Addition Of Neutralizing Antibody Research Articles

Abstract 2253: Proteases and cytokines in the transition from preinvasive DCIS to invasive breast carcinoma

Abstract The transition from preinvasive ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) involves alterations in proteolytic pathways of both epithelial and stromal cells. Infiltration of macrophages, angiogenesis and secretion of chemotactic molecules by DCIS and surrounding stromal cells also facilitate this transition. We have developed a 3D coculture model, MAME (mammary architecture and microenvironment engineering), to study the role of proteases and cytokines in the progression from pre-invasive DCIS to IDC. MAME-A recapitulates the architecture of normal human breast tissue with the epithelial and stromal components growing in overlays of basement membrane and stromal collagen, respectively. In MAME-B, the stromal collagen is eliminated. The cells used in our studies were human MCF10.DCIS cells, WS-12Ti human breast tumor-associated fibroblasts, U937 human monocytes and human umbilical vein endothelial cells (HUVEC). We analyzed proteolysis through live cell imaging of the degradation of quenched fluorescent matrix proteins [DQ-collagen I™ mixed with collagen I and DQ-collagen IV™ mixed with reconstituted basement membrane (rBM)]. DCIS cells in MAME-A cocultures with fibroblasts exhibited invasiveness at 24 days, yet at only 16 days in MAME-B cocultures. Expression and secretion of cytokines, especially IL-6, were elevated in these cocultures. The invasiveness of the DCIS cells in the cocultures with fibroblasts could be reduced by addition of neutralizing antibody to IL-6. Addition of either monocytes or endothelial cells reduced the time at which invasiveness occurred to as early as 4 days in MAME-B cocultures. Addition of monocytes increased secretion of monocyte chemoattractant protein-1 (MCP-1), IL-8 and GRO. The combination of a neutralizing antibody to hMCP-1 and a receptor-blocking antibody to hCXCR2 (a receptor for IL-8 and GRO) reduced invasiveness more effectively than did either of the antibodies used alone. When HUVEC were grown in MAME-B cocultures with DCIS or DCIS and fibroblasts, the HUVEC grew into a network of tubules with distinct lumens over a period of 4 days. In 3D and 4D reconstructions of these same cocultures, DCIS cells could be seen to migrate toward the HUVEC tubules and invade into their lumens. These phenomena occurred in parallel with increases in proteolysis and in expression and secretion of the cytokines MCP-1, IL-8, RANTES and GRO. The MAME models provide a system for the study of proteolytic and cytokine pathways and the interactions between these pathways that are associated with the transition from DCIS to invasive ductal carcinoma. This model could be used as an in vitro tool for screening drugs that target these pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2253.

Adherent lymphokine-activated killer cells suppress autologous human normal bone marrow progenitors

