Galectin-1 mediates radiation-related lymphopenia and attenuates NSCLC radiation response

Abstract

Purpose: Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities.

Experimental design: Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non-small cell lung cancer (NSCLC)] and head and neck cancer patients.

Results: LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8(+) T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients.

Conclusions: Radiotherapy-related systemic lymphopenia appeared to be mediated by radiotherapy-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis.

©2014 American Association for Cancer Research.

Figures

Figure 1

Gal-1 is induced by radiation and mediates T cell apoptosis in vitro. (A) Immunoblots showing intracellular and secreted Gal-1 in LLC-1 and LKR13 mouse NSCLC cell lines at the indicated time points before and after exposure to ionizing radiation (6 Gy). Means ± SE are shown for each condition performed in triplicate. (B) Apoptosis of lymphocytes after treatment with recombinant Gal-1. The percentage of TUNEL-FITC positive, apoptotic lymphocytes after treatment with 500ng/ml, 5ug/ml and 10ug/ml recombinant Gal-1 for 48 hours is shown. (C) The percentage of apoptotic T lymphocytes after culturing for 48 h in LLC-1 scramble conditioned medium in RPMI (1:2 ratio) (~1.2ng/ml Gal-1) with IgG (10ug/ml), anti-Gal-1 antibody (10ug/ml) or TDG (100mM). Representative histograms at left. Means ± SE are graphed (N=3). Experiments were performed at least twice with similar results, *P<0.05.

Figure 2

Tumor secreted Gal-1 facilitates radiation-related T lymphopenia. Four different host/tumor mouse groups, WT/Scr, WT/shGal-1, Gal-1−/−/ Scr and Gal-1−/−/ shGal-1 and two different time points (before radiation (Pre IR) and two weeks after radiation (Post IR)) were used in experiments presented in Figures 3A-D. (A) ELISA quantification of plasma Gal-1 for each host/tumor group before and after RT (N= 5 to 6 mice per group). (B) The percentage of circulating total mature T cells (CD3+), (C) T helper cells (CD3+ CD4+) and (D) cytotoxic T cells (CD3+ CD8+) for each group before and after RT (N= 6 to 7 mice per group). (E) Terminal circulating CD3+, CD4+ and CD8+ T cells in WT/Scr mice with or without tumor irradiation, showing that RT is important in causing a decrease in T lymphocytes (N=5 mice per group). (F) Terminal plasma Gal-1 ELISA from the mice in (E). Inhibition of Gal-1 by Thiodigalactoside (TDG) attenuates radiation-related T lymphopenia. (G) Percentage of CD3+, CD3+CD4+ and CD3+CD8+ circulating T cells in mice treated with intratumoral TDG (120mg/kg) or PBS vehicle injections before and after 20Gy tumor radiation is graphed (N= 5 to 6 mice per group). Data represent the means ± SE, *P<0.05.

Figure 3

Gal-1 mediates tumor radiation response in vitro and in vivo and promotes lung metastases. (A)Tumor growth curves for Gal-1 WT C57Bl/6 mice implanted with scramble (Scr) or Gal-1 knockdown LLC-1 tumor cells (shGal-1) with and without radiation. 20Gy of ionizing radiation was delivered in a single fraction (bolt). Tumor volumes were normalized to size on day of radiation (N=7 to 10 mice per group, P= 0.02). (B) Spontaneous metastases quantified from hematoxylin and eosin stained lungs from mice in (A) (P<0.05). Representative light microscopy images at right (40x objective). (C) Tumor growth curves for Gal-1 WT mice implanted with scramble LLC-1 and treated with intratumoral TDG or PBS injection on the days indicated. Irradiated mice receiving PBS (PBS IR) and TDG (TDG IR) received a single dose of 20Gy ionizing radiation at the indicated time (bolt). Tumor volumes normalized to size on day of radiation (N= 5 mice per group, P= 0.05). (D) Lung metastases of mice from (C) is shown (P = 0.037). Data represent the means ± SE.

Figure 4

Gal-1 induces intratumoral T cell apoptosis and attenuates T cell infiltration and microvessel formation. (A) CD4+ T cell infiltration of scr and shGal-1 tumors, with or without radiation, quantified as counts per field of view (10 random fields at 200x magnification, scale bar=100um, n=5 tumors per group). (B) CD8+ T cell infiltration of scr and shGal-1 tumors, with or without radiation, quantified as counts per field of view (10 random fields at 200x magnification, scale bar=100um, n=5 tumors per group, P<0.05). (C and D) Percentage of TUNEL positive CD4+ and CD8+ T cells within Scr and shGal-1 tumors, with or without radiation, is graphed and representative images are at right (scale bar=50um, n=5 tumors per group, P=0.01. (E) Microvessel density counts of CD31 stained tumors of the indicated Gal-1 status/treatment combinations (N=5 tumors per group, P<0.05). (F) Microvessel density counts of CD31 stained tumors collected from PBS and TDG treated mice, with and without radiation (N=5 mice per group, P<0.05). Representative images at right. Data represent means ± SE.

Figure 5

Relationship between survival and the degree of radiation-mediated drop in lymphocytes counts in 20 patients with early stage NSCLC treated with SABR. Mean (± 95% CI) pre-radiation and post SABR absolute lymphocyte counts is graphed for the entire group (P=0.025). Survival curves based on Cox regression of (B) Overall (P=0.037) and (C) Disease Free (P=0.033) Survival, stratified by the maximum drop in absolute lymphocyte counts before and after radiotherapy. Patients were split by quartiles. The overall survival HR of 1.148 and Disease Free Survival HR of 1.134 indicate that at any specific time t, a patient with a 0.1 unit increase in the maximum drop in absolute lymphocyte count before and after RT will have an increased risk of 14.8% of death or 13.4% of experiencing progression or death. (D) In a separate group of NSCLC patients, plasma Gal-1 pre and post-SABR is graphed. (E) HNC plasma Gal-1 increase after treatment (RT combined with cisplatin or cetuximab) is graphed (N=24) (P=0.05). (F) Decrease in absolute lymphocytes after treatment in HNC is shown (N=24). Mean (±95% CI) is shown (P<0.001).

Figure 6

Proposed mechanism for RT-induced Gal-1 secretion as a potential cause of the poor prognosis linked to RT-related lymphopenia. Radiation-related lymphopenia is associated with worse outcomes in SABR treated, early stage NSCLC patients. This may be due to tumor associated Gal-1 upregulation by radiation, leading to lower circulating T cells, intratumoral T cell viability and microvessel formation, which facilitate tumor growth.