Tumor-dependent differences in the immunohistology of induced tumor regression.

<p>Panel A – Histology of control and treated tumors. Tumors were injected every other day X 5 with PBS as a control or with the triple combination of pSP-D-CD40L + CpG + poly(I:C). As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007334#pone-0007334-g003" target="_blank">Figure 3</a>, the triple combination slowed the growth of tumors, and occasionally led to tumor eradication. Two days after the last injection, tumor tissue was processed for histology by staining with hematoxylin and eosin. Tumors treated with PBS showed areas of spontaneous necrosis suggesting that the rapidly growing tumor cells often outgrow their blood supply. After treatment with the triple combination, large areas of necrotic tissue appeared containing fragmented cells and nuclear remnants consistent with a cell death process that exceeded the availability of phagocytic macrophages to clear the debris (see Panel D). Panel B – CD11c antibody staining for dendritic cells. B16F10 tumors injected with PBS as a control contained identifiable CD11c+ dendritic cells. After treatment with the triple combination, even fewer dendritic cells were found in the tumors. Panel C – CD8 antibody staining. For tumors injected with PBS as a control, relatively few CD8+ T cells were seen. However, following injections with the triple combination, there was a marked increase in intratumoral CD8+ T cells in all tumor sections examined. Panel D – F4/80 antibody staining for macrophages. Tumors injected with PBS as a control contained relatively few F4/80+ macrophages and there was no appreciable increase in F4/80+ macrophages following treatment with the triple combination.</p

PEI nanoparticle delivery of pSP-D-CD40L slowed tumor growth and prolonged survival.

<p>The data shown are representative of three independent experiments. Panel A – Antitumor effects of PEI plasmid DNA nanoparticles prepared with pSP-D-CD40L alone or in combination with CpG or CpG + poly(I:C). The role of DNA transfection efficiency was tested by preparing nanoparticles formed from PEI and pSP-D-CD40L plasmid DNA. Intratumoral injections of PEI pSP-D-CD40L nanoparticles led to significantly slower tumor growth (p<0.05 on day 10) when compared to the injection of naked pSP-D-CD40L plasmid alone. Panel B – Survival benefit of PEI pSP-D-CD40L nanoparticle injections in combination with CpG + poly(I:C). As expected from the tumor growth data, pSP-D-CD40L formulated with PEI was able to enhance mouse survival when combined with CpG and poly(I:C) TLR agonists. This combination therapy resulted in long-term-tumor free survival of 2/5 mice (p<0.01 compared to pcDNA3.1)).</p

Combinations of pSP-D-CD40L, CpG, and poly(I:C) showed strong antitumor effects on established B16F10 melanoma.

<p>Given the promising data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007334#pone-0007334-g002" target="_blank">Fig. 2</a>, further studies were done to determine the relative contributions of pSP-D-CD40L, CpG, and poly(I:C) and the effects of using them in a triple combination. Twelve groups of mice (5/group) were studied in parallel. For display purposes, the data are grouped into three rows of graphs focusing on CpG (top row), poly(I:C) (middle row), and CpG + poly(I:C) (bottom row). Panels A, B, and C – While each individual agent slowed tumor growth, the most significant antitumor effect was produced by the combination of pSP-D-CD40L + CpG + poly(I:C). Panel A shows that CpG alone significantly slowed tumor growth compared to either PBS or pcDNA3.1 alone from day 12 (p<0.01 by Student's t test, mean±SEM, n = 5). In this fully controlled experiment, however, it was clear that the addition of pSP-D-CD40L to CpG produced no further antitumor effects (p>0.05). Similarly, Panel B shows that poly(I:C) alone significantly slowed tumor growth when compared to PBS or pcDNA3.1 alone from day 12 (p<0.01). Again, however, the combination of pSP-D-CD40L + poly(I:C) produced no further antitumor effects (p>0.05). Interestingly, as shown in Panel C, the double combination of CpG + poly(I:C) significantly reduced tumor growth beyond that produced by CpG alone (p<0.05 on day 24 on the combination as compared to CpG alone). The addition of pSP-D-CD40L to the two TLR agonists, CpG and poly(I:C), produced an even stronger antitumor effect (Panel C, p<0.05 on day 24 comparing the triple combination to CpG + poly(I:C)). Panels D, E, and F – For survival, the addition of pSP-D-CD40L did not increase the antitumor effects seen with CpG alone. All three agents (pSP-D-CD40L, CpG, and poly(I:C)) improved survival as single therapies. From pairwise comparisons, the survival benefit was greatest with CpG and less prominent with pSP-D-CD40L and poly(I:C). The combination of CpG + poly(I:C) improved survival further compared to poly(I:C) alone (p<0.05 by log-rank test). Although the effects on tumor growth indicated that the double combination of TLR agonists CpG + poly(I:C) was better than each alone, this was not reflected in the survival data. Similarly, the superiority of the triple combination of pSP-D-CD40L + CpG + poly(I:C) seen in the tumor growth studies was not statistically significant from the survival data.</p