Ibrahim Laniyan

Swainsonine stimulates bone marrow cell proliferation and differentiation in different strains of inbred mice

The immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) has potential for overcoming the bone marrow suppressive effects of cancer chemotherapeutic drugs and radiation. The effect of swainsonine on bone marrow cellularity was evaluated in four different strains (C57BL/6; C3H-HEN; Balb/C and DBA-2 mice) of inbred mice subjected to multiple doses of the alkaloid. Swainsonine treatment stimulated bone marrow cell proliferation in all strains of mice. Examination of the peripheral blood did not reveal any increase in total leukocyte count. In vitro assessment of total colony-forming unit (CFU) capacity of bone marrow cells showed a two- to eight-fold increase in swainsonine treated mice of different strains compared to their corresponding controls given sham injections of physiological saline. Swainsonine induced increase in CFU capacity of bone marrow cells should find clinical application in cancer treatment with chemotherapeutic agents and radiation.

Limits of stimulation of proliferation and differentiation of bone marrow cells of mice treated with swainsonine

The limits of stimulation of the immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) were studied in inbred C57BL/6 mice for potential support of intense high dose cancer chemotherapy and/or radiation because of its attractive pharmacologic profile on the hematopoietic system. Specifically, the effects of swainsonine on bone marrow cellularity and on in vitro progenitor cell proliferation to total colony forming units (CFU) and differentiation to different lineages were studied as a function of number of days post drug administration. The lineages evaluated were colony forming units-granulocyte-macrophage (CFU-GM), erythroid-burst forming units (BFU-e) and CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM or CFU-Mix). Groups of mice were treated with swainsonine or plain vehicle, phosphate buffered saline for 10 consecutive days. The effects of these agents on the hematopoietic system were studied up to 60 days following their discontinuation. The magnitude of the effects of swainsonine on bone marrow system gradually declined with increasing duration of days following its discontinuation. Nevertheless, its residual stimulatory effects on bone marrow cellularity, total CFU, CFU-GM, BFU-e and CFU-Mix continued to be significant (P<0.0001) up to 45, 50, 50, 55 and 50 days, respectively, compared to those of diluent buffer or untreated controls. Since cancer chemotherapeutic agents or radiation are normally given in schedules and/or cycles, these results strongly suggest that swainsonine effects are sustained long enough to potentially support and facilitate hematopoietic recovery during anti-cancer cytotoxic treatment.

Enhanced proliferation of functionally competent bone marrow cells in different strains of mice treated with swainsonine.

The immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) has potential for overcoming the bone marrow suppressive effects of cancer chemotherapy and radiotherapy. An earlier study showed that multiple doses of swainsonine enhanced bone marrow cellularity in four different strains (C57BL/6; C3H-HEN; Balb/C and DBA-2 mice) of inbred mice which were not exposed to any chemotherapeutic agents or radiation. In vitro assessment of total colony formation capacity of bone marrow cells (BM CFUs) showed a 2- to 8-fold increase in swainsonine-treated mice compared to control mice that were given sham injections of physiological saline. In the current study, we have evaluated the functional competence of the bone marrow cells produced in response to swainsonine treatment of normal healthy mice. In particular, colony forming units-granulocyte-macrophage (CFU-GM), erythroid-burst forming units (BFUe) and CFU-Mix (or CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM)) levels, were determined using in vitro assays. The time course of the changes in CFU-GM, BFUe and CFU-Mix (CFU-GEMM) were also followed. Our results demonstrate that swainsonine bolsters the CFU capacity of BM cells without loss of function to levels which are several folds higher than in sham-treated control mice. Swainsonine treatment caused an increase in all lineages of marrow cells without loss of function. This effect was reproduced in all four strains of inbred mice in this investigation. Examination of the peripheral blood did not reveal increase in white blood cells or changes in the hematocrit levels. The long-term effects of swainsonine treatment are not known at present. Nonetheless, swainsonine-induced increase in CFU capacity of bone marrow cells and related cells along the different differentiation paths should find clinical application in cancer treatment with chemotherapeutic agents and/or radiation.

Abstract A83: Lentiviral shRNA-mediated knockdown of eosinophilic Galectin-10/Charcot-Leyden crystals: A novel approach to cancer immunotherapy

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 protein9s 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-109s 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-109s 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.