Cancer stem cells induced migration by CM-1 via integrin αvβ5 as receptor of CM-

Tumor stroma is composed of a variety of cells including cancer stem cells (CSCs).

Cancer stem cells have aggressive and invasive properties and are known to play the important role for cancer metastasis. (Soltysova et al, 2005; Altaner C, 2008) In general pattern, tumor metastasis promotes the spread of tumors to local and distant sites away from primary tumors. (H Jin et al, 2004)

Previous in vivo data showed that tumor cells in 200% NIH 3T3 cells overexpressing 1 (p-NIH 3T3) implanted condition were promoted the gathering of tumors toward CM-1-secreted sites compared with 50% p-NIH 3T3 cells implanted condition. As well as, edge of tumor mass was seemed like clearly assemblage. So, we investigated whether CM-1 induced migration not only NSCs, but also in cancer stem cells. In order to determine the presence of integrin αvβ5 known as receptor of CM-1 in cell membrane, we performed migration assay using functional blocking antibody for human NSCs HB1.F3 and human glioblastoma primary cultured cells, rat glioma cells. (fig. 1) In this result, C6 and human glioblastoma primary cultured cells #18, #24 significantly increased induction of cell migration about 1.7-3.8 fold into pore of membrane via CM-1 as well as F3 and decreased migration by integrin αvβ5 functional blocking antibody though in the presence of CM-1.

This data suggests that CM-1 induces migration not only in cancer stem cells, but also in

NSCs in the presence of integrin αvβ5 as receptor of CM-1.

Fig. 1. Role of alpha v beta 5-integrin receptors in CM-1-induced human neural stem cell, glioma cell lines migration. (A-D) Blocking experiment with integrin αvβ5 antibodies showed the interactions of CM-1-integrin αvβ5. (A) HB1.F3 Human neural stem cells (5x10⁴ cells / well) were pretreated with anti-integrin αvβ5 antibody (10μg/ml) or control antibody (10μg/ml ) for 30min and transferred onto transwells in the presence or absence of

CM-1 (5μg/ml) for 24 hr. Migrated cells were fixed and stained using hematoxylin, followed by quantitative analysis. Human glioblastoma primary cultured #24 cells (B), human glioblastoma primary cultured #18 cells (C), C6 rat glioma cells (D) were assessed by same method. Bottom representative figures of migrated cells under membrane of transwell after 24 hr using bright microscope. Error bars indicate SD. Magnification, X100.

B. Systemic strategy using CM-1 as chemoattractants and CD-5-FC is effective to prolong survival time.

Previous in vivo experiments employed immortalized HB1.F3.CD cells encoding cytosine deaminase (CD) gene to investigate the bystander effect by migrating to tumor via CM-1. As prior result, F3.CD cells were affected to neighborhood cell as induced cell death fairly. So, we determined whether CM-1 was effective to extend survival time in animal model of brain tumor, following described experimental plan via divided 5 groups. (fig. 2) We confirmed the in vivo prolonged survival time of CM-1 in brain tumor animal model, which was made by injection of C6 rat glioma cells into the right striatum of 8-week-old SDS.rats. F3.CD cells were transplanted into left brain hemisphere stereotactically and 5-FC was administrated systemically.

In PET images, tumor mass of group E [C6 + p-NIH 3T3 (2x) + F3.CD + 5-FC] was significantly reduced compared with group A (C6 + HBSS) as a negative control and group C (C6 + F3.CD + 5-FC). (fig. 3) This result showed that F3.CD cells with enhanced mobility via CM-1 made to slow the tumor growth more effectively. To calculate that numerical value of tumor volume, we utilized AMIDE on the basis of PET images. (fig. 4) Tumor volumes of rats implanted with p-NIH 3T3 cells were reduced significantly [group E, an average of tumor volumes = 56.2422 ± 52.8143mm3 (mean ± SD)], compared with those of control group [group A, an average of tumor volumes = 236.233 ± 119.2748mm3 (mean ± SD)]. It means that CM-1 help the targeting of F3.CD cells to tumor region and F3.CD cells hinder to increase tumor volume in established condition of tumor model. To identify whether how much survival time is extended via CM-1-overexpressed condition, we checked dead or alive

