Pancreatic cancer cell/fibroblast co-culture induces M2 like macrophages that influence therapeutic response in a 3D model

Abstract

Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs and monocytes. Using this model, we analyzed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analyzing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated. 3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activated spheroid polarized MDMs or block immune suppressive factors such as Arginase-I.

Fig 1. Formation and viability of tumor cell/fibroblast spheroids.

Tumor cells were cultured alone or with fibroblasts in a poly-Hema-coated 96 well plate for 5 days as described before. 3D spheroid formation and cell viability was measured by CellTiterGlo assays. A) All tumor cell lines showed compact spheroid formation in co-culture with fibroblasts (MRC5) compared to tumor cell monoculture. B) Cell viability of tumor cells was strongly increased in co-culture with MRC5 and reached the maximum on day 5 for most of the cell lines. BxPC3 exhibited the greatest increase of viability upon co-culturing with MRC5, whereas the other cell lines reached a plateau at day 5. Statistical significance was calculated of n = 5 independent experiments by using an unpaired Student´s t test with unequal variances; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 2. Viability of tumor cell/fibroblast co-culture with monocytes.

Tumor cells were co-cultured with fibroblasts for 5 days. Freshly isolated naïve monocytes were added to tumor cell/fibroblast co-culture on day 5 and cell viability was measured every 2 days from day 5 to 11. Addition of monocytes did not influence the co-culture´s viability and tumor cell/fibroblast co-cultures with monocytes were as viable as co-cultures without monocytes for each day and tumor cell line. Represented are n = 5 independent experiments. Using an unpaired Student´s t test with unequal variances, no significances were observed between tumor cell/fibroblast co-cultures with and without monocytes.

Fig 3. Spheroid polarized MDM infiltration into 3D co-culture spheroid.

Tumor cells and fibroblasts were 3D co-cultured for 5 days to form tight spheroids. On day 5 of tumor cell/fibroblast spheroid formation, monocytes were added for 6 days to determine cell migration into spheroids. A) The pan-macrophage marker CD68 on myeloid cells was detected with DAB by performing IHC using a Benchmark XT instrument. Infiltration of spheroid polarized MDMs can be observed for all co-cultures, but was highest in MiaPaCa-2/MRC5 co-culture. One representative picture is shown for each tumor cell line. B) Quantitative analysis of spheroid infiltrated MDMs of n = 4 independent experiments.

Fig 4. 3D co-culture polarized MDMs resemble M2-like macrophages.

Tumor cells and fibroblasts were co-cultured for 5 days. Monocytes were added to co-culture on day 5 to differentiate for 6 days. Spheroids were collected and dissociated by using Accutase to obtain a single cell suspension. A) Single cell suspensions were analysed by flow cytometry using the illustrated gating strategy for MDM phenotyping. B) Cell surface marker expression of 3D myeloid cells was compared to in vitro generated M2c and activated M1 macrophages. Typical M2 and M1 macrophage marker were analyzed by flow cytometry. 3D co-culture MDMs expressed high levels of CD163 and CD14 and low levels of CD86 and HLA-DR comparable to in vitro differentiated M2 macrophages (dotted box). Shown is one representative out of n = 5 experiments, red numbers show geometrical mean values for relevant markers.

Fig 5. 3D tumor cell/fibroblast co-culture with monocytes induces differential secretion of cytokines, chemokines and growth factors.

Tumor cells and fibroblasts were co-cultured for 5 days. Monocytes were added to co-culture on day 5 and further cultivated for 6 days. Supernatants were collected on day 11 from co-cultures without and with monocytes. A panel of 19 soluble factors was measured using Luminex multiplex technology and the most relevant ones at detectable levels are shown. Increased levels of several cytokines and chemokines were detected on day 11 after addition of monocytes. Statistical significance was calculated of n = 3 independent experiments by using an unpaired Student´s t test with unequal variances; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 6. 3D co-culture polarized MDMs suppress CD3+ T cell proliferation.

Tumor cells and fibroblasts were co-cultured for 5 days to form tight spheroids. Freshly isolated monocytes were added on day 5 to differentiate for 6 days. Autologous CD3+ T cells were labeled with CFSE on day 11, added to co-cultures with and without monocytes and stimulated with CD3/CD28 activation beads. Proliferation was measured after 6 days using flow cytometry. A) T cells proliferated strongly in co-culture with tumor cell and fibroblasts, but were suppressed in co-culture with spheroid polarized MDMs. B) Strongest suppression of T cell proliferation was observed in Pa-Tu 8902/MRC5 and HPAC/MRC5 co-cultures with spheroid polarized MDMs. C, D) CD3+ T cell proliferation was more effectively suppressed in co-culture with in vitro generated M2c macrophages compared to co-culture with activated M1 macrophages. Statistical significance was calculated of n = 4 independent experiments by using an unpaired Student´s t test with unequal variances; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 7. Expression of cell surface activation and checkpoint marker on CD8+ and CD4+ T cells decreases in co-culture with 3D co-culture polarized MDMs.

Tumor cells and fibroblasts were co-cultured for 5 days to form tight spheroids. Freshly isolated monocytes were added on day 5 to differentiate for 6 days. Autologous CD8+ and CD4+ T cells were added to co-cultures with and without spheroid polarized MDMs on day 11 and stimulated with CD3/CD28 activation beads. Cell surface marker expression of CD8+ and CD4+ T cells was measured 6 days after T cell activation by flow cytometry. Statistical significance was calculated of n = 3 independent experiments by using an unpaired Student´s t test with unequal variances; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 8. Treatment of spheroid polarized MDMs with immune modulating compounds partially restores T cell proliferation in 3D co-culture.

Tumor cells and fibroblasts were co-cultured for 5 days to form tight spheroids. Freshly isolated monocytes were added on day 5 to differentiate for 6 days. One day prior T cell addition, co-cultures were treated with inhibiting or activating compounds either alone or in combination and compared to untreated T cells. Autologous CD3+ T cells were labeled with CFSE on day 11, added to co-cultures with and without spheroid polarized MDMs and stimulated with CD3/CD28 activation beads. Proliferation was measured after 6 days using flow cytometry. Statistical significance was calculated of n = 3 independent experiments by using an unpaired Student´s t test with unequal variances; *p < 0.05, **p < 0.01, ***p < 0.001.