Silibinin inhibits accumulation of myeloid‐derived suppressor cells and tumor growth of murine breast cancer

Abstract

Myeloid-derived suppressor cells (MDSC)s increase in blood and accumulate in the tumor microenvironment of tumor-bearing animals, contributing to immune suppression in cancer. Silibinin, a natural flavonoid from the seeds of milk thistle, has been developed as an anti-inflammatory agent and supportive care agent to reduce the toxicity of cancer chemotherapy. The goals of this study were to evaluate the effect of silibinin on MDSCs in tumor-bearing mice and antitumor activity of silibinin in a mouse model of breast cancer. 4T1 luciferase-transfected mammary carcinoma cells were injected into in the mammary fat pad female BALB/c mice, and female CB17-Prkdc Scid/J mice. Silibinin treatment started on day 4 or day 14 after tumor inoculation continued every other day. Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Flow cytometry measured total leukocytes, CD11b+ Gr-1+ MDSC, and T cells in the blood and tumors of tumor-bearing mice. The effects of silibinin on 4T1 cell viability in vitro were measured by BLI. Treatment with silibinin increased overall survival in mice harboring tumors derived from the 4T1-luciferase breast cancer cell line, and reduced tumor volumes and numbers of CD11b+Gr-1+ MDSCs in the blood and tumor, and increased the content of T cells in the tumor microenvironment. Silibinin failed to inhibit tumor growth in immunocompromised severe combined immunodeficiency mice, supporting the hypothesis that anticancer effect of silibinin is immune-mediated. The antitumor activity of silibinin requires an intact host immune system and is associated with decreased accumulation of blood and tumor-associated MDSCs.

Poly (I: C) modulates the immunosuppressive activity of myeloid-derived suppressor cells in a murine model of breast cancer

“…4T1-Luc cells were injected s.c into the 1st left mammary fat pad of BALB/c mice as previously described [16]. Mice were treated with Poly (I: C) or PBS on Days 7 and 10 after tumor cell inoculation, as described in ''Materials and methods'' section.…”

“…The samples were analyzed using a FACS Aria instrument (BD Biosciences, Becton, Dickinson, San Jose, CA), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR). MDSC subunits were counted using the scatter pattern and CD11b positive cells gated for Ly6G and Ly6C for granulocytic MDSCs and monocytic MDSCs and using a high side scatter and F4/80 positive gate for macrophages [16]. For intracellular staining, cells were stained first for surface expression of lineage/differentiation markers (CD80, CD86, SCA-1, I-Ad), then fixed and permeabilized by Cyto Fix/Perm Kit (BD Pharmingen).…”

“…Synthetic firefly D-luciferin potassium salt stock solutions (15 mg/ml in PBS) were prepared and injected s.c into anesthetized mice as described [16]. Imaging was performed at two time points in anesthetized mice using an IVIS 100 charge-coupled device imaging system (Xenogen, Alameda, CA, USA).…”

“…Blood samples were drawn from the tail vein and cheek vein according to IACUC guidelines. Peripheral blood was counted using a Coulter AcT diff Analyzer (Beckman) and analyzed by flow cytometry using lyse no-wash method to maximize the numbers of evaluable leukocytes as we have previously described [16].…”

“…The 4T1 mammary carcinoma cell line was maintained as described before [16]. To establish subcutaneous (s.c.) tumors, 1 9 10 6 cells viable 4T1-Luc cells were injected into 1st left mammary fat pad of female BALB/c mice.…”