Novel ocular immunotherapy induces tumor regression in an equine model of ocular surface squamous neoplasia

Abstract

Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of a liposomal TLR complex (LTC) immunotherapy, which previously demonstrated strong antiviral activity in multiple animal models following mucosal application, for ocular antitumor activity in a horse spontaneous OSSN model. In vitro studies demonstrated strong activation of interferon responses in horse leukocytes by LTC and suppression of OSSN cell growth and migration. In a trial of 8 horses (9 eyes), treatment with topical or perilesional LTC resulted in an overall tumor response rate of 67%, including durable regression of large OSSN tumors. Repeated treatment with LTC ocular immunotherapy was also very well tolerated clinically. We conclude therefore that ocular immunotherapy with LTC warrants further investigation as a novel approach to management of OSSN in humans.

Keywords: Horse; Immunotherapy; Ocular surface squamous neoplasia.

Fig. 1

Tumor responses to LTC treatment. Pre- and post-treatment photographs of OSSN effected eye in responder patients—patient 2, patient 4 and patients 5 (OS), patients 5 (OD), patients 7 and 8 from top to bottom. Right column shows tumor area calculations, hand drawn on 3 photographs per visit and calculated using ImageJ as described in methods. A Tumor area in patient 2 from visit 1 to 7, each visit approximately 2 weeks apart. B tumor area calculations from patient 4 from visit 1 to 5. C Tumor area calculations from patient 5 from visit 1 to 6. D Tumor area in patient 5 (OD) from group 1 from visit 4 to 6. Horse 5 developed tumor in right eye after 4 visits. E tumor area calculations from patient 7 from visit 1 to 7. F Tumor area calculations from patient 8 from visit 1 to 3

Fig. 2

LTC uptake by corneal epithelial cells. A corneal explant tissue obtained from a normal horse eye was incubated with fluorescently labeled LTC, as described in Methods. A Explant without the addition of labeled LTC, stained with DAPI (blue) to visualize nuclei. B Explant incubated with labeled (green) LTC for 30 min

Fig. 3

Activation of innate immune responses in blood leukocytes by LTC. PBMC cultures prepared from blood of normal horses were stimulated with LTC in vitro, as described in Methods, and cytokine production assessed by qRT-PCR or by ELISA. In 24 h CM from LTC stimulated cells, there was a significant decrease in concentrations of GM-CSF (A), IL-12 (B) and significant increase of IFN-γ (C). The impact of LTC activation of PBMC on expression of key antitumor cytokines was also assessed by qRT-PCR IFN-a (D) and IFN-b (E), and TNFa (F). Statistical comparisons between two treatment groups were made using nonparametric t tests (Mann–Whitney test). Statistical significance was noted for p < 0.05

Fig. 4

LTC activation of OSSN tumor innate immune responses. Equine OSSN cells were incubated with labeled LTC to assess cell uptake of the complexes, and the impact of LTC stimulation on direct tumor innate immune activation was assessed as well, as described in Methods. A Uptake of labeled LTC visualized in OSSN cells. B, C Assessment of immune activation of OSSN cells by RT PCR. B Increase in expression of IFNa transcript following treatment with 25 uL/mL LTC. (3C) Increase in expression of IFNb transcript. D LTC concentration dependent Increase in secretion of IL-8 and CXCL1 by LTC activated OSSN cells evaluated by cytokine multiplex ELISA. Statistical comparison was done using one‐way ANOVA, followed by Tukey multiple means post‐test. Statistical significance was assigned for p < 0.05

Fig. 5

Suppression of OSSN tumor cell growth and migration by cytokines released from LTC activated PBMC. Indirect effects of leukocyte cytokines on OSSN tumor growth were assessed by incubating tumor cells with cytokines released from LTC activated PBMC 24-h metabolic activity assay, using EQOSSNLC653 (A), and EQOSSNLC914 (C) cell line treated with n = 3 PBMC conditioned medium (CM), from either LTC activated or resting PBMC cultures. B, D Assessment of migration and proliferation using Incucyte scratch assay as described in Methods. Blue line depicts migration of EQOSSNLC653 cell line + untreated PBMC supernatants across the scratch area, red depicts the migration of EQOSSNLC653 cell line + PBMC supernatants treated with 25uL/mL of LTC. 4D Migration of EQOSSNLC914 incubated with PBMC-CM. Comparisons between more than 3 treatment groups were completed using one‐way ANOVA, followed by Tukey multiple means post‐test. Statistical significance was assigned at p < 0.05

Fig. 6

Cytokine concentrations in tears from healthy horses and horses with OSSN. The immunological effects of the presence of OSSN lesions on local cytokine concentrations in tears were assessed using tear samples and a cytokine multiplexing assay, as described in Methods. Tears were collected from n = 10 healthy horses and n = 6 horses with OSSN (collected from the affected eye) and analyzed by multiplex kit. Significant increases in several cytokines were noted in tears from horses with OSSN, whereas no decreases in cytokine concentrations were observed, when healthy horses were compared to horses with OSSN. Cytokine concentrations are depicted in mean fluorescent intensity (MFI) units. A IFN-γ, B fractalkine, C GRO D IL-6 E IL-8, F IL-10, G IL-12, H IL-18, I IL-1a, J IL-1b