rats in everyday. As survival graph, until 85 days, all rats of group A-D died. But, 33.2 % rats of group E were alive without dying continuously over 110 days. There was significantly difference in survival [final survival after tumor cell implantation(minimum period – maximum period); group A (HBSS injection + saline) = 71 days (27-71)days; group B (F3.CD injection + saline) = 69 days (31–69 days); group C (F3.CD injection + 5-FC) = 84 days (32–84 days); group D (p-NIH 3T3 0.5x + F3.CD injection + 5-FC) = 67 days (34–67 days) group E (p-NIH 3T3 2x + F3.CD injection + 5-FC) = alive 3 rats (44- days)]. (fig.5) Interestingly, early tumor volume rate of implanted group D of 0.5 fold p-NIH 3T3 cells was similar with group E. But about 30 days later, rate of tumor volume was rapidly increased and survival time was not different with control group.

These results suggest that CM-1 of the more constant concentration in tumor area can improve the therapeutic effect of gene therapy using NSCs and prolong survival period.

Fig. 2. In vivo Experiment schedule for extensive survival time by using CM-1 treatment. Timeline for the brain tumor model and subsequent treatment using F3.CD cells and 5-fluorocytosine (5-FC). The therapeutic effect was identified by five groups. Each of five groups had been transplanted at different interval of time, C6 rat glioma cell and p-NIH3T3 cells or F3.CD cells were transplanted in different fold. Since then ten days later, 5-fluorosytosine (5-FC) was injected into i.p. for 3weeks. Among that period, Positron emission tomography (PET) was scanned at mentioned days.

Fig. 3. CM-1-induced migration of F3.CD was significantly effective to therapy of tumor in PET analysis. Representative PET (coronal images) data. The rats with C6 tumors (group A : C6 + HBSS + NS) as controls were not treated with 5-FC after tumor inoculation.

Group C (C6 + F3.CD + 5-FC) and E [C6 + p-NIH3T3 (2x) + F3.CD + 5-FC] were implanted with F3.CD after 5 days of tumor inoculation, Followed by daily intraperitoneal injection of 5-FC for 3 weeks. Reduction in signal intensity could be observed after treatment, indicating reduction in uptake of F-FDG in tumor at 15, 22, 29 day.

Fig. 4. Bystander effect of CM-1-induced migration of F3.CD was significantly decreased to tumor volume by tumor volume analysis. Time courses of tumor growth.

Mean tumor size was measured 15, 22, 29, 36, 43 days after transplantation of tumors. The growth rate was monitored by the measurement of tumor volume in PET scan via calculating the FDG uptaken by tumor cells. Group A ; C6 + HBSS + NS (n = 8), Group B ; C6 + F3.CD + NS (n = 8), Group C ; C6 + F3.CD + 5-FC (n = 7), Group D ; C6 + p-NIH3T3 (0.5x) + F3.CD + 5-FC (n = 10), Group E ; C6 + p-NIH3T3 (2x) + F3.CD + 5-FC (n = 9)

Fig. 5. In vivo analysis of extended survival period by therapeutic effect. A survival time plot for the five groups for the 25 to 110 days. Actual survival time of rats after systemic administration with or without 5-FC. At tumor injected day, each rat was transplanted p-NIH3T3 cells (in 3μl HBSS) or HBSS (3μl) with C6 in right striatum. After five days, rats were continuously injected into F3.CD cells (in 3μl HBSS) or HBSS (3μl) in contra-lateral hemisphere cortex. Since then ten days later, each group was given i.p. 5-FC (500mg/kg/day) or 0.9% normal saline for three weeks. The p-NIH3T3 (2x) and CD/5-FC group yielded significantly better survival versus all of the other groups. Rat surviving 120 days were considered cured. Group A (black) ; C6 + HBSS + NS (n = 8), Group B (pink) ; C6 + F3.CD + NS (n = 8), Group C (green) ; C6 + F3.CD + 5-FC(n = 7), Group D (blue) ; C6 + p-NIH3T3 (0.5x) + F3.CD + FC (n = 10), Group E (red) ; C6 + p-p-NIH3T3 (2x) + F3.CD + 5-FC (n = 9)

C. CM-2 was crucial interaction with integrin β1 in induction of NSCs migration.

At the plasma membrane, the members of the tetraspanin family including CD63 are known to interact with cell adhesion molecules such as integrins, regulating intracellular signal transduction pathways including cell adhesion, motility, and survival (Berditchevski, 2001; Hemler, 2001; Yunta and Lazo, 2003). Integrin β1 is one of the main tetraspanin-interacting integrins. Previous results reported that CM-2 induced migration of NSCs by combining CD63. As well as, it complex bound to integrin β1.

