Abstract A83: Lentiviral shRNA-mediated knockdown of eosinophilic Galectin-10/Charcot-Leyden crystals: A novel approach to cancer immunotherapy
Authors: Clarke Christine A. , Lee Clarence M. , Laniyan Ibrahim , Furbert-Harris Paulette 
Published: October 2015

Abstract
Introduction: Despite the discovery of promising anti-cancer immunotherapeutic strategies such as cancer vaccines, cytokines, and T cell-based therapies, curative outcomes remain elusive. We have investigated the eosinophil as a potential anti-cancer effector cell, and have previously reported the ability of the eosinophils and their isolated granular proteins to inhibit prostate and breast cancer cell growth in vitro. In certain tumors, tumor-associated eosinophilia is marked by the deposition of a prominent eosinophil protein, galectin-10/Charcot-Leyden crystals. We have speculated that galectin-10, like other lectin counterparts, is a key player in the anti-cancer immune response. In a large number of studies, a galectin-cancer relationship has been established, and significant roles as tumor promoters or inhibitors have been delineated. Some tumors have also been shown to employ galectins in their tumor-immune evasion mechanisms. Additionally, siRNA-mediated knockdown studies have demonstrated galectin-10 expression in regulatory T cells (Treg)—cells that have been shown to be major players in regulating anti-cancer immune responses—and its mechanistic necessity in maintaining Treg anergy and suppressive function on CD4+ T lymphocytic proliferation. Despite having been documented at numerous tumor sites, the prognostic significance of galectin-10 in tumor resolution requires further investigation. The protein's role in Treg suppression of T lymphocytes, and the involvement of other galectins in the tumor immune response, however, has lent credence to its clinical significance at tumor mileu and, the likelihood that galectin-10, by modulating eosinophil-mediated effects on T-lymphocytes, might impact immunological defense against cancers. In the present study, we have elected to create a galectin-10 knockdown eosinophil sub-line by transfecting GRC.014.24 (an eosinophil cell line established in our laboratory), with shRNA lentiviral transduction particles and, thereafter, conduct further studies to examine eosinophilic galectin-10's potential to increase T- cell homing to tumors.
Experimental Procedure: Briefly, GRC.014.24 (2 x 104 cells/ml) were transfected with galectin-10-specific lentiviral transduction vectors. Puromycin was used to select stable transductants; PCR and immunofluoresence methods, to determine transduction efficiency.
Results: We show that a galectin-10-specific shRNA lentiviral particle effected 100% gene silencing; this eosinophil clone lacked both granular and cytoplasmic protein expression.
Conclusion: The creation of galectin-10 knockdown eosinophils provides a useful model for investigating eosinophilic galectin-10's ability to modulate T cell access and homing to tumors, and a putative role, similar to Treg galectin-10, in regulating tumoral T lymphocytic proliferation can certainly be envisioned. The consideration of galectin-10 knockdown eosinophils as a singular approach to cancer immunotherapy, or in combination with other anti-cancer therapies such as adoptive T cell therapies, or therapeutic cancer vaccines, is intriguing.
Note: This abstract was not presented at the conference.
Citation Format: Christine A. Clarke, Clarence M. Lee, Ibrahim Laniyan, Paulette Furbert-Harris. Lentiviral shRNA-mediated knockdown of eosinophilic Galectin-10/Charcot-Leyden crystals: A novel approach to cancer immunotherapy. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A83.