g(m1)-ganglioside and Carcinoma--Lewis-Lung

Tumor-induced immune suppression involves the accumulation of suppressive infiltrates in the tumor microenvironment such as regulatory T-cells (Tregs). Previous studies demonstrated that NK-dependant increases in CCL22 secretion selectively recruit Tregs toward murine lungs bearing Lewis Lung Carcinoma (LLC). To extend the in vitro studies, the present studies utilized in vivo depletion of NK cells to ascertain the contribution of NK-derived CCL22 toward total CCL22 and subsequent Treg levels in both normal and LLC-bearing lungs. However, NK depletion had the unexpected effect of increasing both CCL22 secretion and Treg levels in the lungs of NK-depleted LLC-bearing mice. This was concurrent with an increase in tumor burden. Flow cytometry and a series of both immunomagnetic and FACS isolations were used to identify the CCL22-producing cellular fractions in LLC-bearing lungs. A novel CD11b(+)CD11c(+) cell population was identified that accumulates in large numbers in NK-depleted LLC-bearing lung tissue. These CD11b(+)CD11c(+) cells secreted large amounts of CCL22 that may overcompensate for the loss of NK-derived CCL22 in the lungs of NK-depleted LLC-bearing mice. Taken together, these data suggest that NK cells play both a positive and negative role in the regulation of CCL22 secretion and, in turn, the recruitment of Tregs toward LLC-bearing lungs.

Topics: Animals; Carcinoma, Lewis Lung; CD11b Antigen; CD11c Antigen; Chemokine CCL22; Female; G(M1) Ganglioside; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Myeloid Cells; T-Lymphocytes, Regulatory

To analyze mechanisms for cancer metastasis, we established high metastatic sublines from mouse Lewis lung cancer (P29) by repeated injection. Sublines established from the two subclones H7 and C4 commonly exhibited increased proliferation and invasion activity and reduced expression of ganglioside GM1, although they showed different preferences in their target organs of metastasis. The high metastatic sublines secreted higher levels of activated matrix metalloprotease (MMP)-9 as well as pro-MMP-9 in the culture medium than the parent lines. Furthermore, they contained MMP-9 at the glycolipid-enriched microdomain (GEM)/rafts fractionated by the sucrose density gradient ultracentrifugation of Triton X-100 extracts, whereas the parent cells showed faint bands at the fraction. When high metastatic sublines were treated with methyl-beta-cyclodextrin, their invasion activities were dramatically suppressed, and the MMP-9 secretion was also suppressed. All these results indicated that GEM/rafts play crucial roles in the increased invasion and high metastatic potential. To clarify the implication of reduced GM1 expression, low GM1-expressing cell lines were established using an RNA interference-expression vector of the GM1 synthase. Low GM1-expressing cell lines showed increased proliferation and invasion, enrichment in the GEM/rafts, and increased secretion of MMP-9. Among adhesion molecules, only integrin beta1 was detected in GEM/rafts with stronger intensity in high metastatic lines and low GM1-expressing cells. Taken together, integrins seemed to be enriched in the GEM/rafts by reduced GM1 levels, and subsequently MMP-9 was recruited to the GEM/rafts, resulting in its efficient secretion and activation, and eventually in the increased invasion and metastatic potentials.

Topics: Animals; beta-Cyclodextrins; Carcinoma, Lewis Lung; Cell Line, Tumor; Down-Regulation; G(M1) Ganglioside; Immunohistochemistry; Integrins; Lung Neoplasms; Matrix Metalloproteinase 9; Membrane Microdomains; Membrane Proteins; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Transplantation; RNA, Small Interfering; Transfection