain tumor sample RNA. 4000~6000 genes were over expressed in each tumor sample (Table. 1). Using microarray data, I analyzed over-expressed or down-expressed 119 secrete or extracellular region located genes in each tumor samples (Fig. 1A). Among of 119 genes, I choose secreted and up regulated gene and except extracellular region or down regulated gene. This study focus on identify of chemoattracters to stem migration. So, chemoattract molecules should be secreted to out of membrane. In each tumor, I found 39, 46 and 47 genes that satisfy above conditions.

Specially, 33genes were commonly up regulated in all tumors and included 13 genes that more than 2 fold up regulated (Fig. 1B).

I selected 12 commonly over expressed genes that expressed more than 2 fold compare normal brain tissue and 2 genes assumed related with tumor formation and development.

Cysteine rich 61 (CYR61), GLI pathogenesis related 1 (GLIPR1), Fibronectin (FN1), Periostin (POSTN) and Tenascin C (TNC) play a important role of tumor growth inducer and angiogenesis inducing factor. Additionally, it promote tumor cell invasion from tumor raised position to near normal tissues. Chemokine (C-X-C motif) ligand 10(CXCL10), glutathione peroxidase 3 (GPX3), Major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) genes were act as homeostasis through reduce oxidative stress and trigger

17 immune response by attract lymphocytes (Table. 2).

Selected 14 genes highly expressed in all tumors over 2 fold and many genes (5~7/14) were ranked upper position of highly over-expressed 20 genes list in each tumor sample. It assumed that chose genes were authentic date for use further in this study. Specially, POSTN expressed in all tumors by more 4 folds and posited in all highly expressed 20 gene list of each brain tumor (Fig. 2B). Microarray result showed that some of these genes participated in antitumor action but many genes acted to help tumor growth and invasion. Specially, periostin expressed intensely in all tumors and play role cancer cell infiltration and angiogenesis.

Table 1. Three tumors samples used for microarray assays Tumor Gender Age Histology Number of

up-regulated gene

Number of down-regulated gene

1 M 32 Glioblastoma 4491 3758

2 F 50 Ganglioglioma 4775 5087

3 N 63 Shwannoma 6119 6824

19 tissues. (A) Comparison of expression patterns of transcripts for over-expressed secretory proteins between brain tumor tissues using the Eisen hierarchical cluster analysis. The cluster contains 119 genes that secreted into ECM and were up- or down-regulated in 3 brain tumor samples. Red indicates increased expressions, and green indicates decreased expressions. The dendrogram on the left shows different clusters of genes segregated according to the pattern of regulation in three tissues. (B) Venn diagram showing the shared and unique genes up-regulated in independently derived human brain tumors. 33 genes are commonly over-expressed in three tumors and 13 genes of these are highly expressed over 2-fold.

Table 2. Function of secretory proteins over-expressed in brain tumors

Fold change Probe ID

Gene

symbol Gene function

Tumor 1 Tumor 2 Tumor 3

Hs.413924 CXCL10 lymphocyte trafficking 3.76 3.16 4.27

Hs.8867 CYR61 cell proliferation, angiogenic factor 2.08 3.23 6.43 Hs.553516 GLIPR1 cell proliferation, invasion, antiapoptic effect 1.53 3.25 5.16

Hs.386793 GPX3 antioxidative function 3.16 2.97 5.20

Hs.409934 HLA-DQB1 immune response 2.08 6.51 6.96

Hs.438102 IGFBP2 regulation of tumor growth and invasion 5.04 1.66 6.07 Hs.136348 POSTN induce cell migration and angiogenesis 4.19 4.13 7.79

Hs.64016 PROS1 Anti-coagulation 3.84 2.19 8.00

Hs.105700 SFRP4 inhibit cell proliferation 2.74 3.37 4.83

Hs.522632 TIMP1 Anti-apoptosis, induce cell growth 2.25 2.26 4.38

Hs.104839 TIMP2 Anti-angiogenesis 2.32 2.07 3.38

Hs.143250 TNC cell proliferation, migration 2.15 7.21 3.87

Hs.279594 TNFRSF1A Apoptosis 2.80 2.32 2.68

Hs.525157 TNFSF13B related with tumor genesis gene 2.69 5.13 3.09

Probe ID is used gene name in microarray assay that indicated unique each gene. Fold change appeared that selected genes expression level in each tumor compare normal tissues as log phase transform.

