PKRA7 decreases subcutaneous and intracranial glioblastoma xenograft tumor growth.

<p>(<b>A</b>) D456MG cells were SC injected into nude mice, and control (n = 5) or PKRA7 (n = 5) treatment was commenced when tumors became visually detectable (14 days). Measurements were taken every 2–3 days. (<b>B</b>) Average tumor weight of control and PKRA7-treated mouse tumors after removal. (<b>C</b>) IHC staining using CD34 endothelial cell marker in D456MG SC tumors from mice treated with control or PKRA7. (<b>D</b>) Cumulative probability of vessel relative density as measured by CD34 staining. Vascular density of tumors decreased with PKRA7 treatment. (<b>E</b>) Representative pictures of H&E staining of sections from control and PKRA7-treated SC tumors (<b>F</b>) Quantification of necrotic regions from 5 slides of each tumor per treatment group, percentages of necrotic areas were measured by ImageJ (*p≤0.05). (<b>G</b>) 1×10⁴ D456MG cells were IC injected into nude mice and treatment started 7 days after tumor implantation. Mice in control (n = 8) or PKRA7 treatment (n = 9) group were sacrificed when they developed severe neurological phenotype indicative of tumor growth intracranially.</p

PKRA7 decreases subcutaneous pancreatic cancer xenograft tumor growth.

<p>(<b>A</b>) AsPc-1 cells were SC injected into nude mice, and control (n = 4) or PKRA7 (n = 5) treatment was commenced when tumors became visually detectable (9 days). Measurements were taken every 2–3 days. (<b>B</b>) Average tumor weight of control and PKRA7-treated mouse tumors after removal (*p≤0.05). (<b>C</b>) Representative H&E slides from control and PKRA7 treated tumors. (<b>D</b>) Quantification of necrotic regions from 5 slides of each tumor per treatment group, percentages of necrotic areas were measured by ImageJ (p = 0.205719). (<b>E</b>) IHC staining using F4/80 mouse macrophage marker of AsPc-1 SC tumors treated with control or PKRA7. (<b>F</b>) Quantification of average macrophage infiltration of AsPc-1 tumors treated with control (n = 4) or PKRA7 (n = 5), 5 slides of each tumor per treatment group (*p≤0.05).</p

PKRA7 enhances the efficacy of chemotherapeutic drugs to reduce glioblastoma and pancreatic xenograft tumor growth.

<p>(<b>A</b>) Kaplan-Meier curve of nude mice after temozolomide and PKRA7 treatment following IC injection of 1×10⁴ D456MG cells. Treatment with 10 mg/kg temozolomide or control started 3 days after IC injection for a total of 5 consecutive daily treatments. Treatment with PKRA7 or control started 7 days after IC injection and continued for the duration of experiment. 5 mice per condition. (<b>B</b>) AsPc-1 cells were SC injected into nude mice, and control (n = 10) or PKRA7 (n = 10) treatment was commenced when tumors were visible (7 days). Treatment with 100 mg/kg gemcitabine (n = 10) or control (n = 10) started 7 days after tumor implantation and was administered every 4 days for two weeks for a total of 4 treatments (#). Measurements were taken every 3 days. 5 mice per condition. (<b>C</b>) Average tumor weight of control, gemcitabine, PKRA7 and gemcitabine plus PKRA7-treated mouse tumors after their removal (*p≤0.05).</p

PKRA7 blocks endothelial cell branching, myeloid cell migration, and PK2-induced expression of specific chemokine/chemokine receptors.

<p>(<b>A</b>) IHMVECs were plated on Matrigel in indicated treatment groups. Representative photographs were taken at 8 hours after plating. (<b>B</b>) Average number of connections between cells was counted and analyzed. Results are normalized data from 3 independent experiments. Addition of PKRA7 significantly blocks PK2-induced capillary branching (*p≤0.05). (<b>C</b>) 1×10⁵ THP-1 cells on top chamber of transwells were allowed to migrate for 4 hours. Cells were fixed, stained and the number of cells per field of view counted. Results are the normalized average of 3 independent experiments. Addition of PKRA7 significantly blocked PK2-induced monocyte migration (*p≤0.05). (<b>D</b>) 7.5×10⁴ RAW264.7 cells on top chamber of transwells were allowed to migrate for 18 hours. Cells were fixed, stained and the number of cells per field of view counted. Results are the normalized average of 3 independent experiments. Addition of PKRA7 significantly blocked PK2-induced macrophage migration (*p≤0.05). (<b>E</b>) Average measured luminescence of tumor site after IP injection of luciferase-labeled RAW cells into control (n = 4) or PKRA7 (n = 4) treated mice with SC AsPc-1 tumors 30 days after implantation. Average total luciferase counts were lower in mice treated with PKRA7 compared to control (*p≤0.05). (<b>F</b>) qPCR assay to measure the effect of PKRA7 on the expression of chemokines and chemokine receptors that were identified to be induced by PK2 treatment. Data on the mRNA level changes were shown as log2 of the Ct value changes. PKRA7 inhibits upregulation of CCL27, CCR10, CCR4, CCR5, and CCR6 (*p≤0.05).</p

c-Myc knockdown abolishes xenograft tumor formation by glioma cancer stem cells.