To explore the essential role of integrin β1 by which CM-2 promotes induction of NSCs migration, we used functional blocking antibody for integrin β1 in migration assay. As shown in fig. 6, migrated NSCs were 2.26-2.44 fold compare with control in treatment condition of CM-2. In the presence of functional blocking antibody for integrin β1, NSCs did not induce migration and moved cells were no almost.

Its results supported that induction of NSCs migration by CM-2 was mediated to interact with CM-2 and integrin β1 as well as CD63.

Fig. 6. Integrin β1 mediated functional cell migration by induction ofCM-2. HB1.F3 (5 x 10⁴ cells/well) were preincubated with anti-integrin β1 antibody (10μg/ml) or control antibody (10μg/ml) for 30min and were cultured in a 24-well plate medium with or without CM-2 (100ng/ml) for 24hr. Migrated cells on the lower side of the membrane were fixed and stained with hematoxylin and counted under a bright microscope using a ×100 objective.

Error bars indicated SD.

D. FAK and PI3K signal pathway were included in the NSCs migration.

The binding of ECM proteins to the extracellular domains of integrins leads to integrin activation, which recruits and activates signaling molecules such as focal adhesion kinase (FAK). FAK signaling pathway is known as cell motility related signal pathway. FAK signal pathway connected with many kinases in its downsteam and convey signal. In cell migration, phosphoinositol-3-kinase (PI3K) signal pathway was mainly used that located in FAK downstream. (Wozniak et al., 2004; Zhao et al, 2009)

To investigate PI3K signal pathway is continuous with induction of migration by CM-2, we performed boydenchamber assay using PI3K inhibitor, LY294002 (10μM/ml). We observed that activation of PI3K signaling by CM-2 was interrupted by PI3K inhibitor, LY294002 (10μM/ml) and moving ratio was reduced in dose-dependent manner (70% in the migrated cells). (fig. 7)

Also, we identified that the present or absent condition of CM-2, the tyrosine 397 phosphorylated level of FAK in CD63-knockdown F3 cells and pREP4 vector F3 cells (pREP4.F3 cells). CD63-knockdown F3 cells (shCD63. F3 cells) was established by using a vector-based small hairpin RNA (shRNA) strategy. As shown western blot results, phosphorylated FAK levels of CD63-knockdown F3 cells were decreased compare with pREP4 vector F3 cells in the condition of treatment CM-2. (fig. 8) To check total FAK level, same membrane with phospho-FAK detected membrane deproved and then detected again.

These results suggest that CM-2 lead to NSCs migration via tyrosine 397 phosphorylation of FAK and activation of PI3K signaling.

Fig. 7. PI3K signal pathway control NSC-induced migration by CM-2. HB1.F3 cells were pre-incubated with medium containing 10% FBS for 24hr and then treated with or without LY294002. Other cell groups were treated dose-dependently PI3K inhibitor (LY294002) with CM-2. In all cases, migration was measured after 12h and was expressed relative to the migration of stimulated cells. Bottom images were representative migrated F3.

Error bars indicated SD. Magnification, X100.

Fig. 8. CM-2 controlled activation of FAK in induction of NSC migration. After incubation of pREP4.F3 cells and shCD63.F3 cells in the presence or absence of CM-2 for 30min, cell lysates were prepared by using RIPA buffer. Equal volumes of cell lysates were analyzed by Western blot using antibodies directed against the phosphorylated state of FAK at Tyrosine 397. The membranes were further analyzed by Western blotting using anti-FAK antibody to detect FAK expression levels. As controls, β-actin was detected. The graph of each relative activity was analyzed by densitometric quantification using Image J software.