We have generated a homogeneous population of recombinant interleukin-2 (rIL-2)-stimulated effector cells termed adherent lymphokine-activated killer cells (A-LAK) from peripheral blood mononuclear cells (PBMNC) of 14 normal individuals and tested the effect of A-LAK cells on autologous hematopoietic bone marrow (BM) progenitor growth. Enrichment of A-LAK from PBMNC depended on the propensity of A-LAK precursors to adhere to plastic and proliferate in the presence of rIL-2. The resultant population had the morphologic appearance of large granular lymphocytes, and the majority of cells (73% +/- 4%) expressed the CD56+/CD3- phenotype associated with rIL-2-stimulated natural killer (NK) cells. The A-LAK population had potent lytic activity in chromium release assays against both NK-sensitive (K562) and NK-resistant (Raji) targets. When BM mononuclear cells (BMMNC) were coincubated with autologous A-LAK and rIL-2 (1,000 U/mL) added at the start of culture, dose-dependent suppression of burst-forming unit-erythroid (BFU-E) and colony-forming unit mix (CFU-MIX) colony growth was observed at effector to target ratios (E:T) ranging from 0.25:1 to 5:1 (maximal suppression BFU-E = 85% +/- 6%; CFU-MIX = 95% +/- 3%). This suppression was rIL-2 dose-dependent, and no suppression was seen in the absence of rIL-2. Depletion of BM monocytes and T lymphocytes did not alter A-LAK suppression of progenitors coincubated with A-LAK cells. Addition of polyclonal neutralizing antibodies against both interferon-gamma (IFN- gamma) and tumor necrosis facto alpha (TNF-alpha) to the coincubation culture completely abrogated the suppressive effect of A-LAK on BFU-E and CFU-MIX colony growth while each neutralizing antibody used alone had intermediate effects. In contrast to coincubation studies, 36 hours of preincubation of A-LAK cells with autologous BM (E:T 2.2:1) and rIL- 2 (1,000 U/mL) followed by plating of preincubated BM cells in hematopoietic progenitor culture produced significant suppression of day 14 BFU-E (47% +/- 5%), but spared the more primitive CFU-MIX (7% +/- 9%), suggesting a divergent effect of A-LAK cells on hematopoietic progenitors at different stages of differentiation. Addition of neutralizing antibodies against IFN-gamma and TNF-alpha in preincubation failed to abrogate the suppressive effect of A-LAK on BFU- E colony growth, suggesting that this suppression occurs by a different mechanism than that seen in coincubation studies. Previous studies have demonstrated that the A-LAK population has cytotoxic and proliferative advantages over other killer cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Adherent Lymphokine-Activated Killer Cells Suppress Autologous Human Normal Bone Marrow Progenitors

We have generated a homogeneous population of recombinant interleukin-2 (rIL-2)-stimulated effector cells termed adherent lymphokine-activated killer cells (A-LAK) from peripheral blood mononuclear cells (PBMNC) of 14 normal individuals and tested the effect of A-LAK cells on autologous hematopoietic bone marrow (BM) progenitor growth. Enrichment of A-LAK from PBMNC depended on the propensity of A-LAK precursors to adhere to plastic and proliferate in the presence of rIL-2. The resultant population had the morphologic appearance of large granular lymphocytes, and the majority of cells (73% +/- 4%) expressed the CD56+/CD3- phenotype associated with rIL-2-stimulated natural killer (NK) cells. The A-LAK population had potent lytic activity in chromium release assays against both NK-sensitive (K562) and NK-resistant (Raji) targets. When BM mononuclear cells (BMMNC) were coincubated with autologous A-LAK and rIL-2 (1,000 U/mL) added at the start of culture, dose-dependent suppression of burst-forming unit-erythroid (BFU-E) and colony-forming unit mix (CFU-MIX) colony growth was observed at effector to target ratios (E:T) ranging from 0.25:1 to 5:1 (maximal suppression BFU-E = 85% +/- 6%; CFU-MIX = 95% +/- 3%). This suppression was rIL-2 dose-dependent, and no suppression was seen in the absence of rIL-2. Depletion of BM monocytes and T lymphocytes did not alter A-LAK suppression of progenitors coincubated with A-LAK cells. Addition of polyclonal neutralizing antibodies against both interferon-gamma (IFN-gamma) and tumor necrosis facto alpha (TNF-alpha) to the coincubation culture completely abrogated the suppressive effect of A-LAK on BFU-E and CFU-MIX colony growth while each neutralizing antibody used alone had intermediate effects. In contrast to coincubation studies, 36 hours of preincubation of A-LAK cells with autologous BM (E:T 2.2:1) and rIL-2 (1,000 U/mL) followed by plating of preincubated BM cells in hematopoietic progenitor culture produced significant suppression of day 14 BFU-E (47% +/- 5%), but spared the more primitive CFU-MIX (7% +/- 9%), suggesting a divergent effect of A-LAK cells on hematopoietic progenitors at different stages of differentiation. Addition of neutralizing antibodies against IFN-gamma and TNF-alpha in preincubation failed to abrogate the suppressive effect of A-LAK on BFU-E colony growth, suggesting that this suppression occurs by a different mechanism than that seen in coincubation studies. Previous studies have demonstrated that the A-LAK population has cytotoxic and proliferative advantages over other killer cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)