<p>(A) T3359 CD133+ cells were infected and selected as described. After selection, cells were injected into brains of athymic BALB/c nu/nu mice (5000 cells per mouse). Four mice were injected for each group. Mice in the control group were sacrificed upon the development of neurologic signs. All the mice bearing c-Myc knockdown glioma cells did not develop neurologic signs and were sacrificed after 100 days without evidence of tumor. Kaplan-Meier survival curves are displayed. (B) Representative photographs of hematoxylin and eosin staining of intracranial xenograft tumors (10×). (C) Xenograft tissue of the control group composed of pleomorphic cells featuring high nuclear to cytoplasmic ratios, prominent nucleoli with minimal cytoplasm, brisk mitotic activity and central geographic necrosis (asterisk, 600×). (D) The control glioma xenograft exhibited focal areas of better differentiated tumor cells with relatively more eosinophilic cytoplasm and cells with eccentric cytoplasmic profiles suggestive of a gemistocytic appearance (arrows, 400×). (E) Xenograft tumor of the control group exhibits infiltration of tumor cells into the surrounding brain tissue along the margin. The mitotically active (arrowhead) infiltrating tumor cells exhibit high nuclear to cytoplasmic ratios and elongated fibrillar cytoplasm (arrow) (600×). (F) Mouse brain injected with T3359 cells expressing c-Myc shRNA showed no evidence of tumor at the needle injection site (arrows, 200×).</p

c-Myc modulates cell cycle regulators of glioma cancer stem cells.

<p>(A) Early passage (WAF1/CIP1, cyclin D₁ (cycD1) and cyclin D₂ (cycD2) were determined by quantitative real-time PCR 3 days after infection. (B) Protein levels of c-Myc, p53, cyclin D₁, cyclin D₂, cyclin E and p21^WAF1/CIP1 were determined by immunoblotting.</p

c-Myc is highly expressed in glioma cancer stem cells.

<p>(A) CD133− and CD133+ cells were isolated from glioma surgical biopsy specimens passaged short-term in immunocompromised mice and briefly cultured. Total RNA was isolated from both CD133− and CD133+ cells. cDNA was prepared by reverse transcription. Expression of c-Myc was then determined by quantitative real-time PCR and normalized to β-actin and HPRT1. Relative mRNA levels of c-Myc in CD133− cells were assigned a value of 1. Data are represented as mean±S.E.M in this and all subsequent graphs (#: p<0.001). (B) Total cellular lysates were resolved by SDS-PAGE. Protein levels of c-Myc and Olig2 were determined by immunoblotting. Actin was blotted as the loading control. (C) Glioma cells were isolated directly from human surgical biopsy specimens, fixed in 4% paraformaldehyde following dissociation, labeled with anti-CD133-APC and anti-c-Myc-FITC, and subjected to FACS analysis. (D) Percentage of cells expressing high levels of c-Myc within either the CD133− fraction or the CD133+ fraction was demonstrated (#: p<0.001). (E) Sections of freshly frozen human glioma surgical biopsy specimens were fixed and co-stained for c-Myc (green) and Nestin (red). Nuclei were counterstained with Hoechst 33342. Representative images (630×) were demonstrated.</p

c-Myc induces cell cycle arrest of glioma cancer stem cells.

<p>(A, B) Early passage (0 phase; M2-G₀/G₁ phase; M3-S phase; M4-G2/M phase.</p

Depletion of c-Myc induces apoptosis in glioma cancer stem cells.

<p>(A, B) T3359 and (C, D) T4597 CD133− and CD133+ cells were isolated and infected as described. (A, C) Six days after infection, cells were trypsinized and quantified for apoptosis using the Annexin V-FITC Apoptosis Detection kit (Calbiochem). The percentage of FITC positive cells was determined by FACS analysis, and dead cells were excluded by propidium iodide staining. (B, D) These cells were also plated in 96-well plates at 5000 cells per well for CD133− cells and 1000 cells per well for CD133+ cells after infection and selection. Six days after infection, combined activities of caspase 3 and caspase 7 were determined by the Caspase 3/7 Luminescence Assay kit (Promega), and were normalized by the viable cell numbers determined by the CellTiter-Glo assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003769#pone-0003769-g004" target="_blank">Figure 4</a>. *, p<0.05 with one-way ANOVA comparison of the control groups to the corresponding c-Myc knockdown groups.</p

Depletion of c-Myc inhibits growth of glioma cancer stem cells.

<p>(A) T3359, (B) T3832, and (C) T4597 CD133− and CD133+ cells were isolated and infected as described. Cells were then plated in 96-well plates in triplicate at 5000 cells per well for CD133− cells or 1000 cells per well for CD133+ cells. Total viable cell numbers were then determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega) daily. *, p<0.05; #, p<0.001 with one-way ANOVA comparison of the control groups to the corresponding c-Myc knockdown groups on the same day.</p