Fold changes CXCL10 CYR61 GLIPR1 GPX3 HLA-DQB1 IGFBP2 POSTN PROS1 SFRP4 TIMP1 TIMP2 TNC TNFRSF1A TNFSF13B

CXCL10 CYR61 GLIPR1 GPX3 HLA-DQB1 IGFBP2 POSTN PROS1 SFRP4 TIMP1 TIMP2 TNC TNFRSF1A TNFSF13B

Fig. 2. Comparison of genes encoding secretory proteins in three brain tumor samples.

(A) Expression pattern of the 14 shared genes up-regulated more than 2-fold in all brain tumors as designated in Venn diagram of Fig. 1. (B) Genes exhibiting the greatest magnitude of up-regulated changes in each brain tumor tissue. Red indicate

genes over-expressed in all tumors over 2-fold. All genes or ESTs were identified as being statistically significant by the SAM algorithm. For all tumors, the false discovery rate (FDR) were set at 10%.

2. Differential expression of 14 Candidate genes in several human brain tumor tissues From microarray date, we selected 14 candidates that secreted from each tumor commonly and related to tumor metastasis, growth and cell migration. To confirm the candidate genes expression pattern, I examined real time PCR in 11 different kinds of tumor that classified benign and malignant tumors (Table. 3). Those candidates showed very various expression patterns. Most genes –CXCL10, FN1, GLIPR1, GPX3, PROS1, SFRP4, TIMP1, TIMP2, TNFRSF1A, TNFSF13B - were commonly over-expressed in each tumor. Those genes may play same role in most tumors that indistinct tumor group. TNC appeared any tumor specific expression pattern. However, POSTN was showed very different expression pattern. It was highly expressed in malignant tumors but not in benign tumors. It could be participated in malignant tumor development mainly (Fig. 3). Additionally, POSTN gene expression manner in real time PCR data matched with microarray data that presented commonly over-expressed in 3 different brain tumors. Those tumors have malignancy character. This result support that periostin could important factor in tumor development especially malignant tumor. Its discrimination with other genes was important to choose candidate for malignant targeting cancer therapy.

Table. 3. Information of tumors used in real-time PCR assays

Sample Gender Age Histology

Tumor1 F 59 adenoma

Tumor2 F 56 adenoma

Tumor3 F 36 Choroid plexus papilloma

Tumor4 M 36 meningotheliomatous meningioma

Tumor5 F 48 meningioma

Tumor6 M 45 Fibroblastic meningioma

Tumor7 F 46 Fibroblastic meningioma

Tumor8 F 56 meningioma

Tumor9 M 63 schwannoma

Tumor10 M 45 Anaplastic oligodendroglioma

Tumor11 M 33 Glioblastoma

25 different human brain tumor tissues. Expression of 14 genes was analyzed by real-time RT-PCR, and fold changes in expression are expressed as the ratio of GAPDH-normalized brain tumor tissue/normal brain tissue values. Asterisk indicates the periostin, the only gene specifically expressed in malignant tumor samples.

3. Periostin more intensely expressed human brain tumor tissues than normal brain Periostin usually expressed in osteoblast cells and found to be overexpressed in various types of human cancer, such as non-small-cell lung carcinoma, breastcancer, colon cancer, head and neck cancer, ovarian cancer, and pancreatic ductal adenocarcinoma. Whether brain tissues expressed periostin in protein level, i carried out immunohistochemistry with periostin antibody in normal or tumor tissues. In normal brain tissues, periostin was very weakly expressed compare tumor tissues intensely expressed (Fig. 4 C-E). Periostin was expressed broadly in entire tumor tissues but not all tumor cells expressed it. Furthermore, periostin expression manner was changed followed tumor. Low grade tumor (meningioma) rarely expressed periostin but high grade malignancy tumors (Astrocytoma and Glioblastoma) significantly expressed it (Fig. 4F). It expression pattern in tumor tissues supported by real time PCR result that periostin highly expressed mainly malignant tumor. It means that periostin could be important candidate for malignant tumor specific cancer therapy.

Normal

Tumor

B

C C’

F F’

D D’

E E’

A A’

B’

Fig. 4. GBM tumors have high periostin expression. Periostin was hardly detected in normal brain. Human brain tumor (C-F) and normal brain tissues (A and B) were fixed with 4% paraformaldehyde followed sectioned at 30mm on a cryostat. In each slices, periostin was detected using DAB staining method with periostin antibody (1:500). Slices of normal brain (A-B) or low grade tumor (F) presented rarely expressed periostin. However, Periostin intensely expressed only malignant tumors (C-E). Scale bar, Left panels (A-F) : 500mm, Right panels (A’-F’) : 100mm.

4. Periostin strongly attracted neural stem cell than VEGF

Whether periostin attract human neural stem cell, i checked migrated NSCs number with boyden chamber assay. Cultured NSCs in transwell upper chambers transferred to periostin treated lower chamber. 12 hours after, we counted migrated NSCs that passed boyden chamber membrane from upper position of membrane to periostin contained low chamber.

