- Authors: Aznar MA. et al.
- Year: 2019
- Journal: J Immunother Cancer 7 116
- Applications: in vitro / in vivo / Poly (I:C) / in vivo-jetPEI
- Cell types:
- Name: 4T1
Description: Malignant neoplasms of the mouse mammary gland cells, also known as 4T1-A
- Name: B16
Description: Mouse melanoma cells.
Known as: B-16; B16 melanoma; B16 subline B78; B78.
- Name: B16-F10
Description: Murine melanoma cells
- Name: BT-474
Description: Human breast ductal carcinoma cells
- Name: HCT 116
Description: Human colon carcinoma cells
Known as: HCT116
- Name: HT-29
Description: Human colon adenocarcinoma cells
- Name: ICNI
- Name: MC-38
- Name: SK-BR-3
Description: Human mammary gland adenocarcinoma
- Name: UMBY
- Name: 4T1
B16-F10 and B16-OVA melanoma, MC38 colon carcinoma or 4 T1 breast carcinoma cells were injected subcutaneously (5 × 105–106) into the right flank of 8- to 10-week-old female C57BL/6 or BALB/c (6–11 mice/group) on day 0. Tumors were measured twice per week with calipers and the volume calculated (length x width2/2). When tumors reached a volume of 80–100 mm3 (day 0) mice were randomized into different groups of treatment according to the experiment. Poly I:C or BO-112 formulation (2.5 mg/Kg, 100 μl), was administered by intratumoral injection twice per week for three weeks (six doses in total). The control group received intratumoral injections of 5% glucose (BO-112 vehicle, identical volume) or PEI (identical amount per dose as present in each dose of BO-112). Survival was monitored daily, and tumors were measured twice per week until the animals died or the tumor volume reached the maximum allowed size. The in vitro cytotoxicity of BO-112 in mouse and human cell lines was continuously assessed by measuring electric impedance in an xCELLigence machine (ACEA). Tumor cells (1.5-2 × 105) were seeded on specific 8-well plates to measure electric impedance. After 4-5 h, BO-112 or Poly I:C (Sigma) was added to culture media at identical concentrations in a final volume of 200 μL per well. PEI (Polyplus-transfection®) was added to culture media at the same concentrations as it is present in BO-112 formulation. Electric impedance was measured every five minutes for 48 h. In vitro BO-112 cytotoxicity was also assessed.
Poly I:C is a powerful immune adjuvant as a result of its agonist activities on TLR-3, MDA5 and RIG-I. BO-112 is a nanoplexed formulation of Poly I:C complexed with polyethylenimine that causes tumor cell apoptosis showing immunogenic cell death features and which upon intratumoral release results in more prominent tumor infiltration by T lymphocytes. Intratumoral treatment with BO-112 of subcutaneous tumors derived from MC38, 4 T1 and B16-F10 leads to remarkable local disease control dependent on type-1 interferon and gamma-interferon. Some degree of control of non-injected tumor lesions following BO-112 intratumoral treatment was found in mice bearing bilateral B16-OVA melanomas, an activity which was enhanced with co-treatment with systemic anti-CD137 and anti-PD-L1 mAbs. More abundant CD8+ T lymphocytes were found in B16-OVA tumor-draining lymph nodes and in the tumor microenvironment following intratumoral BO-112 treatment, with enhanced numbers of tumor antigen-specific cytotoxic T lymphocytes. Genome-wide transcriptome analyses of injected tumor lesions were consistent with a marked upregulation of the type-I interferon pathway. Inspired by these data, intratumorally delivered BO-112 is being tested in cancer patients (NCT02828098).