Search

MCP-1/IL-6 in vitro monocyte secretion upon coculture with autologous fragment spheroids was studied in relation to patient 5- and 10-year overall survival rates in head and neck squamous cell carcinoma (HNSCC) patients (n = 65) diagnosed between 1998 and 2005, nine of whom had an human papilloma virus (HPV) tumour infection. The spheroids were harvested from malignant or benign tissue during primary surgery. Two weeks following surgery, freshly isolated autologous monocytes and benign or malignant spheroids were cocultured 24 h in vitro. The IL-6 secretion was expressed as a fraction of the lipopolysaccharide (LPS) response from the same batch of monocytes. HPV status was obtained by employing PCR analyses of primary diagnostic blocks. IL-6/MCP-1 response levels were not found to be dependent on HPV infection status. MCP-1 secretion did not predict prognosis, nor did in vitro IL-6 monocyte background or LPS-stimulated IL-6 secretion. At 5-year observation, dichotomized IL-6 levels following monocyte coculture, with both malignant and benign spheroids, showed a strong trend towards predicting survival, that is a low monocyte malignant coculture response showed a survival of 31 ± 17 versus 58 ± 17% with a high such response (P = 0.057). When studying monocyte IL-6 coculture responses evaluating benign and malignant spheroid results statistically together, a prediction of survival up to 10 years was found (hazard ratio = 0.48; confidence interval = 0.24-0.96; P < 0.05) with double low IL-6 responses. This survival prediction was also present after an adjustment for HPV tumour infection status. In conclusion, monocyte IL-6 in vitro secretion in cocultures with autologous spheroids/serum from HNSCCs predicted 5- and 10-year survivals, both with and without tumour HPV tumour adjustment., (© 2016 The Foundation for the Scandinavian Journal of Immunology.)

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma (HNSCC) patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro augment their secretion of monocyte chemotactic protein-1 (MCP-1). Presently, the aims of the present work were to study whether the metabolic activity, secreted products and/or specific receptor/ligand on the surface of the F-spheroids and monocytes are necessary for stimulation of the monocyte MCP-1 secretion upon F-spheroid co-culture. Actinomycin D (1 mug/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte MCP-1 co-culture response. Co-culture of monocytes and F-spheroids separated by a semi-permeable membrane showed a decreased, but still present, monocyte MCP-1 co-culture response. Conditioned medium from F-spheroids stimulated allogenous monocytes to secrete MCP-1. The addition of glucose or galactose, but not mannose, to co-cultures partially inhibited the monocyte MCP-1 co-culture response. The addition of anti-CD14 antibody diminished the MCP-1 co-culture response. In conclusion, the monocyte MCP-1 co-culture response is dependent on metabolically active spheroids, secreted stimuli, and is augmented by direct contact with F-spheroids, possibly via lectin-like receptors and the CD14 receptor.

Monocyte fragment (F)-spheroid-stimulated and F-spheroid IL-6 and monocyte chemotactic protein (MCP)-1 secretion are related to inflammatory state, macrophage density and the TNM stage of patients with head and neck squamous cell carcinoma (HNSCC). Fragment (F)-spheroids from HNSCC patients in vitro secrete and stimulate autologous monocytes to secrete IL-6 and MCP-1. The aim of this investigation was to study this cytokine secretion in relation to other cytokines, spheroid composition and host factors.In series I (n=14) the densities of epithelial cells, fibroblasts and macrophages were determined in sections from F-spheroids and donor tissue. In series II (n=17) the TNM stage, donor inflammatory state, macrophage density and the secretion of F-spheroid- and monocyte F-spheroid-stimulated IL-6, MCP-1 and tumor necrosis factor (TNF)-alpha were determined. Epithelial cells were partly replaced by interstitial tissue during spheroid formation. Malignant (M) F-spheroids secreted more MCP-1 than benign (B) F-spheroids. No F-spheroid secreted measurable amounts of TNF-alpha. Monocytes secreted more IL-6 when co-cultured with MF- compared to BF-spheroids. Monocyte IL-6 MF- and MCP-1 MB-spheroid-stimulated secretion correlated with macrophage density. In addition, there was an association between MF- and BF-spheroid-stimulated monocyte cytokine secretion, as well as between BF- and MF-spheroid-stimulated MCP-1 secretion. An inverse relation was also noted between the erythrocyte sedimentation rate at monocyte harvest and the monocyte MCP-1 F-spheroid responses.