NSCs motility was promoted by periostin dose dependently about 3 times than periostin no treated condition. Also, periostin induced NSC attraction effect was picked at 10ug/ml periostin concentration but higher concentration decreased its effect. (Fig. 5A spot line)

To confirm stem cell attraction capacity by periostin, i compared periostin to VEGF which used as positive control. VEGF was highly expressed factor in cancer and have angiogenesis ability. Besides, it was well known as cell attract molecule. Various stem cell lines controlled by VEGF in migration procedure. VEGF induced NSC migration about 50%

compare VEGF no treated condition but periostin induced about 100%. Periostin has 2 times NSC attraction ability than VEGF (Fig. 5A). Compared two factor results indicated that NSC migration ability was strongly increased in periostin treated condition compare VEGF treated it. Additionally, i experiment cell type motility discrimination between HB1.F3 and HB1.F5 that has strong tropism for tumor. In VEGF treated same condition, both cell lines showed same migration rate in dose dependent manner. However, they showed difference of stem cell attraction ability in comparison with VEGF and periostin. When both factors were treated for same time, HB1.F3 cells migration ability was increased 92% by periostin and VEGF increased 26%. Its discrimination rate is about 3 times. Even HB1.F5 has no difference by both factors (Fig 5 A and B). It means that periostin could be powerful neural

stem cell attraction molecule than others and very unique to HB1.F3.

And i checked that NSC motility about periostin treated time. NSCs were cultured in transwell upper chambers for 12hr followed transferred to periostin contained lower chamber.

In each time point (4-24hours), i counted migrated NSCs from upper chamber to periostin contained low chamber. The number of migrated NSC was increased as time passes in periostin treated condition (Fig. 6 A and B). Periostin treated 12 hour after, the number of migrated NSC was noticeably increased and continued 24 hour time point. To confirm that cell migration pattern by different cell type, i used two types of NSC (HB1.F3 and HB1.F5) (Fig 6 C and D). Both cell lines showed not difference in cell migration by periostin. In summary, periostin promote NSC migration as time and concentration dependent manner and show very powerful attraction of NSC that more unique in HB1. F3 cell line.

50 100 150 200 250 50 100 150 200 250

0 5 10 20

Number of m

i

grated cellNumber of m

i

grated cell VEGF (ng/ml)

POSTN (mg/ml) HB1.F3

VEGF (ng/ml)

POSTN (mg/ml) HB1.F5

concentration

A

B

Fig. 5. Periostin significantly promote NSC migration ability compare with VEGF.

NSC chemotactic motility measured using in vitro migration assay using boyden cahmber. NSCs (5 x 10⁴ cells) inoculated to upper chamber. 1 day later, upper chamber transferred to VEGF or POSTN contained low chamber. VEGF or POSTN of different concentration were applied to HB1.F3 cells (A) or HB1.F5

cells (B) as concentration rising pattern. 12 hours after, migrated NSCs were stained with hematoxylin and counted. Black continues line indicated VEGF induced NSC migration level and spot line indicated POSTN that. POSTN promoted migration of F3 cells 3 times higher potency. However, F5 cells showed similar migration induction by both factors. The results represented as means ±SD

33 cells was inoculated in transwell upper chamber and cultured for 1day. After then, upper chamber transferred to lower chamber filled with VEGF (10ng/ml). Migrated upper side to lower side of upper transwell HB1.F3 cells was stained with H&E and counted at each time point (B). HB1.F5 cells tropism was performed as same method. (C), H&Estain result. (D), Migrated NSCs was calculated. Both neural stem cell lines showed same migration pattern that NSC migration capacity was increased time dependently. Scale bar : 200mm

5. Neural stem cell has motility to Periostin expressing NIH3T3 in vivo condition To check NSC migration activation by periostin in vivo condition, i constructed periostin over expressing NIH3T3 cell line (p-NIH3T3) using retroviral vector system (Fig. 7 A and B). Periostin gene and amphotropic envelop gene encoding were confirmed with enzymatic gene restriction assay. Periostin gene transduce cells were selected by blastidine S contained medium culture for 2 week. We confirmed periostin expression and release to culture medium with ELISA method and western blotting (Fig. 7 C and D). The p-NIH3T3 cell line (1-8) that most intensely expressed a periostin than others was chose for further studies.

