Abstract: We aimed to determine the role that integrin 4 plays in glioma cell migration as well as glioma stem cell differentiation. Evidence indicates a Cancer Stem Cell (CSC) method of tumor propagation in gliomas. In this model, a small subpopulation of cancer cells, which display stem-cell-like qualities, initiate tumor proliferation and are required for metastasis. While CSCs are not necessarily derived from normal stem cells, the cells share similar qualities. Increased levels of integrin 4 promote tumor invasion and normal neural stem cell (NSC) differentiation through a complex series of ligand based signaling. Therefore, we investigated the effects of an upregulation of integrin 4 on CSC proliferation as well as migration. We also attempted to isolate the sub population of CSCs by washing cells in serum free medium containing Vincristine. We found that an upregulation of integrin 4 increased glioma cell migration and caused the migratory cells to form distinct colonies. The increase in migration was approximately 24%. Also, washing cells in serum free medium increased the presence of tight colonies that are indicative of CSCs. Finally, transfection of integrin 4 increased the average number of tumor spheres from one to four spheres per well.

Washing C6 Glioma cells in serum free stem cell medium with Vincristine increases the presence of tumor spheres

4 promotes differentiation of Neural stem cells, not glioma stem cells

Conclusions:

1. Growing cells in serum free stem cell medium plus Vincristine is an effective method of increasing the number of Nestin positive tumor spheres in culture

2. Upregulation of integrin 4 increases the rate of migration of C6 glioma cells

3. Transfecting C6 cells with integrin 4 increases the number of tumor spheres per well

4. Although high levels of integrin 4 promoted migration and proliferation of CSCs, it did not induce differentiation of C6 cancer stem cells

5. Upregulation of integrin 4 can be discounted as an effective form of cancer therapy

Cancer Stem cells can be identified via morphology

Cancer stem cells are precursors to the development of tumors in metastasis but resist chemotherapy

Integrin 4 is involved in cellular migration and stem cell differentiation

Cells with upregulated levels of 4 migrate at higher rates and form distinct colonies

An upregulation of integrin 4 increases frequency of Nestin positive tumor spheres in C6 cells

Determining the effects of integrin 4 in migration and differentiation of C6 glioma stem cells

Garrett LeeInstructor: Dr. Christine Marshall-Walker

Phillips Academy Biology 600

A) B)

A) A control population of cells grown in DMEM and FBS

B) A Nestin positive tumor sphere found in the serum free stem cell medium plus Vincristine

B) Regulation of intracellular signaling by integrins. Integrin ligation induces a complex network of signaling pathways to control cell migration (Hood et al. 2002, Nature Reviews Cancer).

A) The simple scheme for the functions of integrin 4 in neural cells (Su et al. 2009, Neuromol Med)

According to the cancer stem cell hypothesis, tumor burden is maintained by a small group of self-renewing and differentiating cells, cancer stem cells (CSCs).

A) Under a neighborless environment, cancer stem cells divide symmetrically, which generates identical progeny. Committed cancer cells get more differentiated after each cell division (Zhou et al. 2009, Cancer Letters).

B) A graph showing the increase in cell migration and tumor sphere frequency following a transfection. The cell

migration was an average of three fields of view from the bottom of a control and transfected transwell filter. The number of tumor spheres per well was averaged from six

transfected wells and four control wells.

A) A tight colony of glioma stem cells observed following a transfection of

integrin 4

B) A sample of cells transfected with integrin 4 and stained with Hoechst following a transwell migration.

A) These cells from the control population were stained with Hoecsht following a transwell migration.

B)A)

A)

B)

Conventional therapy targeting tumor bulk leads to tumor regression, however, presence of CSCs in the residual tumor leads to tumor recurrence. (Dey, 2010, Cancer Letters)

A) B)

C) This is an observed tumor sphere from a population of cells transfected with integrin 4. The remarkable overlap of Nestin and 4 creates the orange hue. The larger, round dark spot around the mass are cells while the interior of the sphere is dominated by the 4 marker.

A) Overexpression of integrin 4 induces differentiation in neural stem cells based on frequency of an astrocyte marker.

A)

Control Transfection B) Differentiation of NSCs under phase-contrast microscopy (Ye et al. 2009, Biochemistry & Cell Biology)

B)

C) Tight Medium Loose

B) Examples of tight, medium, and loose colonies in vitro

C) An example of a large tumor sphere

A)B)

C)