Galectin‐1 is implicated in making tumor cells immune privileged, in part by regulating the survival of infiltrating T cells. Galectin‐1 is strongly expressed in activated pancreatic stellate cells (PSCs); however, whether this is linked to tumor cell immune escape in pancreatic cancer is unknown. Galectin‐1 was knocked down in PSCs isolated from pancreatic tissues using small interfering RNA (siRNA), or overexpressed using recombinant lentiviruses, and the PSCs were cocultured with T cells. CD3⁺, CD4⁺ and CD8⁺ T cell apoptosis was detected by flow cytometry; T cell IL‐2, IL‐4, IL‐5 and INF‐γ production levels were quantified using ELISA. Immunohistochemical analysis showed increased numbers of PSCs expressed Galectin‐1 (p < 0.01) and pancreatic cancers had increased CD3⁺ T cell densities (p < 0.01) compared to normal pancreas or chronic pancreatitis samples. In coculture experiments, PSCs that overexpressed Galectin‐1 induced apoptosis of CD4⁺ T cells (p < 0.01) and CD8⁺ T cells (p < 0.05) significantly, compared to normal PSCs. Knockdown of Galectin‐1 in PSCs increased CD4⁺ T cell (p < 0.01) and CD8⁺ T cell viability (p < 0.05). Supernatants from T cells cocultured with PSCs that overexpressed Galectin‐1 contained significantly increased levels of Th2 cytokines (IL‐4 and IL‐5, p < 0.01) and decreased Th1 cytokines (IL‐2 and INF‐γ, p < 0.01). However, the knockdown of PSC Galectin‐1 had the opposite effect on Th1 and Th2 cytokine secretion. Our study suggests that the overexpression of Galectin‐1 in PSCs induced T cell apoptosis and Th2 cytokine secretion, which may regulate PSC‐dependent immunoprivilege in the pancreatic cancer microenvironment. Galectin‐1 may provide a novel candidate target for pancreatic cancer immunotherapy.

Leptomeningeal carcinomatosis (LC) is the third most common metastatic complication of the central nervous system. However, the current modalities to reliably diagnose this condition are not satisfactory. Here, we report a preclinical proof of concept for a metabolomics-based diagnostic strategy using a rat LC model incorporating glioma cells that stably express green fluorescent protein. Cytologic diagnoses gave 66.7% sensitivity for the 7-day LC group and 0% for the 3-day LC group. MR imaging could not diagnose LC at these stages. In contrast, nuclear magnetic resonance–based metabolomics on cerebrospinal fluid detected marked differences between the normal and LC groups. Predictions based on the multivariate model provided sensitivity, specificity, and overall accuracy of 88% to 89% in both groups for LC diagnosis. Further statistical analyses identified lactate, acetate, and creatine as specific for the 7-day LC group, with glucose a specific marker of the normal group. Overall, we showed that the metabolomics approach provided both earlier and more accurate diagnostic results than cytology and MR imaging in current use. Cancer Res; 72(20); 5179–87. ©2012 AACR.

Metabolomic isotopic tracing can provide flux information useful for understanding drug mechanisms. For that, NMR has the unique advantage of giving positional isotope enrichment information, but the current ¹³C 1D NMR approach suffers from low sensitivity and high overlaps. We developed a new 2D heteronuclear NMR experiment incorporating J-scaling and distortion-free elements that allows for quantitative analysis of multiplets with high sensitivity and resolution. When applied to an old chemotherapeutic drug, the approach provided a quantitative estimation of TCA-cycle turns, confirming the conventional mechanism of its mitochondrial metabolic enhancement. Additionally, the approach identified a new mechanism of the higher contribution of the pentose phosphate pathway to serine synthesis in the cytosolic compartment, possibly explaining the broad pharmacological activities of the drug. Our approach may prove beneficial in helping to find new usages or metabolic mechanisms of other drugs.