Myeloid dendritic cells (CD11b⁺CD11c⁺) are increased in TDLN and are required for tumor regression.

<p>(A) C57BL/6 mice were challenged with 1×10⁶ MB49 on day 0 and treated with Val-boroPro (closed bars) or saline (open bars) during week one (n = 4/group). Tumor-draining lymph nodes were harvested and analyzed by flow cytometry on day 7 (p<0.05, Mann-Whitney) (B) C57BL/6 mice were inoculated with 10⁶ MB49 and injected with clodronate IP (0.1 mL/10 g body weight) every other day from day –1 to 9 following tumor inoculation. Control groups were injected with empty lysosomes or saline (sham). Val-boroPro was administered orally for 1 week (days 3–7). Survival was significantly different in Val-boroPro treated mice given clodronate (thick solid line) compared to those treated with Val-boro-Pro and given empty lysosomes (gray solid line) (*p<0.05, Logrank test, n = 5/group). (C) CD11c-diphtheria toxin (DT) chimeric mice were generated by transplanting bone marrow from CD11c-DT transgenic mice into lethally irradiated C57BL/6 recipients (n = 5/group). Female chimeras were inoculated with 10⁶ MB49 on day 0 and treated with Val-boroPro or saline during week one (days 3–7) with or without IP injections of DT (8 ng/1 g body weight) every other day from day –1 to 9. Tumor volumes were significantly larger in Val-boroPro treated chimeric mice receiving DT (closed circles) compared to Val-boro-Pro treated chimeric mice receiving saline (closed squares) (p<0.001 for all timepoints beyond day 10). Tumor volumes were not statistically different between saline treated (open squares) and DT treated (open circles) chimerics not treated with Val-boroPro. Experiments displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g007" target="_blank">Figures 7A–C</a> were each conducted twice.</p

Val-boroPro treatment is associated with an acceleration of tumor-induced priming.

<p>(A) Female mice were inoculated with MB49 and treated with Val-boroPro (closed bars) or saline (open bars) (n = 12/group). Spleens and lymph nodes were harvested from mice on days 10, 17, and 24 for IFN-gamma ELISPOT analysis (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney). (B) Mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g003" target="_blank">Figure 3A</a>. On day 17, greater numbers of HY-reactive CD8⁺ T cells were observed in the lymph nodes of saline-treated (open bars) mice compared to Val-boroPro treated (closed bars) mice (*p<0.05, **p<0.01, Mann-Whitney, n = 5/group). (C) Composite data showing combined (UTY+SMCY+DBY) relative to tumor volume from mice treated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">figure 1A</a> with saline (open squares top, open bars bottom) or Val-boroPro (closed squares top, closed bars bottom) (n = 12/group). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g003" target="_blank">Figures 3A–C</a> are representative of 3 experiments.</p

Antitumor activity with Val-boroPro is associated with increased trafficking of DCs.

<p>(A–B) B6CD45.1 congenic mice were treated with 20 µg Val-boroPro for three days, and CD11c⁺ cells from spleens and LNs were magnetic bead purified and injected at a dose of 5×10⁶ into lateral tarsals of C57BL/6 (CD45.2⁺) recipients. Immediately following CD11c⁺ cell injection, recipients were treated with Val-boroPro or saline (n = 3/group). Popliteal lymph nodes were harvested 15 hours following Val-boroPro treatment for flow cytometric analysis. (A) Representative dot plots. (B) Bar graph showing statistically more CD45.1⁺ adoptive transferred DCs in the popliteal LN in recipients of DCs from Val-boroPro treated donors also receiving a single injection of Val-boroPro compared to all other group (*p<0.05, **p<0.01, Mann-Whitney). (C) Purified CD11c⁺ cells from Val-boroPro treated GFP⁺ mice were injected intratumorally into established MB49 tumors. Mice were treated immediately thereafter with one dose of Val-boroPro (n = 3/group). Tumor-draining inguinal lymph nodes were harvested 15 hours later and imaged by fluorescent microscopy. Representative lymph nodes from mice receiving CD11c⁺ cells from Val-boroPro treated donors and Val-boroPro post injection. Experiments displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g008" target="_blank">Figures 8A–C</a> were conducted 2 times.</p

Val-boroPro mediated tumor regression is T cell dependent.

