Figure 1.
PKRA7 decreases subcutaneous and intracranial glioblastoma xenograft tumor growth.
(A) 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) Average tumor weight of control and PKRA7-treated mouse tumors after removal. (C) IHC staining using CD34 endothelial cell marker in D456MG SC tumors from mice treated with control or PKRA7. (D) Cumulative probability of vessel relative density as measured by CD34 staining. Vascular density of tumors decreased with PKRA7 treatment. (E) Representative pictures of H&E staining of sections from control and PKRA7-treated SC tumors (F) Quantification of necrotic regions from 5 slides of each tumor per treatment group, percentages of necrotic areas were measured by ImageJ (*p≤0.05). (G) 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.
Figure 2.
PKRA7 decreases subcutaneous pancreatic cancer xenograft tumor growth.
(A) 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) Average tumor weight of control and PKRA7-treated mouse tumors after removal (*p≤0.05). (C) Representative H&E slides from control and PKRA7 treated tumors. (D) Quantification of necrotic regions from 5 slides of each tumor per treatment group, percentages of necrotic areas were measured by ImageJ (p = 0.205719). (E) IHC staining using F4/80 mouse macrophage marker of AsPc-1 SC tumors treated with control or PKRA7. (F) 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).
Figure 3.
PKRA7 blocks endothelial cell branching, myeloid cell migration, and PK2-induced expression of specific chemokine/chemokine receptors.
(A) IHMVECs were plated on Matrigel in indicated treatment groups. Representative photographs were taken at 8 hours after plating. (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). (C) 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). (D) 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). (E) 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). (F) 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).
Figure 4.
PKRA7 enhances the efficacy of chemotherapeutic drugs to reduce glioblastoma and pancreatic xenograft tumor growth.
(A) 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) 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. (C) Average tumor weight of control, gemcitabine, PKRA7 and gemcitabine plus PKRA7-treated mouse tumors after their removal (*p≤0.05).