Topical Calcipotriol Plus Imiquimod Immunotherapy for Nonkeratinocyte Skin Cancers

Abstract

Nonkeratinocyte cutaneous malignancies, including breast cancer cutaneous metastasis and melanoma in situ, are often poor surgical candidates. Imiquimod (IMQ), a toll-like receptor 7 agonist that activates innate immunity in the skin, is used to treat these cutaneous malignancies. However, IMQ's modest effect on the activation of adaptive immunity limits its efficacy as a monotherapy. In this study, we demonstrate that topical TSLP cytokine inducers-calcipotriol and retinoic acid-synergize with IMQ to activate CD4⁺ T-cell immunity against nonkeratinocyte cutaneous malignancies. Topical calcipotriol plus IMQ treatment reduced breast tumor growth compared with calcipotriol or IMQ alone (P < 0.0001). Calcipotriol plus IMQ-mediated tumor suppression was associated with significant infiltration of CD4⁺ effector T cells in the tumor microenvironment. Notably, topical calcipotriol plus IMQ immunotherapy enabled immune checkpoint blockade therapy to effectively control immunologically cold breast tumors, which was associated with induction of CD4⁺ T-cell immunity. Topical treatment with calcipotriol plus IMQ and retinoic acid plus IMQ also blocked subcutaneous melanoma growth. These findings highlight the synergistic effect of topical TSLP induction in combination with innate immune cell activation as an effective immunotherapy for malignancies affecting the skin.

Figure 1

Calcipotriol plus imiquimod blocks breast tumor growth. (a) Schematic diagram of the experimental protocol used to determine the efficacy of calcipotriol plus imiquimod as a topical immunotherapy for breast cancer. PyMt^tg primary breast cancer cells were implanted subcutaneously into the inguinal region of WT C57BL/6 mice. When tumors reached 5 mm in diameter (day 10), the following topical treatments were applied to the tumor sites three times every 3 days: (a) 80 nmol calcipotriol in 20 μl of 100% EtOH followed by 5% imiquimod cream, (b) 80 nmol calcipotriol in 20 μl of 100% EtOH followed by moisturizing (control) cream, (c) 20 μl 100% EtOH followed by 5% imiquimod cream, and (d) 20 μl 100% EtOH followed by moisturizing (control) cream. (b) Serum TSLP level of WT C57BL/6 mice 24 hours after the last topical treatments listed earlier (n = 6 per group, one-way ANOVA with Dunn’s multiple comparisons test). (c) Representative macroscopic images of breast tumors in each treatment group at the endpoint (bar = 1 cm). (d) Tumor volume measurements over time in each treatment group. Purple arrows mark the treatment time points (n = 5 per group, two-way ANOVA with Sidak’s multiple comparison test). Graphs show mean + SD; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.0001. Experimental data were verified in two independent experiments. EtOH, ethanol; ns, not significant; WT, wild-type.

Figure 2

Calcipotriol plus imiquimod induces T-cell immunity in the breast tumor microenvironment. (a) Representative immunofluorescence images of CD3⁺, CD4⁺ T cells, and FOXP3⁺ Tregs in calcipotriol plus imiquimod cream (n = 7), calcipotriol plus moisturizing (control) cream (n = 7), EtOH plus imiquimod cream (n = 8), and EtOH plus moisturizing (control) cream (n = 5) (upper row) and their magnified images (lower row). White arrows point to CD4⁺ CD3⁺ T cells, and yellow arrows point to FOXP3⁺ CD4⁺CD3⁺ Tregs (bars = 100 μm). (b–e) Quantification of (b) CD3⁺ T cells, (c) CD4⁺ CD3⁺ T cells, (d) FOXP3⁻ CD4⁺ CD3⁺ effector T cells, and (e) FOXP3⁺CD4⁺CD3⁺ Tregs in the breast tumors in each treatment group. Cells were counted in 10 HPF images per tumor. Each dot represents cell counts from an HPF image. Experimental data were verified in two independent experiments. Graphs show mean + SD; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.0001; one-way ANOVA with Dunn’s multiple comparisons test was performed. HPF, high power field; ns, not significant; Treg, regulatory T cell.

Figure 3

Topical calcipotriol and imiquimod immunotherapy synergize with ICB therapy to suppress breast tumor growth. (a) Schematic diagram of the experimental protocol used to determine the efficacy of calcipotriol plus imiquimod and ICB as a combination immunotherapy for breast cancer. PyMt^tg primary breast cancer cells were implanted subcutaneously into the inguinal region of WT C57BL/6 mice. When tumors reached 5 mm in diameter (day 10), the following treatments were applied to the tumor sites three times every 3 days: (a) 80 nmol of calcipotriol in 20 μl of 100% EtOH followed by topical application of imiquimod cream and 250 μg anti–PD-1 antibody in 200 μl PBS IP injection (n = 6), (b) 80 nmol of calcipotriol in 20 μl of 100% EtOH followed by topical application of imiquimod cream and 250 μg IgG isotype in 200 μL PBS IP injection (n = 7), (c) 20 μL 100% EtOH followed by moisturizing cream and 250 μg anti-PD1 Ab in 200 μl PBS IP injection (n = 9), and (d) 20 μl 100% EtOH followed by control cream and 250 μg IgG isotype in 200 μl PBS IP injection (n = 9). (b) Representative macroscopic images of breast tumors in each treatment group at the endpoint (bar = 1 cm). (c) Tumor volume measurements over time in each treatment group. Purple arrows mark the treatment time points (n = 10 per group; two-way ANOVA with Sidak’s multiple comparison test). (d) The endpoint tumor weights in each treatment group (n = 10 per group; one-way ANOVA with Dunn’s multiple comparisons test). EtOH, ethanol; ICB, immune checkpoint blockade; IP, intraperitoneal; ns, not significant; WT, wild-type.

