Combined thermal ablation and liposomal granulocyte-macrophage colony stimulation factor increases immune cell trafficking in a small animal tumor model

Abstract

Purpose: To characterize intratumoral immune cell trafficking in ablated and synchronous tumors following combined radiofrequency ablation (RFA) and systemic liposomal granulocyte-macrophage colony stimulation factor (lip-GM-CSF).

Methods: Phase I, 72 rats with single subcutaneous R3230 adenocarcinoma were randomized to 6 groups: a) sham; b&c) free or liposomal GM-CSF alone; d) RFA alone; or e&f) combined with blank liposomes or lip-GM-CSF. Animals were sacrificed 3 and 7 days post-RFA. Outcomes included immunohistochemistry of dendritic cells (DCs), M1 and M2 macrophages, T-helper cells (Th1) (CD4+), cytotoxic T- lymphocytes (CTL) (CD8+), T-regulator cells (T-reg) (FoxP3+) and Fas Ligand activated CTLs (Fas-L+) in the periablational rim and untreated index tumor. M1/M2, CD4+/CD8+ and CD8+/FoxP3+ ratios were calculated. Phase II, 40 rats with double tumors were randomized to 4 groups: a) sham, b) RFA, c) RFA-BL and d) RFA-lip-GM-CSF. Synchronous untreated tumors collected at 7d were analyzed similarly.

Results: RFA-lip-GMCSF increased periablational M1, CTL and CD8+/FoxP3+ ratio at 3 and 7d, and activated CTLs 7d post-RFA (p<0.05). RFA-lip-GMSCF also increased M2, T-reg, and reduced CD4+/CD8+ 3 and 7d post-RFA respectively (p<0.05). In untreated index tumor, RFA-lip-GMCSF improved DCs, M1, CTLs and activated CTL 7d post-RFA (p<0.05). Furthermore, RFA-lip-GMSCF increased M2 at 3 and 7d, and T-reg 7d post-RFA (p<0.05). In synchronous tumors, RFA-BL and RFA-lip-GM-CSF improved DC, Th1 and CTL infiltration 7d post-RFA.

Conclusion: Systemic liposomal GM-CSF combined with RFA improves intratumoral immune cell trafficking, specifically populations initiating (DC, M1) and executing (CTL, FasL+) anti-tumor immunity. Moreover, liposomes influence synchronous untreated metastases increasing Th1, CTL and DCs infiltration.

Fig 1. Effect of liposomal GM-CSF and RFA on antigen presenting cells in R3230 rat model on 3 and 7 days post ablation in the periablational rim.

(A) At 3 days post treatment TAM (M2: CD163⁺ and M1: CD68⁺) infiltration in the tumor periablational in RFA-lip-GM-CSF group was higher than other groups, but not at 7 days. M1⁺/M2⁺ ratio in the tumor periablational rim (B) demonstrated no differences between groups. Immunohistochemistry representative images of infiltrating APCs in the periablational rim (C). *, p< 0.05, RFA-lip-GM-CSF vs all groups, **, p< 0.05, RFA-lip-GM-CSF vs all groups, except RFA-BL, ns, not significant.

Fig 2. Effect of liposomal GM-CSF and RFA on antigen presenting cells in R3230 rat model on 3 and 7 days post ablation in untreated index tumor.

RFA-lip-GM-CSF group expression of TAMs and DCs (A) was higher at 3 and 7 days post treatment, and 7 days only, respectively, when comparing RFA-lip-GM-CSF to all other groups. M1⁺/M2⁺ ratio (B) demonstrated no differences between groups. Immunohistochemistry representative images of infiltrating APCs in untreated index tumor (C). *, p< 0.05, RFA-lip-GM-CSF vs all groups, **, p< 0.05, RFA-lip-GM-CSF vs all groups, except RFA-BL, ns, not significant.

Fig 3. Effect of liposomal GM-CSF and RFA on tumor infiltrating lymphocytes in R3230 rat model on 3 and 7 days post ablation in the periablational rim.

In the periablational rim (A) TILs expression (CD4⁺: Th1, FoxP3⁺:Treg, CD8⁺:CTL and FasL⁺: Activated CTL) increased in RFA-lip-GM-CSF group compared to other groups at 7 days (7d), but not at 3 days (3d). CD4⁺/CD8⁺ ratio in the periablational rim (B) decreased in in RFA-lip-GM-CSF group compared to other groups at 7 days, but not at 3 days. For CD8⁺/FoxP3⁺ ratio, in the periablational rim (C) RFA-lip-GM-CSF group demonstrated a higher ratio than other groups at 7 days, but not at 3 days. Immunohistochemistry representative images of infiltrating TILs in the periablational rim (C). *, p< 0.05, RFA-lip-GM-CSF vs all groups, **, p< 0.05, RFA-lip-GM-CSF vs all groups, except RFA-BL, ˟˟, p< 0.05, RFA-lip-GM-CSF, RFA-BL, RFA alone vs. lip-GM-CSF alone, fr-GM-CSF and sham, ns, not significant.

Fig 4. Effect of liposomal GM-CSF and RFA on tumor infiltrating lymphocytes in R3230 rat model on 3 and 7 days post ablation in the untreated index tumor.

In the untreated index tumor (A), FoxP3⁺, CD8⁺, and FasL⁺ increased in RFA-lip-GM-CSF group compared to other groups at 7 days, but not at 3 days. CD4⁺/CD8⁺ ratio in all RFA treatment groups (RFA-lip-GM-CSF, RFA-BL, RFA alone) was lower than all non-RFA arms (lip-GM-CSF, fr-GM-CSF, and sham) at 7 days, but not at 3 days. For CD8⁺/FoxP3⁺ ratio, no differences in were noted in all groups at both 3 days and 7 days. Immunohistochemistry representative images of infiltrating TILs in the untreated index tumor (D). *, p< 0.05, RFA-lip-GM-CSF vs all groups, **, p< 0.05, RFA-lip-GM-CSF vs all groups, except RFA-BL, ˟˟, p< 0.05, RFA-lip-GM-CSF, RFA-BL, RFA alone vs. lip-GM-CSF alone, fr-GM-CSF and sham, ns, not significant.

Fig 5. Effect of RFA and lip-GM-CSF on APCs and TILs infiltration in synchronous metastasis 7 days post ablation.

(A) Expression of TAMs markers and DCs marker in a synchronous metastasis was examined 7 days post ablation of a primary tumor. CD11c⁺ expression, a DC marker, in RFA-lip-GM-CSF and RFA-BL group was higher than other groups, but CD163⁺ and CD68⁺ expression, markers for M2 and M1, showed no difference between groups. Expression of TILs markers in a synchronous metastasis (B) 7 days post ablation of a primary tumor demonstrated that CD4⁺ and CD8⁺ expression in RFA-lip-GM-CSF and RFA-BL group was higher than other groups, but FoxP3⁺ expression showed no difference between groups. All ratios, CD4⁺/CD8⁺, CD8⁺/FoxP3⁺, and M1⁺/M2⁺ did not demonstrate any differences between any of the treatment groups. Immunohistochemistry representative images of infiltrating APCs and TILs in synchronous metastasis. **, p< 0.05 RFA-lip-GM-CSF vs all groups, except RFA-BL, ns, not significant.