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro secrete interleukin (IL)-6 upon 24h in co-culture. Presently, the aim was to study the mechanisms of this monocyte secretion. Paraformaldehyde (0.1% for 2min) or actinomycin-D (1 microg/ml for 24h) pre-treatment of the F-spheroids abolished the monocyte IL-6 co-culture response. Addition of glucose (100mM) or mannose (100mM), and to some extent galactose (100mM), but not fructose (100mM) to the co-cultures, partly inhibited the monocyte IL-6 co-culture response, but such addition did not inhibit the in vitro monocyte lipopolysaccharide (LPS)-generated IL-6 secretion. When mannose was added to the co-cultures, monocyte IL-6 mRNA expression was eradicated in malignant co-cultures and reduced to a low level in benign co-cultures. Addition of mouse anti-human beta(1)-integrin (anti-CD29) antibody (2 microg/ml) diminished the IL-6 co-culture response but not the monocyte LPS-generated IL-6 response. In conclusion, the monocyte IL-6 co-culture response is dependent on live spheroids and to some extent on direct contact with the F-spheroids, possibly via lectin-like receptor(s), the mannose receptor and beta(1)-integrin.

The secretion of interleukin (IL)-1 beta, IL-6 and tumour necrosis factor (TNF)-alpha were compared when freshly isolated autologous monocytes or monocytederived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from a series of head and neck squamous cell carcinoma (HNSCC) patients. F-spheroids were generated from the malignant tumour (M-spheroids) or from benign mucosa (B-spheroids) from which the tumour originated control. If monocytes maturated towards MDMs before co-culture, the IL-6 secretion declined dependent on the extent of the MDM maturation by both M- and B-spheroid stimulation. When MDMs maturated in continuous co-culture, a steady-state secretion of IL-6 continued for several days but diminished when the culture medium was changed every 24 h. No co-culture-induced IL-1 beta or TNF-alpha was determined. Both the cytokine secretion and the mRNA gene expression revealed a different monocyte/MDM activation when co-culture and lipopolysaccharide (LPS)-stimulation were compared. Addition of anti-CD14 (10 microg/ml) decreased monocyte LPS-stimulated, but increased monocyte co-culture stimulated IL-6 secretion. In conclusion, M- and B-spheroids similarly stimulated monocytes and to a lesser extent MDMs. MDMs that maturated with F-spheroids present, retained responsiveness at the monocyte level. Co-culture-induced monocyte stimulation, as measured by IL-6 secretion, was not dependent on activation via the CD14 molecule.

Biopsies from tumour and benign mucosa were removed from patients with head and neck squamous-cell carcinoma (HNSCC), chopped into cubes and transferred to a nonadhesive culture system where in vitro fragment (F)-spheroids were established. The F-spheroids stabilized within 14 days of culture in vitro with epithelial cells and fibroblasts on the surface. F-spheroids were co-cultured with freshly isolated autologous monocytes. The monocytes of 10 of 11 patients secreted interleukin (IL)-6 at a level similar to that of the average monocyte endotoxin-stimulated response. Secreted IL-1beta or tumour necrosis factor-alpha (TNF-alpha) levels greater than 0.1 times the endotoxin-stimulated secretion were determined in one and two of the 11 co-culture experiments, respectively. This different monocyte response to F-spheroids compared with endotoxin stimulation was also present at the mRNA expression level. HNSCC monocytes secreted no IL-6 after co-culture with autologous fibroblasts. When monocytes and F-spheroids were cultured separated by a semipermeable membrane, the IL-6 supernatant level was only approximately 25% of that observed during co-culture with direct contact. F-spheroids secreted only trace amounts of IL-6. In conclusion, monocytes of HNSCC patients generally secrete IL-6, but not IL-1beta or TNF-alpha, after stimulation with epithelial-associated components of F-spheroids upon direct contact and in part by a soluble substance.