Tumor-penetrating peptide internalizing RGD enhances radiotherapy efficacy through reducing tumor hypoxia

Abstract

Resistance to irradiation (IR) remains a major therapeutic challenge in tumor radiotherapy. The development of novel tumor-specific radiosensitizers is crucial for effective radiotherapy against solid tumors. Here, we revealed that remodeling tumor tissue penetration via tumor-penetrating peptide internalizing arginine-glycine-aspartic acid RGD (iRGD) enhanced irradiation efficacy. The growth of 4T1 and CT26 multicellular tumor spheroids (MCTS) and tumors was delayed significantly by the treatment with IR and iRGD. Mechanistically, iRGD reduced hypoxia in MCTS and tumors, resulting in enhanced apoptosis after MCTS and tumors were treated with IR and iRGD. This is the first report that shows enhanced radiation efficacy by remodeling tumor-specific tissue penetration with iRGD, implying the potential clinical application of peptides in future tumor therapy.

Keywords: hypoxia; irradiation; multicellular tumor spheroids; radiosensitivity; tumor-penetrating peptides.

FIGURE 1

Tumor‐penetrating peptide iRGD enhanced the efficacy of radiation therapy in vitro. (A) Model of the treatment with iRGD and IR. (B) Growth curves of MCTS treated with 2 Gy radiation and peptides. (C) Growth of MCTS treated with various concentrations of iRGD peptide. (D) Relative area of colonies formed under different treatments. (E) Representative pictures of colonies. (F) Migrated cells under different treatments. (G) Growth curves of CT26 MCTS treated with iRGD and control peptides with or without 1.5 Gy IR

FIGURE 2

Tumor‐penetrating peptide iRGD enhanced the efficacy of radiation therapy in vivo. (A, E) Schematic description of tumor‐penetrating peptide iRGD combined with radiation treatment. (B) Combination therapy with IR and iRGD greatly delayed 4T1 tumor growth compared with single treatments. (C) Combination therapy greatly inhibited 4T1 tumor weight compared with single treatments. (D) Combination therapy dramatically reduced lung metastasis compared with single treatments. Metastatic tumor area was quantified by the percentage of metastatic area relative to total lung area. (F) Combination therapy greatly delayed CT26 tumor growth compared with IR treatment alone. (G) Combination therapy greatly reduced CT26 tumor weight compared with IR treatment alone

FIGURE 3

Body weight change of 4T1 tumor‐bearing mice that received the iRGD and IR treatment

FIGURE 4

Tumor‐penetrating peptide iRGD promoted IR‐induced apoptosis in vitro. (A) Model for treatments of MCTS. (B–D) Percentages of early apoptosis (B), late apoptosis and dead (C) and total apoptosis (D) cells in MCTS treated with IR and peptides. (E) Representative scatterplots of annexin V/PI staining

FIGURE 5

Tumor‐penetrating peptide iRGD promoted IR‐induced apoptosis in vivo. (A) Percentages of apoptotic cells in tumor tissues treated with IR and peptides. (B) Representative TUNEL immunofluorescence of the tumor tissues

FIGURE 6

Tumor‐penetrating peptide iRGD reduced hypoxia in MCTS. (A) Schematic description of three‐dimensional (3D) MCTS culture. (B) Representative immunofluorescence of MCTS stained with anti‐pimo (green) antibody and DAPI (blue). (C) Relative hypoxic cells (pimo⁺) in MCTS after administration of iRGD for the indicated times. (D) Relative hypoxic cells (pimo⁺) in MCTS after administration of peptides for 4 h. (E) Percentages of hypoxic cells (pimo⁺) in MCTS under different treatments. (F) Relative mRNA expression of four hypoxia‐related genes in MCTS under different treatments

FIGURE 7

Tumor‐penetrating peptide iRGD reduced tumor hypoxia in vivo. (A) Model for tumor hypoxia detection. (B) Representative immunofluorescence of the tumor tissues stained with anti‐pimo (green), anti‐mouse CD31 (red) and DAPI (blue) (C) Percentages of hypoxic area (pimo⁺) in tumors treated with iRGD and control peptides

FIGURE 8

NRP‐1 is the critical molecules for iRGD to reduce hypoxia and to enhance the efficacy of IR. (A) Hypoxic cell percentages of MCTS treated with t‐iRGD or PBS. (B) Relative hypoxic cells in MCTS treated with iRGD after NRP‐1 blocking. (C) Growth of MCTS treated with iRGD after NRP‐1 blocking. (D) Mechanistic model of the enhancement of IR efficacy by iRGD. Tumor‐penetrating peptide iRGD increases the penetration of molecular oxygen and reduce hypoxia in MCTS and tumors through NRP‐1 pathway, leading to increased sensitivity of tumor cells to IR. As a result, the apoptosis of tumor cells increased and tumor burden and metastasis decreased