Fig 1: CTLA-4 blockade enhances the therapeutic effect of cancer immunotherapy using PLGA-MP enabling tumor growth retardation and prolonged survival.a Scheme of PLGA-microparticles (MP) and immune checkpoint blockade combination therapy. b E.G7-OVA-luc+ subcutaneous (s.c.) tumor-bearing C57BL/6J mice were treated with PLGA-MP (250 µg OVA, 2.5 µg Riboxxim, OVA/Rib, red squares, n = 10), PLGA-MP (250 µg OVA, 2.5 µg Riboxxim) in combination with systemic anti-CTLA-4 (250 µg/mouse) (OVA/Rib+aCTLA4, orange hexagons, n = 10), or with anti-CTLA-4 monotherapy (250 µg/mouse, aCTLA4, green triangles, n = 5). Mice immunized with respective isotype control monoclonal antibody (mAb) (ctrl mAb, black diamonds in dotted line, n = 5) or MP Empty (Empty, black diamonds, n = 10) served as control. CD8+ T-cell depletion (100 µg/mouse of anti-CD8 mAb, CD8 depl OVA/Rib, black squares in dotted line, n = 5) was initiated 3 days before and on the day of microparticle injection and lasted until the end of the experiment with a weekly i.p. injection, demonstrating CD8+ T-cell indispensability in PLGA-MP-mediated tumor vaccination. Tumor growth is presented over time. Survival rates are expressed using Kaplan–Meier overall survival over time. Data are presented as mean ± S.D. with dotted lines in corresponding colors demonstrating individual data variances. Data were replicated in two independent experiments with a similar outcome. c Representative bioluminescence images of treated mice at indicated time points are shown. Scaling of the bioluminescent pseudo-color code is depicted and presented as photons/seconds/cm2/steradian (p/sec/cm/sr). d 60 days after tumor challenge, tumor-free mice of the MP-OVA/Riboxxim (OVA/Rib, red, n = 1) and the MP-OVA/Riboxxim plus anti-CTLA-4 combination therapy group (OVA/Rib + aCTLA4, orange, n = 5), as well as naive C57BL/6 mice (n = 3, Ø) were subcutaneously re-challenged with 5 × 105 E.G7-OVA-luc+ cells and tumor rejection was monitored for additional 30 days, demonstrating memory CD8+ T-cell formation causing rejection of re-implanted tumor cells.