Figure 1.
PTT stimulates the expression of proinflammatory cytokines and chemokines.
Serum from B16-F10 tumor bearing mice was isolated at 24 hours (n = 10 per group) and 96 hours (n = 8 per group) post-PTT and analyzed using a 32-plex cytokine/chemokine array. PTT induced the expression of the pro-inflammatory cytokines (A) IL-6, TNF-α, IL-1β, and IL-12p70, cytokines that induce stem cell differentiation (B) GM-CSF, G-CSF, M-CSF, and chemokines (C) CXCL-1, CCL-2 and CCL-4. Grey bars represent untreated tumor-bearing mice. Black bars represent PTT-treated mice. Data are presented as the mean +/− SEM. *p<0.05, **p<0.01, ***p<0.001; Student's t-test.
Figure 2.
PTT induces the maturation of DC within TDLN.
. B16-F10 tumor bearing mice were treated with PTT. 48 hours post-PTT, TDLN were isolated and stained with antibodies to CD11c, CD80, CD86, CD40 and I-A/I-E and analyzed by flow cytometry. DC maturation was assessed by gating on viable CD11c+ cells. Untreated tumor-bearing mice were used as controls (n = 3 per group). (A) Representative FACS plots of CD80, CD86, CD40 and I-A/I-E expression. (B) Corresponding mean fluorescent intensity (MFI) quantification of CD80 and I-A/I-E expression. Data represent the mean +/− SD. *p<0.05; Student's t-test.
Figure 3.
PTT induces systemic effects that influence T cell and MDSC tumor infiltration.
B16-OVA tumors were established on opposing flanks on days 0 and 7, respectively. Primary tumors were ablated by PTT on day 14. Mice were euthanized on day 23 for analysis of contralateral tumors. The control group consisted of untreated tumor-bearing mice (Ctrl: n = 4, PTT: n = 5). (A) IFN-γ ELISpot assay using CD8+ splenocytes stimulated using LPS-matured DC pulsed with tumor-lysate. (B) Representative FACS plots and corresponding quantitative data demonstrating (C) CD8+ and (D) CD4+ T cells infiltrating contralateral tumor sites. (E) Representative FACS plots and corresponding quantitative data demonstrating (F) CD4+FoxP3+ Treg cells and (G) CD4+FoxP3− Thelper cells within contralateral tumor sites. (H) Teffector:Treg and (I) Thelper:Treg ratios within contralateral tumors. (J) Representative FACS plots and corresponding quantitative data measuring (K) CD11b+Ly-6G/C− macrophages and (L) CD11b+Ly-6G/C+ MDSC within contralateral tumor sites. Red line represents the mean. The data is representative of two or more experiments. *p<0.05; z-test.
Figure 4.
PTT induces systemic MDSC expansion capable of suppressing T cell proliferation and function.
B16-F10 tumors were established on the flanks of C57BL/6J mice on day 0. Tumors were ablated by PTT on day 7. Mice were euthanized on day 12 for analysis. The control group consisted of untreated tumor-bearing mice (Ctrl: n = 4, PTT: n = 5). (A) Representative FACS plots and (B) corresponding quantitative data demonstrating CD11b+Ly6G/C+ cells within the spleens of control and PTT-treated mice. (C) Thymidine incorporation assay to measure T cell proliferation 72 hours following CD3/CD28 stimulation of splenocytes at various ratios of splenocytes to Gr-1HighLy-6G+ cells (T cell: MDSC). (D) IFN-γ secretion to measure T cell function in response to CD3/CD28 stimulation at various ratios of splenocytes to Gr-1HighLy-6G+ cells (T cell: MDSC). The data is representative of two or more experiments. *p<0.05, **p<0.01, ***p<0.001; ANOVA followed by Student's t-test with multiple comparison adjustment.
Figure 5.
PTT of a single tumor site can slow the growth of distant pre-established B16-OVA tumors but not less immunogenic B16-F10 tumors.
(A) B16-OVA or (B) B16-F10 tumor-bearing mice were treated with PTT then rechallenged with corresponding tumor cells 10 days post-ablation. Naïve mice were used as controls (n = 5 per group). Primary and secondary (C) B16-OVA or (D) B16-F10 were established on opposing flanks on days 0 and 6, respectively. Primary tumors were ablated with PTT once they reached a size of 5–8 mm (day 12–14). Control mice did not receive PTT (n = 5 per group). Data represent tumor growth over time and is displayed as mean +/− SD. The data is representative of two or more experiments. *p<0.05, **p<0.01; Student's t-test.
Figure 6.
PTT promotes the expansion of adoptively transferred pmel T cells.
Primary B16-F10 tumors were established on day 0, and contralateral tumors were established on day 6. Primary tumors were ablated by PTT on day 10 followed by pmel ATCT on day 11. On day 20, mice were euthanized and tissues were harvested for analysis. IFN-γ secretion in response to hgp100 by cells isolated from the (A) spleen and (B) TDLN. Treatment groups consisted of untreated tumor bearing mice (n = 5), PTT alone (n = 4), ATCT alone (n = 5), and dual PTT/ATCT (n = 5). The data is representative of two or more experiments. *p<0.05, **p<0.01; ANOVA followed by Student's t-test with multiple comparison adjustment.
Figure 7.
ATCT prevents tumor recurrence post-PTT, while PTT enhances the antitumor efficacy of ATCT.
Primary B16-F10 tumors were established on day 0, and contralateral tumors were established on day 6. Primary tumors were ablated by PTT on day 10 followed by pmel ATCT on day 11. (A) Primary tumor growth and (B) primary tumor recurrence in mice treated with PTT alone or dual PTT/ATCT (n = 23 per group). *p<0.05; Z-test. (C) Growth of contralateral B16-F10 secondary tumors (n = 5 per group). Grey indicates tumor volume of individual mice. Red represents mean tumor volume. The data is representative of two or more experiments. *p<0.05; Student's t-test.
Figure 8.
ATCT abrogates the outgrowth of lung metastases induced by PTT.
B16-F10 tumors were established on day 0. Contralateral tumors were established s.c., and lung metastases were established by the administration of tumor cells i.v. on day 6. Primary tumors were ablated by PTT on day 10 followed by pmel ATCT on day 11. On day 20, mice were euthanized and lungs were harvested for analysis. (A) Representative lung photographs. (B) Lung mass and (C) quantification of lung metastases. Treatment groups consisted of untreated tumor bearing mice (n = 5), PTT alone (n = 4), ATCT alone (n = 5), and dual PTT/ATCT (n = 5). *p<0.05; ANOVA followed by Student's t-test with multiple comparison adjustment.