Ⅳ. DISCUSSION

The present study identified CM-1 and CM-2 as novel chemoattractants molecules for mobility inducers in activating tumor tropism of NSCs. According to prior results, CM-1 and CM-2 were over-expressed in human glioblatomas tissue and possessed ability of induction of NSCs migration. Furthermore, when human NSC line HB1.F3 carrying cytosine deaminase (CD) enzyme gene (F3.CD) was transplanted intracranially at distant sites from the tumor, the NSCs migrate through normal tissue and selectively “ home in” to the glioblastoma tumor mass and upon administration of prodrug 5-FC, significant reduction in tumor volume was demonstrated in rat brain tumor model. (Kim et al, 2011; Black, 1991;

Heese et al, 2005)

In previous results, tumor mass was assembled to tumor area and non-invasive signature relatively. Cancer stem cells, one of the cells that comprise the tumor, have aggressive and invasive features are related to metastasis. So, we hypothesized that cancer stem cells had integrin αvβ5 as a receptor of CM-1 and induced cell migration via CM-1. As shown in our result, human glioblastoma primary cultured cells and C6 rat glioma cells had integrin αvβ5 and led to migration toward CM-1.

As well as in this study, we identified that use of CM-1 prolonged survival time with advanced strategy using CD and 5-FC suicide gene therapy for brain tumor model. Among experimental group D of CM-1-over expressing NIH 3T3 cells 0.5x and experimental group E of CM-1-over expressing NIH 3T3 cells 2x (compare with C6 number), tumor volume rate

of group D was similar with group E until 30 days. But, after then, rate of tumor volume was rapidly increased and survival time was not different with control group. In contrast, the group E showed distinctly slowed the tumor growth rate as well as increased survival rate compared with other groups. It means that CM-1 of the more constant concentration was significantly effective to brain tumor treatment.

Other previous results showed that CM-2 induced migration specifically in NSCs and interacted with CD63 and integrin β1 for induction of migration. When we blocked integrin β1 using functional blocking antibody, induction of migration was significantly decreased despite the presence of CM-2. Some researchers demonstrated phosphoinositol 3-kinase (PI3K), which has been shown to be an important regulator of directed NSCs migration.

(Wozniak et al, 2004; Zhao et al, 2009) These observations revealed when CM-2 induced NSCs migration, PI3K signaling pathway was important shown by migration assay. As well as, CM-2 induced phosphorylation of FAK in tyrosine 397 site but not in CD63 knockouted F3 cells. The present results support that interaction of integrin β1 and PI3K pathway and phosphorylation of FAK are related to induce NSCs migration via CM-2.

Overall, our results reveal that a novel tumor tropism signature of neural stem cells was induced by CM-1 and CM-2. As a result, these chemoattractants improve ability that the inherent tumor-tropism of NSCs to primary and invasive brain tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. (Imitola et al, 2004) Thus, identification and characterization of such chemoattractants will become to use crucial for brain tumor therapy more efficiently.

For Further studies, we have to establish CM-2 over-expressing NSCs and check whether

CM-2 is also effective to induce cell migration in vivo. As well as, when CM-1 and CM-2 is introduced in the therapy of actual human brain tumor, we think about that mobility inducers can be used most effectively over a long period of time for therapeutic effect.

Ⅴ. CONCLUSION

This study showed that both NSCs and CSCs has receptor of CM-1 as integrin αvβ5 and induced migration by CM-1. Moreover, CM-1 enhanced migration ability and elevated tumor targeting for F3.CD. Such F3.CD cells decreased growth rate of tumor as well as prolong survival period in tumor model by converting 5-FC to cytotoxic agents, accordingly it made suicide gene therapy was more effective. Another inducer of NSCs migration, CM-2 induced migration via interaction with integrin β1 and PI3K signaling pathway. The existence of CD63 in NSCs upon interaction with CM-2 activated FAK and then, NSCs induced migration. These results demonstrated that CM-1 and CM-2 as chemoattractants led to migration of NSCs & CSCs and can improve the effect of neural stem cell-based gene therapy for brain tumor.

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문서에서 Characteristic analysis of mobility inducers for neural stem cells (페이지 21-0)