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Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. Cancer Res; 74(1); 119-29. Ó2013 AACR.

For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer.

Chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR Ts) produced impressive clinical results against selected hematological malignancies, but the extension of CAR T cell therapy to the challenging field of solid tumors has not, so far, replicated similar clinical outcomes. Many efforts are currently dedicated to improve the efficacy and safety of CAR-based adoptive immunotherapies, including application against solid tumors. A promising approach is CAR engineering of immune effectors different from αβT lymphocytes. Herein we reviewed biological features, therapeutic potential, and safety of alternative effectors to conventional CAR T cells: γδT, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells. The intrinsic CAR-independent antitumor activities, safety profile, and ex vivo expansibility of these alternative immune effectors may favorably contribute to the clinical development of CAR strategies. The proper biological features of innate immune response effectors may represent an added value in tumor settings with heterogeneous CAR target expression, limiting the risk of tumor clonal escape. All these properties bring out CAR engineering of alternative immune effectors as a promising integrative option to be explored in future clinical studies.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR+.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR+.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities.We report first evidence of CAR+.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR+.CIK in clinical trials against high grade STS.

The term “cancer stem cells” (CSCs) commonly refers to a subset of tumor cells endowed with stemness features, potentially involved in chemo-resistance and disease relapses. CSCs may present peculiar immunogenic features influencing their homeostasis within the tumor microenvironment. The susceptibility of CSCs to recognition and targeting by the immune system is a relevant issue and matter of investigation, especially considering the multiple emerging immunotherapy strategies. Adoptive cellular immunotherapies, especially those strategies encompassing the genetic redirection with chimeric antigen receptors (CAR), hold relevant promise in several tumor settings and might in theory provide opportunities for selective elimination of CSC subsets. Initial dedicated preclinical studies are supporting the potential targeting of CSCs by cellular immunotherapies, indirect evidence from clinical studies may be derived and new studies are ongoing. Here we review the main issues related to the putative immunogenicity of CSCs, focusing on and highlighting the existing evidence and opportunities for cellular immunotherapy approaches with T and non-T antitumor lymphocytes.

This work reports the effective antitumor activity of patient-derived cytokine-induced killer (CIK) cells against autologous chemo-resistant melanoma Cancer Stem Cells (CSCs).CSCs are clinical relevant targets, associated with disease relapse. We demonstrate that chemotherapy kills indeed proliferating melanoma cells but spares tumorigenic CSCs, in vitro and in vivo. The MHCindependent immunotherapy with CIK cells was proved successful in this challenging framework.Consistent findings were obtained in selected cases of BRAF mutated melanoma treated with small molecule BRAFi. Our data, generated within an autologous system, support the exploration of CIK cells in clinical trials. Cost effectiveness, safety profile and ability to overcome tumor MHC-downregulation are favorable issues to be considered in clinical perspective. CIK cells may be integrated at different levels in the composite therapeutic scenario of metastatic melanoma, offering an additional weapon to control tumor spread and promote its eradication. ABSTRACT PurposeThe MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSCs) was explored, as CSCs are considered responsible for chemo-resistance and relapses. Experimental designPutative mCSCs were visualized by engineering patient-derived melanoma cells (MCs) with a lentiviralvector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays.We explored the sensitivity of eGFP CIK cell activity against chemoresistant mCSCs was confirmed vivo in two distinct immunodeficient murine models. ResultsWe visualized eGFP + mCSCs (14±2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP-positive MCs (1/42) compared with the eGFP-negative counterpart (1/4870).In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells ( ConclusionsThese findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving chemotherapy or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided.

BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti-PD-1 antibody. A portion of melanoma cells may express PD-1, and anti-PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1 melanoma cells, supporting an additional-lymphocyte-independent-basis for their therapeutic combination with anti-PD-1 antibody. With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, = 61; validation cell lines, = 7) and melanoma tumors (TCGA, = 214). We explored how BRAF/MEKi affect rates of PD-1, PD-L1/2 melanoma cells, and characterized the proliferative and putative stemness features of PD-1 melanoma cells. We tested the functional lymphocyte-independent effect of anti-PD-1 antibody alone and in combination with BRAF/MEKi and in an immunodeficient murine model. PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1 cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6-14.2) vs. 1.5% (0.7-3.2), = 0.0156; = 7], together with PD-L2 melanoma cells [8.5% (0.0-63.0) vs. 1.5% (0.2-43.3), = 0.0312; = 7]. PD-1 cells proliferate less than PD-1 cells (avg. 65% less; = 7 days) and are preferentially endowed with stemness features. , the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse. BRAF/MEKi increase the rates of PD-1 melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti-PD-1 antibody. .