Figure 4

The combination of ICB therapy with topical calcipotriol plus imiquimod increases the effector T cells in breast tumors. (a) Representative immunofluorescence images of CD4⁺ CD3⁺ T cells in ICB plus calcipotriol plus imiquimod cream (n = 6), IgG plus calcipotriol plus imiquimod cream (n = 7), ICB plus control cream (n = 9), and EtOH plus control cream (n = 9). Red arrows point to CD3⁺ T cells, and yellow arrows point to CD4⁺CD3⁺ T cells (bars = 100 μm). (b–e) Quantification of (b) CD3⁺ T cells, (c) CD4⁺ CD3⁺ T cells, (d) FOXP3⁻ CD4⁺ CD3⁺ effector T cells, and (e) FOXP3⁺CD4⁺CD3⁺ Tregs in the breast tumors in each treatment group. Cells were counted in 9–12 HPF images per tumor. Each dot represents cell counts from an HPF image (one-way ANOVA with Dunn’s multiple comparisons test). Graphs show mean + SD; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.0001. EtOH, ethanol; HPF, high power field; ICB, immune checkpoint blockade; ns, not significant; Treg, regulatory T cell.

Figure 5

Calcipotriol plus imiquimod and retinoic acid plus imiquimod block melanoma growth. (a) Schematic diagram of the experimental protocol used to determine TSLP induction after topical retinoic acid treatment in WT C57BL/6 mice. A total of 20 nmol retinoic acid in 20 μl of 100% EtOH (test) versus 20 μl of 100% EtOH (carrier control) was applied on the back skin of mice three times at 3 days apart. Blood was collected 24 hours after the last topical treatment. (b) Serum TSLP levels in WT mice after treatment with topical retinoic acid versus EtOH control (n = 5 per group; Mann–Whitney U test). (c) Schematic diagram of the experimental protocol used to determine the efficacy of calcipotriol plus imiquimod and retinoic acid plus imiquimod immunotherapy for melanoma. B16-F10 melanoma cells were implanted subcutaneously into the flanks of WT C57BL/6 mice. When tumors become palpable (day 5), the following treatments were applied three times at 3 days apart: 20 nmol calcipotriol in 20 μl of 100% EtOH followed by 5% imiquimod cream, 20 nmol retinoic acid in 20 μl of 100% EtOH followed by 5% imiquimod cream, 20 nmol calcipotriol in 20 μl 100% EtOH followed by moisturizing (control) cream, 20 nmol retinoic acid in 20 μl 100% EtOH followed by control cream, 20 μl 100% EtOH followed by 5% imiquimod cream, and 20 μl 100% EtOH followed by control cream. (d) Representative macroscopic images of melanoma tumors in each treatment group at the endpoint (bar = 1 cm). (e) Tumor volume measurements over time in each treatment group. Purple arrows mark the treatment time points. n = 5 per group; two-way ANOVA with Sidak’s multiple comparison test was performed. P-values are in comparison with EtOH plus control cream group. EtOH, ethanol; WT, wild-type.

Figure 6

Calcipotriol plus imiquimod and retinoic acid plus imiquimod induce T-cell immunity in the melanoma microenvironment. (a) Representative immunofluorescence images of CD4⁺ CD3⁺ T cells in the following treatment groups: calcipotriol plus imiquimod cream (n = 6), retinoic acid plus imiquimod cream (n = 6), calcipotriol plus moisturizing (control) cream (n = 6), retinoic acid plus control cream (n = 5), EtOH plus imiquimod cream (n = 5), and EtOH plus control cream (n = 6). Red arrows point to CD3⁺ T cells, and yellow arrows point to CD4⁺CD3⁺ T cells. (b–e) Quantification of (b) CD3⁺ T cells and (c) CD4⁺ CD3⁺ T cells, (d) FOXP3⁻ CD4⁺ CD3⁺ effector T cells, and (e) FOXP3⁺CD4⁺CD3⁺ Tregs in CD3/CD4/Foxp3-stained melanoma sections in each treatment group. Each dot represents cell counts from an HPF image (one-way ANOVA with Dunn’s multiple comparisons test, bar = 200 μm). Graphs show mean + SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.0001. HPF, high power field; ns, not significant; Treg, regulatory T cell.

Figure 7

Schematic diagram demonstrating the synergistic effect of ICB with topical calcipotriol plus imiquimod in activating adaptive immunity against malignancies affecting the skin. The combination of ICB with topical calcipotriol plus imiquimod leads to a synergistic effect to induce CD4⁺ T-cell immunity because these T cells directly interact with antigen-presenting cells in the tumor microenvironment. ICB, immune checkpoint blockade.