Analysis of tumor cell invasion.

<p>A: Immunofluorescent staining for laminin (red), a component of the basement membrane was performed in OTCs cultured for one week. Laminin is disrupted by islands of tumor cells (white arrow heads) in OTCs containing macrophages that were stimulated with IL-4. B: Enzymatic degradation of collagen-IV was analyzed performing <i>in situ</i> collagen zymography on OTC cryosections. Collagen-IV can be degraded by MMP-2 and MMP-9. C: Immunofluorescent staining of MMP-2 shows increased signal in macrophage containing OTCs that were stimulated with IL-4 and, to a lesser extent in fibroblast containing OTCs. D: Immunofluorescent staining of MMP-9 reveals increased expression in macrophage containing OTCs stimulated with IL-4 as well as in fibroblast-containing OTCs. E: Thickness of dermal equivalents were quantified. Dermal equivalents are significantly reduced in OTCs containing macrophages or fibroblasts and macrophages stimulated with IL-4. Bar 10 µm.</p

Detection of macrophages in OTCs.

<p>Immunofluorescent staining of cryosections against the pan-macrophage marker F4/80 (red) after one and three weeks. F4/80 positive cells are detectable in OTCs in which macrophages had been incorporated alone (week one A, week three D) or together with fibroblasts (week one B, week three E). OTCs in which fibroblasts were integrated were negative for F4/80 (week one C, week three F). Bar 10 µm.</p

Polarization of macrophages in OTCs.

<p>Immunofluorescent staining of cryosections from OTCs against M1 and M2 markers after one and three weeks. M2 polarization of macrophages was detected by staining for the M2 markers CD-206 (A, red). High CD-206 signals were detected in OTCs containing fibroblasts and macrophages or upon treatment with IL-4. CD-206 was detected on macrophages in all 3 week setups (B). Quantification demonstrated that not only signal intensity but also the number of M2 macrophages increased upon stimulation with IL-4 and over time (C). Stimulation of OTCs with IFN-gamma and LPS results in increased numbers of macrophages positive for iNOS, an M1 marker (D), while the number of CD-206+ M2 macrophages decreased (D). Further, the signal intensity of iNOS expression of macrophages in IFN-gamma + LPS stimulated OTCs clearly increased (E), while CD-206 was not detected at all (E). Bar 10 µm.</p

Adaptation of macrophage containing tumor OTCs to a human system.

<p>Human PBMC-derived macrophages and human primary dermal fibroblasts were included in OTCs with the human SCC cell line A-5RT3. Immunofluorescent staining of the pan macrophage marker CD-68 (green) and the M2 polarization marker CD-209 (red) in week one (A) and week three (B) shows that macrophages are viable and that in week one, M2 polarization is induced upon co-culture with fibroblasts or upon stimulation with IL-4 (A). Prolonged cultivation for three weeks results in enhanced CD-209 expression in all setups (B). Quantification of CD-209+ macrophages further underlines this dynamics (C). Addition of IFN-gamma and LPS in week two of culture results in increased production of the M1 marker CD-127 (quantification: D, immunofluorescent staining in red: E) whereas unstimulated or IL-4 stimulated OTCs contain less CD-127 macrophages with a lower expression level. Only few CD-209+ macrophages can be detected in OTCs stimulated with IFN-gamma and LPS (F). Bar = 100 µm.</p