<p>(A) Female C57BL/6 mice were challenged on day 0 with 1×10⁶ MB49 and treated with 20 µg Val-boroPro during weeks 1 through 4 (5× per week). Mice that were treated with 20 µg Val-boroPro and subsequently rejected MB49 were then rechallenged with MB49 (1×10⁶) or 76-9 rhabdomyosarcoma (5×10⁵) on day 56 post-primary challenge (n = 5/group). Saline control, initial treatment (closed squares), naïve mice (that did not previously reject tumors) injected with MB49 (closed triangles) or 76-9 (closed diamonds) at time of rechallenge, mice that previously rejected MB49 during Val-boroPro and rechallenged with MB49 (open circles) or 76-9 (inverted open triangles). (B) Mice were inoculated with MB49 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1A</a> and treated with Val-boroPro or saline (closed squares) (n = 5/group). CD4⁺ and/or CD8⁺ T cells were depleted with anti-CD4 and/or anti-CD8 monoclonal antibodies as outlined in methods. Tumor volumes in Val-boroPro treated mice without T cell depletion (closed squares) were significantly smaller than either CD4 (closed triangles) or CD8 (inverted open triangles) depleted groups (p<0.05) and CD4/CD8 depleted group (closed circles) (p<0.001) Open circles represent anti-CD4/anti-CD8 without Val-boroPro treatment. (C) Male (closed circles) and female (closed squares) mice were challenged subcutaneously with 10⁶ HY-expressing tumor MB49 on day 0 and were treated with 20 µg Val-boroPro 5×/week for two weeks (n = 5/group). P<0.01 for all timepoints beyond day 15. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g002" target="_blank">Figures 2A–C</a> are representative of 3 experiments.</p

Treatment with Val-boroPro alters lymphocyte and myeloid cell population in secondary lymphoid tissues.

<p>C57BL/6 mice were treated with 20 µg Val-boroPro (solid bars) or saline (open bars) 5×/week for four weeks, and spleens (n = 15/group) and lymph nodes (n = 8/group) were harvested and analyzed by flow cytometry. <b>(</b>*p<0.05, **p<0.01, ***p<0.001 and reflect comparisons between Val-boroPro treated and Saline treated groups by Mann-Whitney). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g006" target="_blank">Figure 6</a> was conducted twice.</p

Val-boroPro induces complete regression in multiple tumor models and requires inhibition of intracellular PPCE.

<p>(A) C57BL/6 female mice were inoculated with 1×10⁶ MB49 on day 0. Mice were treated with saline (open squares) or 20 µg Val-boroPro 5×/week during weeks 1 through 4 (days 3–28, closed squares), week 1 only (days 3–7, open triangles), or weeks 2 through 4 (days 10–28, inverted solid triangles) (n = 6/group). All treatment groups are statistically different than saline (Anova, p<0.05 for late treament vs saline and p<0.01 for early treatment groups vs saline). The days 10–28 group is also statistically different than both early treatment groups (p<0.05). (B) C57BL/6 mice were inoculated with 1×10⁶ M3-9-M on day 0 and treated with 20 ug Val-boroPro (dashed line) or saline (solid line) for four weeks (n = 5/group). Tumor volumes are statistically different between the Val-boroPro and saline groups at p<0.001 for all times beyond day 10. (C) Mice were inoculated with MB49 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1A</a> and treated with 20 ug Val-boroPro (open squares), 20 ug PT-630 (open triangles), or saline (closed squares) (n = 5/group). (D) Mice were inoculated with M3-9-M as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1B</a> and treated with PT-630 (open triangles) or saline closed squares) (n = 5/group). Mean tumor volumes show that PT-630 treatment was no different from saline. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figures 1A–D</a> are representative of 3 or more experiments.</p

Val-boroPro is an effective adjuvant to tumor primed dendritic cell vaccination.

<p>(A) C57BL/6 female mice were challenged with MB49 (1×10⁶) on day 0 and treated with 20 µg Val-boroPro or saline from days 3–7 (closed squares) or 10–14 (n = 5/group). On day 12, late-treatment groups received either a male-derived HY-expressing DC vaccine or a female-derived DC vaccine IP (1×10⁵). Tumors were significantly smaller in mice receiving male DC vaccine plus Val-boroPro during week 2 (open circles) compared to no treatment (open squares), male DC alone (closed triangles) or female DC plus Vale-boroPro during week 2 (closed circles) (p<0.01, Anova with Tukey post-test) (B) C57BL/6 mice were injected intramuscularly with M3-9-M and treated with Val-boroPro starting on day 10. Bone marrow-derived DCs pulsed with irradiated M3-9-M were given IP on day 12 (n = 8/group). Tumors were significantly smaller in mice receiving combined treatment (open triangles) compared to no treatment (closed squares) or either treatment alone (open squares or closed triangles) (p<0.01, Anova). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g007" target="_blank">Figures 7A and 7B</a> are both representative of 2 experiments.</p