Whether NSC has tropism for periostin over expressing NIH3T3 cells, i transplanted p-NIH3T3 cells to rat right hemisphere and followed NSC transplantation to same rat hemisphere cortex on 5 day. 2~4 weeks after, we removed rat brain and observed NSC tropism for NIH3T3 cells in vivo condition. NSC migrated from transplanted site to p-NIH3T3 injected position passed corpus callosum and infiltrated to p-p-NIH3T3 cell. It looks like that p-NIH3T3 cells were raped by NSCs (Fig. 8 A and B). Indeed, many NSC cells reached and posited near the p-NIH3T3 cells in injected original site (Fig. 8 C and D). This situation was not raised in NIH3T3 cell transplanted environment. NSC migrated with any direction or located in original transplanted site.

NSC mobile routes were similar to each cell inject needle track. In procedure of each cell were injection, needle could madden tunnel in brain tissues that may be used by NSC for their efficiency. Additionally, i observed that p-NIH3T3 expression was discontinued for long time using immunohistochemistry assay with periostin antibody in the experiment of NSC migration condition for 4 weeks. To avoid possibility of mechanical support by needle

and periostin discontinuously expression, i performed modified in vivo model experiment that P-NIH3T3 cells were injected to right hemisphere and NSCs were transplanted to left hemisphere cortex. 1~2 weeks later, rat brains were removed and observed. Like to NSC migration pattern in ipsi-lateral transplantation condition, NSC chased to P-NIH3T3 cell followed corpus callosum route that connect construct of right and left hemisphere and attained original P-NIH3T3 injection site and near site of it (Fig 9.). Also, periostin continuously expressed (Fig. 9 D and I).

These results suggest that periostin induced NSC tropism powerfully and give some cue about their migration direction. Also, it is possible in both in vitro and in vivo situation.

36 293T cell

pCXbsr (2455 bp) pCL-Ampho (4070bp)

1Kb DNA EcoR1 Sal1 EcoR1

Sal1 100 bp 1Kb DNA Cla1 Sal1 Cla1 Sal1 100 bp

3 Kb 10 Kb

2 Kb 3 Kb 10 Kb

2 Kb

A B

NIH3T3 cell

1-20 1-16 2-2

1-12 1-8 1-24

WB : POSTN 50 100 150 200 250

POSTN level in culture medium(%)

Fig. 7. Construction of periostin over expressing NIH3T3 cell line. 293T cell transfected with periostin gene encoding pCXbsr vector, containing the blastcidine S resistant gene, and pCL-Ampho vector which expresses an amphotropic envelope.

Transfection two days after, the culture medium collected and incubated with NIH3T3 cells. After then, cells were selected with blasticidin S (5 mg/ml). (A, B), Enzymatic gene restriction was performed to confirm that existence of periostin gene and amphotropic envelope gene in each plasmid. Among of constructed periostin overexpressing cell lines, 1-8 cell line selected using ELISA assay that evaluate secreted periostin amount to culture medium (C) and confirmed with western blotting (D).

POSTN POSTN--NIH3T3NIH3T3

2 week 4 week

B C

D E

Fig. 8. Ipsi-laterally transplanted NSCs have tropism for periostin over-expressing NIH3T3 cells. DiI labeled NSC transplanted to p-NIH3T3 or NIH3T3 implanted same hemisphere cortex. DiI labled NSC located in original injected site and showed not tropism for NIH3T3 that at time point of transplanted 2 weeks later (A).

However, NSC migrated through corpus callosum and reached Vybrant Dyecycle Green labeled p-NIH3T3 (B) that continuously expressed periostin (small box in Fig. B). NSC transplanted 2 weeks later, NSCs distributed entire p-NIH3T3 cells which spread from implanted position to near striatum tissues and intermixed with p-NIH3T3 cell (D). In more times was given to NSC condition (4 weeks), NSCs

have motility to p-NIH3T3 (C) and constantly maintained (E). In addition, periostin expressed continuously (small box in Fig. C). Coronal section diagram of rat brain indicate both cell injected site. Scale bar : (A-C), 200mm; (D,E), 50mm

F3 POSTN- NIH3T3

POSTN- NIH3T3 F3

2 week 1 week

I J

H E C

Fig. 9. Contra-laterally transplanted NSCs reached to p-NIH3T3 cell. NSC was labeled with DiI and injected contra-laterally to p-NIH3T3 cell implanted rat hemisphere cortex. Even both cells have long distance between that, DiI labeled NSC moved follow corpus callosum (C) and surrounded p-NIH3T3 cells (A, B). For 2 weeks after NSC cell injection, NSCs traveled to p-NIH3T3 cells which located far from NSC injected site and infiltrated themes (F, G) through corpus callosum route (H).

문서에서 Glioma produced periostin attracts human neural stem cells (페이지 25-0)