To evaluate the necessity of forming a nanoparticle before intratumoral injection of the PIC components (PLL, ION and CpG) in enhancing the anti-tumor immune response to RT + anti-CTLA-4, we injected the three components (PLL, ION and CpG) separately using a technique in which needle entry was made into three different sides of a single B78 melanoma tumor to minimize the possibility of spontaneously forming PIC nanoparticle in vivo after injection. Here, CTLA4 is linked to melanoma.