Galectin-1: a link between tumor hypoxia and tumor immune privilege

Abstract

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression.

Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CD3 (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes.

Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CD3 staining (P = .01). Expression of galectin-1 and CD3 were significant predictors for overall survival on multivariate analysis.

Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege.

Fig 1.

(A) Representative surface-enhanced laser desorption ionization time-of-flight mass spectra of concentrated FaDu culture media after 6, 12, and 24 hours (hrs) of either normoxia (N) or hypoxia (H). The mass/charge values are along the x-axis, and relative intensity is along the y-axis. The arrow indicates a 15-kDa protein peak that is increased under hypoxia compared with normoxia. (B) Immunoblots of FaDu concentrated culture media and cell lysates showing increased galectin-1 expression after of hypoxia treatment (6, 12, and 24 hrs for the media and 24 hrs for the lysates) relative to normoxia. (C) Quantitative real-time polymerase chain reaction results showing a two-fold induction of galectin-1 mRNA after 24 hrs of hypoxia treatment (< 0.01% O₂). Experiments were performed in triplicates. (D) Enzyme-linked immunoabsorbent assay results showing increased galectin-1 protein in the culture media of several different cell lines by hypoxia treatment (24 hrs of < 0.01% O₂).

Fig 2.

(A) Increased plasma levels of galectin-1 in severe combined immunodeficiency–mouse xenografts after 48 to 72 hours of hypoxia (H; 10% O₂) compared with normoxia (N; 21% O₂). Each group is represented by pooled data from three to four mice. Plasma galectin-1 levels (expressed in mg/mL) were corrected for tumor volume (expressed in mL). Data are expressed as the mean galectin-1 level ± standard deviation (P =.15 for entire group; P = .06 for N versus H72; analysis of variance analysis [ANOVA]). (B) Increased urinary β-hCG levels in the same mice after 48 to 72 hours of hypoxia compared with normoxia. Urinary β-hCG was corrected for urinary creatinine and tumor volume (ANOVA P = .64).

Fig 3.

Comparison between galectin-1 and CD3 staining in consecutive sections of the same tumor. Note the inverse relationship between the two markers. Tumors that stained strongly for galectin-1 (A, solid arrow) had minimal CD3 staining (B), and tumors that stained minimally for galectin-1 (C) had strong CD3 staining (D, clear arrow).

Fig 4.

Kaplan-Meier estimates of (A-C) freedom from relapse by galectin-1, CD3 and CA IX tumor staining (D-F) Cancer-specific survival by galectin-1, CD3 and CA IX tumor staining and (G-I) Overall survival by galectin-1, CD3 and CA IX tumor staining in 101 head and neck squamous cell carcinoma patients included in the tissue microarray.

Fig 5.

Kaplan-Meier estimates of (A) freedom from relapse by galectin-1 and CD3 grouping; (B) cancer-specific survival by galectin-1 and CD3 grouping; (C) overall survival by galectin-1 and CD3 grouping. The favorable group has negative to weak (neg-weak) galectin-1 and strong CD3 staining; intermediate group has either strong both galectin-1 and CD3 staining or neg-weak both galectin-1 and CD3 staining; unfavorable group has strong galectin-1 and neg-weak CD3 staining.