Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.


F-actin/G-actin assay
Macrophages (1 × 10 6 cells/ml RPMI 1640, 5% FCS) were allowed to adhere for 1 h onto glass coverslips (Roth, Karlsruhe, Germany) in a 12-well plate and then incubated at 37 °C with vehicle (0.1% DMSO) or test compounds. Cells were then collected and lysed with 100 µl of pre-warmed lysis and F-actin stabilization buffer (50 mM PIPES pH 6.9, 50 mM NaCl, 5 mM MgCl2,5 mM EGTA, 5% (v/v) glycerol, 0.1% (v/v) Nonidet P40, 0.1% (v/v) Triton X-100, 0.1% (v/v) Tween 20, 0.1% (v/v) β-mercaptoethanol, 0.001% (v/v) Antifoam C plus freshly added 10 μl/ml of 100 mM ATP, and 10 μl/ml Protease inhibitor cocktail). Cells were homogenized using a 200 µl pipette tip with a fine orifice (up and down, eight times) and incubation at 37 °C for 10 min. Cell lysis was checked by trypan blue staining and light microscopy. The lysate was centrifuged (600 × g, 5 min, 4 °C) to remove unbroken cells, and the homogenate was then centrifuged at 100,000 × g for 60 min at 4 °C. The resulting supernatants were immediately put on ice, the pellets were resuspended with ice cold Milli-Q water plus 10 μM cytochalasin D and left on ice for 1 h to dissociate F-actin, and pipetted once 3 every 15 min with a fine 200 µl pipette tip. Samples were then mixed with 4×SDS-PAGE sample loading buffer, heated to 95°C for 5 min and analyzed by SDS-PAGE and Western blot.
Human recombinant MCP-1 (100 ng/ml, PeproTech) in RPMI 1640 medium was added to the lower compartment of the disposable 96-well chemotaxis chamber in a total volume of 29 μl.
Resuspended monocytes (1 × 10 5 cells/well in 25 µl) were added to the upper compartment of the chamber that had been pre-coated with FCS for 2 h and incubated at 37°C and 5% CO2 for another 2 h. Then, the filter was removed and the number of migrated monocytes was evaluated by ATP analysis. ATP was analyzed according to the manufacturer´s protocol (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Madison, WI). Luminescence was measured (Novostar, BMG Labtechnologies, Offenburg, Germany) and the values were compared to a simultaneously obtained standard curve of monocytes from a range of zero to 1 × 10 5 cells.
Images were taken with an AxioCam MR3 camera and were acquired, cut, linearly adjusted in the overall brightness and contrast, and exported to TIF by the AxioVision 4.8 software.

Microfluidically supported biochips assay: preparation of biochips
Biochips were made by injection moulding from cyclo olefin polymer (COP) Zeonor®, obtained from microfluidic ChipShop GmbH (Jena, Germany), and manufactured as described previously [1]. Briefly, a 12.2 µm thick PET membrane with a pore diameter of 8 µm and a pore density of 1 × 10 5 pores/cm² (Sabeu, Radeberg, Germany) was integrated. Chips and channel structures were sealed on top and bottom side with an extruded 140 µm thick COP foil using a low temperature proprietary bonding method. Oxygen plasma treatment for hydrophilisation of the whole chip surface was performed to support cell growth and to reduce in-chip air bubble formation. Additionally, equilibration of the medium under perfusion conditions was performed to reduce air bubble formation. Gas permeable silicon tubing was used for perfusion allowing oxygen equilibration during experiments.
In each sterilized biochip 1.3×10 5 HUVEC/cm² and 0.43×10 5 monocytes / cm² were mixed and cultured for 72 h in ECGM supplemented with 10% autologous serum and Pen/Strep. 10 ng/ml M-CSF was added for macrophage differentiation. On day four, 1.3×10 5 MCF-7 cells/cm² were seeded in the lower compartment and cultured for two more days in DMEM-HG plus 10% FCS.
Macrophage polarization was induced on day five by replacing the medium with ECGM plus 10% autologous serum, 10 ng/ml M-CSF, Pen/Strep and 10 ng/ml IFN-γ for M1 or 10 ng/ml IL-4 for M2 polarization for 24 h. Medium containing 1 µM ChA or vehicle was perfused over the vascular layer with a shear stress rate of 3 dyn/cm² for 2 h. Medium was replaced with ECGM plus 10% autologous serum, 10 ng/ml M-CSF, Pen/Strep and 10 ng/ml IFN-γ for tumours models using M1 or 10 ng/ml IL-4 for M2; tumour models were cultured for additional 48 h. Where indicated, 1 µg/ml TNFα capture antibody (R&D systems, MN) was added. For subsequent analysis, supernatants were collected on a daily basis. Images were analyzed using ImageJ2/Fiji. After perfusion, the medium within the lower chamber containing MCF-7 cells was collected, and cytokines were measured using a multiplexed bead-based immunoassay (Cytometric bead array (CBA), BD Bioscience/Pharmingen, Heidelberg, Germany). Cytokine kits used: human enhanced sensitivity TNFα Flex Set, human enhanced sensitivity IL-1β Flex Set, human IL-6 Flex Set, human IL-8 Flex Set and human IL-10 Flex Set (BD Bioscience/Pharmingen).
Analysis was performed with a FACS Canto II flow cytometer (BD Bioscience/Pharmingen) and data were quantified using FlowJo v10 software (TreeStar, Ashland OR).

Cytokine and chemokine assays
Cytokine and chemokine levels were determined by sandwich ELISA using the DuoSet Kit from R&D Systems (Minneapolis). Maxisorp Immunomodules (Nunc, Roskilde, Denmark) were coated with the primary antibody over night at 4 °C. After washing with PBS containing 0.05% Tween 20 the plate was blocked with 1% BSA in PBS for 1 hour at RT. The cell culture supernatants were added to the plate and incubated for 2 h. Then, the secondary antibody was added for additional 2 h followed by 30 min incubation of Streptavidin-HRP. Afterwards, 1step Ultra-TMB-ELISA (Thermo Scientific) was added for 30 min and the reaction was terminated by addition of 2 M H2SO4. The absorbance was measured at 450 nm (Multiskan Spektrum).

Total internal reflection fluorescence (TIRF) microscopy
7 Nucleation of actin in a cell-free assay was visualized by TIRF microscopy. The Actin-toolkit TIRM containing Atto488-labelled actin (Hypermol, Bielefeld, Germany) was used according to the manufacturer´s instructions. Coverslips were coated with 0.1 mg/ml NEM-myosin in order to tether actin nuclei or filaments. Actin polymerization in a 1 µM solution was started by adding a respective high salt buffer. Due to sample handling and focusing on the TIRF microscope there is a delay of approx. 30 seconds. Images were obtained on a Leica TIRF MC microscope equipped with a 100× oil immersion TIRF lens at an excitation of 488 nm and an emission of 505 -520 nm every 10 seconds for 15 minutes.