Fig 2: Neoadjuvant irradiation augments the efficacy of checkpoint therapy in HTM with JIMT-1 and MDA-MB-231 tumors possibly by systemic upregulation of T cell abundance. JIMT-1, MDA-MB-231, and MCF-7 breast cancer cells were transplanted orthotopically into humanized NSG mice. Therapy was started when tumors were palpable (about 50 mm3). In the case of irradiation, only tumor areal was irradiated with 6 Gy. Anti-PD-L1 antibody (5 mg/kg body weight) was administered i.p. weekly, for treatment regimen see also Supplementary Figure 1A . (A) Tumor weight was measured for evaluation of response rate to therapy at the end of the experiment. (B, C) The tumors and spleens were processed to a single-cell suspension and the cells were subsequently analyzed by flow cytometry. (B) Percentage of CD45+ cells infiltrated into tumors is shown (median, each symbol represents one individual tumor). (C) Composition of immune cell populations among CD45+ cells was analyzed by CD3, CD19, CD33 and CD56/Nkp46 staining in spleens and tumors. (D) PD-L1 surface expression was analyzed by PD-L1 staining on CD33+ cells in spleens and tumors. Due to too little events of CD33+ cells in the tumor of JIMT-1 and MDA-MB-231 mice, only spleen is depicted. (A, B, D) One-way ANOVA, Tukey’s multiple comparisons test was applied, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. The mean ± SD is depicted, each symbol represents an individual tumor under indicated conditions. (C) Data are shown as mean ± SD, n is given in the lower left of each bar and two-way ANOVA, Tukey’s multiple comparisons test was applied, *p ≤ 0.05, **p ≤ 0.01.

Fig 3: RNA sequencing and bioinformatic analysis.A Gene expression fold change in osteoclasts following different treatments. B Gene expression overlap of upregulated (left) and downregulated (right) genes in the dataset of control vs PD-L1 treatment and nivolumab vs PD-L1 treatment. C Top 15 upregulated and downregulated canonical pathways in osteoclast differentiation. D Canonical pathways overlap of upregulated (left) and downregulated (right) pathways in the dataset of control vs PD-L1 treatment and nivolumab vs PD-L1 treatment.

Fig 4: PD-1/PD-L1 signaling and osteoclast function.A-D TRAP activity and CTSK concentration in cell medium and intracellularly. E Toluidine blue staining of pit formation assay and SEM imaging of bone slices following in vitro osteoclast culture. Scale bar for pit assay: 500 µm, for SEM: 100 µm. F Quantification of pit formation assay.

Fig 5: Changes in immune cell populations during racotumomab-alum treatment in long-term (LS) and short-term survivors (SS). (A) gating strategy to analyze NK, NKT and T cell subsets is shown. Lymphocytes were subjected to doublet discrimination. Singlet lymphocytes were separated according CD3 and CD56 expression to identify NK (CD56+CD3−), NKT (CD56+CD3+) and T cells (CD56−CD3+). T cells were plotted using CD4 and CD8 to further divide the population. CD8+T cells were further resolved according to CCR7 and CD45RA expression: CCR7+CD45RA+ as naïve (N), CCR7+CD45RA− as central memory (CM), CCR7−CD45RA− as effector memory (EM) and CCR7−CD45RA+ as terminal effector memory T cells (EMRA). CD4+T cells were plotted for CD25 and CD127 to identify Tregs as CD25+CD127−. (B–D) Behavior of CD8+T cell subpopulations, (E) and (F) CD4+Tregs cells and CD8+T/CD4+Tregs cells ratio and (G) NKT lymphocytes in long- and short-term survivors during racotumomab-alum vaccination. The black triangles and circles show the values for each patient. The asterisks indicate statistically significant differences between the groups (* p < 0.05, Mann–Whitney U test).