ABSTRACT
BACKGROUND AND PURPOSE: In BALB/c mice with transplantable breast tumors, we showed that CD200R1KO mice were cured of local and metastatic growth of EMT6 cells following surgical resection of localized tumor and immunization with irradiated cells along with CpG as adjuvant. On the other hand, wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. To develop an in vitro system which would mimic the in vivo model and allow exploration of factors controlling tumor invasion as a precursor to in vivo metastasis, we have developed and characterized a two-phase culture system. METHODS: Bone marrow mesenchymal stromal cells (BMMSCs) from WT, CD200KO or CD200R1KO mice were admixed with T lymphocytes from tumor-immunized mice and cultured in collagen gels. Tumor cells were subsequently seeded in fresh medium above this gel 1d later. We then investigated the regulation of tumor invasion from the liquid to the gel layer. Tumor cells were measured in the gel layer following collagenase digestion and cultured at limiting dilution-an aliquot of the digest was also analyzed for cytokine levels in ELISA. RESULTS: BMMSCs from WT, CD200KO and CD200R1KO mice all augmented seeding/growth of EMT6 and 4THM tumor cells into the collagen matrix. Inclusion of IL-6 and IL-17 in the gel matrix was associated with increased invasion of tumor cells into this layer. Inclusion of DLN cells from EMT6 immune or 4THM immune mice further modified tumor invasion, with increased tumor numbers seen using stromal elements from CD200 and CD200R1KO mice and DLN from 4THM immune, while CD200R1KO-derived DLN of EMT6 immune mice attenuated tumor invasion, despite inclusion of IL-6/IL-17 in the gel layer. CONCLUSION: Multiple factors can regulate tumor invasion, including micro-environmental stromal elements, IL-6/IL-17, and signals from tumor-derived DLN cells.
Subject(s)
Antigens, CD/genetics , Breast Neoplasms/pathology , Cell Culture Techniques/methods , Lymph Nodes/pathology , Membrane Glycoproteins/genetics , Animals , Antigens, CD/metabolism , Breast Neoplasms/genetics , Collagen , Female , Humans , Interleukin-17/immunology , Interleukin-17/metabolism , Interleukin-6/blood , Interleukin-6/immunology , Interleukin-6/metabolism , Membrane Glycoproteins/metabolism , Mice, Inbred BALB C , Mice, Knockout , Stromal Cells/pathology , T-Lymphocytes/pathologyABSTRACT
We examined expression of genes associated with cytokine production, and genes implicated in regulating bone metabolism, in bone stromal and osteoblast cells incubated under standard ground conditions and under conditions of neutral buoyancy, and in the presence/absence of serum from normal or sleep-deprived mice. We observed a clear interaction between these two conditions (exposure to neutral buoyancy and serum stimulation) in promoting enhanced osteoclastogenesis. Both conditions independently altered expression of a number of cytokines implicated in the regulation of bone metabolism. However, using stromal cells from IL-1 and TNFx cytokine(r) KO mice, we concluded that the increased bone loss under microgravity conditions was not primarily cytokine mediated.
