Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages

Abstract

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.

Figure 1. Detection of macrophages in OTCs.

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.

Figure 2. Polarization of macrophages in OTCs.

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.

Figure 3. Histology of OTCs containing macrophages.

OTCs were fixated with formaldehyde and stained with hemalaun/eosin. PDVA tumor cells form a tumor epithelium (TE) on top of the dermal equivalents (DE). Spindle-shaped fibroblasts (f) and round macrophages (m) were detected within the dermal equivalents. Islands of tumor cells protrude into the dermal equivalents in OTCs containing macrophages in combination with fibroblasts (E) or alone (F) which had been stimulated with IL-4 (bold arrows). After three weeks, infiltrating tumor cells are also observed in OTCs with macrophages in absence of IL-4 (H, I). Bar 10 µm.

Figure 4. Analysis of tumor cell invasion.

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 in situ 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.

Figure 5. Adaptation of macrophage containing tumor OTCs to a human system.

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.

Figure 6. Proteolytic activity in human OTCs.

A: Conditioned media from human OTCs were analyzed by gelatin zymography and degradation of co-polimerized gelatin in a SDS PAA gel was investigated. Active MMP-9 is increased in macrophage containing OTCs stimulated with IL-4. B: Thickness of dermal equivalents were quantified. Dermal equivalents are significantly reduced in OTCs containing macrophages or fibroblasts and macrophages stimulated with IL-4.