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Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug.
High-risk rhabdomyosarcoma (RMS) occurring in childhood to young adulthood is associated with a poor prognosis; especially children above the age of 10 with advanced stage alveolar RMS still succumb to the disease within a median of 2 years. The advent of chimeric antigen receptor (CAR)-engineered T cells marked significant progress in the treatment of refractory B cell malignancies, but experience for solid tumors has proven challenging. We speculate that this is at least in part due to the poor quality of the patient's own T cells and therefore propose using CAR-modified cytokine-induced killer (CIK) cells as effector cells. CIK cells are a heterogeneous population of polyclonal T cells that acquire phenotypic and cytotoxic properties of natural killer (NK) cells through the cultivation process, becoming so-called T-NK cells. CIK cells can be genetically modified to express CARs. They are minimally alloreactive and can therefore be acquired from haploidentical first-degree relatives. Here, we explored the potential of ERBB2-CAR-modified random-donor CIK cells as a treatment for RMS in xenotolerant mice bearing disseminated high-risk RMS tumors. In otherwise untreated mice, RMS tumors engrafted 13–35 days after intravenous tumor cell injection, as shown by in vivo bioluminescence imaging, immunohistochemistry, and polymerase chain reaction for human gDNA, and mice died shortly thereafter (median/range: 62/56–66 days, n = 5). Wild-type (WT) CIK cells given at an early stage delayed and eliminated RMS engraftment in 4 of 6 (67%) mice, while ERBB2-CAR CIK cells inhibited initial tumor load in 8 of 8 (100%) mice. WT CIK cells were detectable but not as active as CAR CIK cells at distant tumor sites. CIK cell therapies during advanced RMS delayed but did not inhibit tumor progression compared to untreated controls. ERBB2-CAR CIK cell therapy also supported innate immunity as evidenced by selective accumulation of NK and T-NK cell subpopulations in disseminated RMS tumors, which was not observed for WT CIK cells. Our data underscore the power of heterogenous immune cell populations (T, NK, and T-NK cells) to control solid tumors, which can be further enhanced with CARs, suggesting ERBB2-CAR CIK cells as a potential treatment for high-risk RMS.
Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD.