Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy

Abstract

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-X_L inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-X_L or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

Fig. 1. Spheroid formation in RMS cells.

RH30-GFP cells were cultured in 96-well ultra-low attachment plates at different cell densities. To enable spheroid formation, plates were centrifuged at 1 000 g for 10 min directly after seeding. A GFP fluorescence signal of RH30-GFP spheroids was acquired for 7 days following spheroid formation. B-D Spheroid size was quantified at selected time points in RH30-GFP (B), RD-GFP (C), or Kym-1 (D) cells. Data shown are mean + S.D. (n = 2). Scale bar equals 500 μm.

Fig. 2. Cell death induced by BH3 mimetics in 2D and 3D cultures.

A RH30-GFP, RD-GFP and Kym-1 cells were cultured as adherent monolayer cells (2D) before treatment with different concentrations of BH3 mimetics for 48 h. Viability was analyzed using Cell Titer Glo® assay. B RH30-GFP, RD-GFP and Kym-1 cells were cultured as spheroids (3D) for 3 days before treatment with different concentrations of BH3 mimetics for 48 h. Viability was analyzed using CellTiter-Glo® assay. Data shown are mean + S.D. (n = 3). Synergy was quantified using Synergyfinder and expressed as Bliss Score with a score >0 indicating synergy on a scale to 100.

Fig. 3. Microscopic analysis confirms cell death induced by BH3 mimetics.

A mRNA expression levels of BCL2 family members were compared in 3D spheroids and 2D culture of RH30-GFP and RD-GFP cells by qRT-PCR. Data shown are mean + SEM (n = 4). B–E RH30-GFP or RD-GFP cells were cultured as 3D spheroids for 3 days before exposure to A1331852 (0.25 μM RD-GFP, 0.1 μM RH30-GFP) or S63845 (0.3 μM RD-GFP, 0.03 μM RH30-GFP) either alone or in combination for 48 h. Cell death was assessed by PI staining for 30 min before analysis. B, C PI and GFP fluorescence was quantified and expressed as the ratio of PI/GFP. Data shown are mean + S.D. (n = 3), **p < 0.01; ***p < 0.001. D–E Exemplary images are shown for PI and GFP fluorescence. Scale bar equals 500 μm.

Fig. 4. Morphological assessment of spheroids.

A, B RD-GFP cells cultured as 3D spheroids were left untreated (A) or exposed to the combination of A1331852 (0.25 μM) and S63845 (0.3 μM) (B) for 48 h before PI staining and light-sheet microscopy. C, D RD-GFP cells were cultured as large spheroids containing 50,000 cells for 7 days and either left untreated (C) or treated with the combination of A1331852 (0.25 μM) and S63845 (0.3 μM) (D) for 6 h before formalin fixation and H/E staining (lower right side) or analysis of Ki-67 staining by immunohistochemistry (large image and upper right side).

Fig. 5. NK cells migrate into tumor spheroids to induce tumor cell killing.

A RH30-GFP or RD-GFP cells were cultured as spheroids for 4 days. Activated NK cells were stained with CellTrace™ Violet and added to the spheroids at an effector to target (E:T) ratio of 5:1. Infiltration of NK cells into tumor spheroids was monitored by continuous live-cell microscopy for 8 h (see supplementary videos) and representative images for the indicated time points are displayed. B Modification of the E:T ratio influences the NK cell infiltration within the initial 8 h, measured by CellTrace™ Violet intensity within RH30-GFP or RD-GFP spheroids (mean + SEM, n = 3, normalized to only spheroids as background fluorescence). C NK cells are able to induce tumor cell killing in an E:T ratio-dependent manner. Selected images are displayed for the different E:T ratios after 5 days of NK cell addition. To visualize cell death spheroids were counterstained with PI. D Quantification of GFP and PI fluorescence intensity of RMS spheroids co-cultured with NK cells at different E:T ratios and calculation of the PI/GFP ratio as cell death indicator, exemplary images shown in (C). Data shown are mean + SEM (n = 5), **p < 0.01. Scale bar equals 500 μm.

Fig. 6. BH3 mimetics increase NK cell-mediated killing of tumor spheroids.

A Experimental setup of combined BH3 mimetics and NK cell treatment. B Addition of the Mcl-1 inhibitor S63845 (0.3 μM) or the Bcl-X_L inhibitor A1331852 (0.3 μM) increases the NK cell-mediated effect (E:T ratio of 1:1) on spheroid growth inhibition. Data shown are mean + SEM (n = 5–7). ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. C Exemplary images of the combination of S63845 or A1331852 with NK cells showing increased cell death in tumor spheroids, indicated by PI counterstaining of GFP fluorescent RMS spheroids after 72 h of NK cell co-culture. Scale bar equals to 500 μm.

Fig. 7. Caspase dependency of BH3 mimetic sensitization towards NK cell killing.

A RH30 and RD wild-type spheroids were pretreated with either S63845 (0.3 μM) or A1331852 (0.3 μM) for 4 h, before addition of NK cells. After 24 h of NK cell co-cultivation, caspase activity was stained using a CellEvent™ Caspase 3/7 green detection substrate. TL: transmitted light channel. B To inhibit caspase activity or TRAIL, GFP expressing RMS spheroids were co-cultured with zVAD.fmk (50 μM) or anti-TRAIL blocking antibody (1 μg/ml) in combination with BH3 mimetics for 4 h before addition of NK cells (E:T ratio of 1:1). To visualize cell death, spheroids were counterstained with PI after 72 h of NK cell co-culture. Data shown are mean + S.D. (n = 3). Scale bar equals to 500 μm.