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-XL 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-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

How to build a tumor: An industry perspective

“…Ranging from a 96-well to a maximum of 1536-well format together with a broad range of readouts it is an ideal format to perform mechanistic studies and to screen for new indications or combinations. 32 .…”

Mitochondrial apoptosis: facilitator of NK cell-mediated immunotherapy

“…This observation is supported by another recent study demonstrating synergy between BH3 mimetics and NK cell-mediated killing in spheroids of pediatric solid tumors. 4 A requirement for safe and beneficial incorporation of BH3 mimetics into cellular immunotherapy is that the given cells are not functionally impaired or killed by the BH3 mimetics. Importantly, Pan et al show that activation by IL-2 renders NK cells resistant to different BH3 mimetics.…”

“…1 Similarly, a previous report showed comparable protection of NK cells by IL-15. 4 The molecular mechanism underlying this protection is currently not well described, but may involve the upregulation of several BCL2 proteins upon activation. 1 Taken together, the combination of BH3 mimetics and NK cells is capable of inducing apoptosis at lower concentrations than either approach alone, and thus may be beneficial in overcoming the toxicity of BH3 mimetics while maintaining efficient tumor cell killing.…”

Molecular dissection of BV6 induced sensitization of rhabdomyosarcoma spheroids towards NK cell attack

“…Hence, novel treatment strategies are needed, particularly for the hard to target and more aggressive alveolar/fusion-positive RMS subtype [3,18]. In contrast to the ever optimizing chemotherapeutic regiments [19], novel therapies and targeted approaches, like immune-checkpoint inhibitors [20], cellular-immunotherapies (CAR T/NK cells) [21], blockade of PAX-FOXO1 co-regulators by epigenetic targeting [22], sensitizing pro-cell death pathways (autophagy, apoptosis) [23,24] or combination of mentioned strategies [25,26] are being investigated. Focusing on a cellular based immunotherapeutic approach might be challenging, due to an immunosuppressive tumor microenvironment (TME) within the solid RMS tissue [27].…”

“…Again, these studies either used 2D flow cytometric cytotoxicity assays [25,117,118,121], and/or used artificial models of immunocompromised mice [119,168]. Following the 3R principles of animal research (Replacement, Reduction, Refinement), we utilized a multicellular RMS spheroid model, as an in-vivo-like model resembling the solid rhabdomyosarcoma tumor phenotype more closely [26,[170][171][172].…”