Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D–NKG2DL dependent manner

Highlights

• NK cells ability to target osteosarcoma cells and the pathways involved were studied.

• We explored NK cell elimination of osteosarcoma (OS) Tumor Initiating Cells (TICs).

• NKG2D–NKG2DL pathway is involved in NK cell elimination of OS cells, including TICs.

• NK cells reduce tumor volume, metastasis and improve survival in an OS animal model.

Abstract

Current therapies fail to cure most metastatic or recurrent bone cancer. We explored the efficacy and the pathways involved in natural killer (NK) cells' elimination of osteosarcoma (OS) cells, including tumor initiating cells (TICs), which are responsible for chemotherapy resistance, recurrence, and metastasis.

The expression of ligands for NK cell receptors was studied in primary OS cell lines by flow cytometry. In vitro cytotoxicity of activated and expanded NK (NKAE) cells against OS was tested, and the pathways involved explored by using specific antibody blockade. NKAE cells' ability to target OS TICs was analyzed by flow cytometry and sphere formation assays. Spironolactone (SPIR) was tested for its ability to increase OS cells' susceptibility to NK cell lysis in vitro and in vivo.

We found OS cells were susceptible to NKAE cells' lysis both in vivo and in vitro, and this cytolytic activity relied on interaction between NKG2D receptor and NKG2D ligands (NKG2DL). SPIR increased OS cells' susceptibility to lysis by NKAE cells, and could shrink the OS TICs.

Our results show NKAE cells target OS cells including the TICs compartment, supporting the use of NK-cell based immunotherapies for OS.

Introduction

Osteosarcoma (OS) is the most common type of solid bone cancer, mainly affecting children and young adults. OS usually affects long bones, in particular the distal femur and proximal tibia. For the last three decades, mainstream treatments consisting of radical surgery and chemotherapy have yielded a 5-year survival rate of 67% [1]. About 20% of patients present metastases at diagnosis primarily in the lungs. Once the metastases have occurred, the prognosis is very poor, and the current therapeutic strategies have shown limited efficacy. Furthermore, prognosis of patients with recurrent disease is also dismal. OS remains the second leading cause of cancer-related death in children and young adults [2], therefore, there is a need for new therapeutic approaches. Current treatment failure could be related to the inability to target OS TICs [3]. TICs, also called cancer stem cells, are a subset of tumor cells characterized by self-renewal capacity, increased tumorigenicity, and resistance to chemotherapeutic agents [4]. TICs from OS have been described by their capacity to grow as spheres (sarcospheres) under serum-starvation conditions [5], by their expression of different surface markers such as c-kit, CD133, or Stro-1 [6], [7], or as a “side population” through the exclusion of the vital dye Hoechst 33342 [8]. OS TICs also express CXCR4, a metastasis-associated stem cell marker. CXCR4 expressing cells metastasize to organs such as lung that produce high levels of SDF-1 (or CXCL12). Clinically, greater expression of CXCR4 correlates with disease severity [9], [10], [11].

NK cells are lymphocytes of the innate immune system that can eliminate virally infected or transformed cells without prior sensitization [12]. The interactions between inhibitory and activating NK cell receptors and their ligands are determinants of NK cell activation and anti-tumor activity [13]. The main NK cell activating receptors are NKG2D, DNAM-1, and the natural cytotoxicity receptors (NCRs). NKG2D receptor recognizes human MICA/B and ULBP1-6 [14], [15], [16] ligands, whereas DNAM-1 recognizes CD112 (Nectin-2) and CD155 (PVR) [17]. NKG2D ligands (NKG2DLs) are expressed selectively or at low levels by normal cells but are upregulated during stress and cellular transformation. NKG2D stimulation of NK cells leads to strong activation and tumor cell rejection. Although NKG2DLs are up-regulated in most tumor cells, sarcomas are the tumor type most sensitive to NK cell cytotoxicity [18]. However, during tumor progression, tumors evade immunosurveillance by different mechanisms such as shedding or downregulating NKG2DL. Different therapeutic agents such as spironolactone (SPIR) [19], irradiation [18], and gemcitabine [20], [21] enhance NKG2DL expression in different tumor cell lines, which then become more sensitive to NK cell-mediated lysis.

In this report we demonstrate that activated and expanded NK (NKAE) cells can efficiently eliminate primary and metastatic OS cells, including OS TICs. The elimination of OS by NK cells relies in NKG2D–NKG2DL interaction. We also found OS cells being highly responsive to SPIR, triggering up-regulation of surface NKG2DL and increasing their susceptibility to NKAE cell lysis both in vitro and in vivo. Finally we found NKAEs and SPIR could reduce OS TICs ability to form sarcospheres and to express c-kit and CXCR4. Together our data support the use of NKAE cells in combination with SPIR as a new treatment for OS.