heparitin-sulfate and Carcinoma--Lewis-Lung

While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCre + mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCre + mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I - T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer.

Topics: Animals; Carcinoma, Lewis Lung; CD11c Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dendritic Cells; Heparitin Sulfate; Histocompatibility Antigens Class I; Humans; Immunity, Cellular; Loss of Function Mutation; Lymphocytes, Tumor-Infiltrating; Major Histocompatibility Complex; Mice; Polysaccharides; Proteoglycans; Sulfotransferases; T-Lymphocytes

Inflammation and cancer are related pathologies acting synergistically to promote tumor progression. In both, hematogenous metastasis and inflammation, P-selectin participates in interactions involving tumor cells, platelets, leukocytes and endothelium. Heparin has been shown to inhibit P-selectin and as a consequence it blunts metastasis and inflammation. Some heparin analogs obtained from marine invertebrates are P-selectin inhibitors and do not induce bleeding effects. The present work focuses on the P-selectin blocking activity of a unique heparan sulfate (HS) from the bivalve mollusk Nodipecten nodosus. Initially, we showed that the mollusk HS inhibited LS180 colon carcinoma cell adhesion to immobilized P-selectin in a dose-dependent manner. In addition, we demonstrated that this glycan attenuates leukocyte rolling on activated endothelium and inflammatory cell recruitment in thioglycollate-induced peritonitis in mice. Biochemical analysis indicated that the invertebrate glycan also inhibits heparanase, a key player in cell invasion and metastasis. Experimental metastasis of Lewis lung carcinoma cells was drastically attenuated by the mollusk HS through a mechanism involving inhibition of platelet-tumor-cell complex formation in blood vessels. These data suggest that the mollusk HS is a potential alternative to heparin for inhibiting P-selectin-mediated events such as metastasis and inflammatory cell recruitment.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Blood Platelets; Carcinoma, Lewis Lung; Cell Adhesion; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glucuronidase; Heparitin Sulfate; Humans; Inhibitory Concentration 50; Leukocyte Rolling; Lung Neoplasms; Mollusca; Neoplasm Transplantation; P-Selectin

Tumor-bearing (TB) patients and TB animal models show a wide array of immunologic deficits. Heparan sulfate (HS) has been shown to both improve immune cell proliferative responses and to induce Th1 cytokine responses in normal animals. These HS effects, if harnessed, would be of great benefit to TB patients. The present study focused on replicating previous HS-induced Th1 and proliferative response results as well as extrapolating the beneficial immunomodulatory effects to an experimental model derived from TB animals of Lewis lung cell carcinoma. Lewis Lung Carcinoma (LLC)-TB and control mouse splenocytes were assessed for proliferation and cytokine response to concanavalin A (Con A) with 1 and 3 days' exposure to HS. Our results found HS treatment stimulated splenocyte proliferation to Con A in control mice splenocytes after 1 and 3 days of treatment, although HS proliferative effects were not seen in unfractionated TB cultures. Furthermore, cytokine studies revealed normal splenocytes treated with HS had increased levels of both Th1 and Th2 cytokines. Surprisingly, HS treated TB-splenocytes showed suppressed cytokine levels. Of particular interest was the decreased levels of the Th2 cytokine IL-4 in TB-derived samples. In conclusion, we found that HS did show immune-modulator properties in both normal and TB environments. Our studies reinforced the possibility that HS could one day be used as an immune-modulating therapeutic agent.

Topics: Animals; Antigen Presentation; Carcinoma, Lewis Lung; Concanavalin A; Cytokines; Dendritic Cells; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Female; Heparitin Sulfate; Immunologic Factors; Interleukin-4; Macrophages; Mice; Mice, Inbred C57BL; Spleen; T-Lymphocytes; Th1 Cells; Th2 Cells

Heparin, a widely used anticoagulant, is known to have anti-metastatic activity, although the mechanism is not fully understood. In the present study, we investigated the mechanism of this anti-metastatic activity using periodate-oxidized and borohydride-reduced heparin with low anticoagulant activity (LAC heparin). The anticoagulant activity of LAC heparin is markedly reduced to almost the control level in terms of prothrombin time in vitro, and no hemorrhagic complication was observed with injection of LAC heparin into mice in vivo. LAC heparin injected intravenously with Lewis lung carcinoma cells or 10 min before tumor cell injection significantly inhibited, to the same extent as intact heparin and in a dose- and time-dependent manner, the lung colonization that develops after intravenous injection (i.v.) of tumor cells. Flow cytometric analysis revealed that Lewis lung carcinoma cells strongly express heparan sulfate on their surface. Both the LAC heparin and intact heparin inhibited the adhesion and invasion of tumor cells to Matrigel-coated dishes in vitro without significant effect on the tumor cell growth. LAC heparin also significantly diminished tumor cell retention in the lung after i.v. of LacZ gene-tagged Lewis lung carcinoma cells. These results suggest that LAC heparin may prevent tumor cells from attachment to the subendothelial matrix of lung capillaries by competitively inhibiting cell surface heparan sulfate functions and suppress lung colonization.

Topics: Animals; Anticoagulants; Borohydrides; Carcinoma, Lewis Lung; Cell Adhesion; Cell Division; Dose-Response Relationship, Drug; Flow Cytometry; Heparin; Heparitin Sulfate; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Periodic Acid; Survival Rate; Time Factors; Tumor Cells, Cultured