Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 19 (1), 1172

Biological Treatment of Pediatric Sarcomas by Combined Virotherapy and NK Cell Therapy

Affiliations

Biological Treatment of Pediatric Sarcomas by Combined Virotherapy and NK Cell Therapy

Chihab Klose et al. BMC Cancer.

Abstract

Background: In pediatric sarcomas, outcomes of established therapies still remain poor, especially due to high-grade resistances to chemotherapeutic compounds. Taking novel biological approaches into account, virotherapy was found to be efficient in many pediatric sarcoma types. Also NK cell therapy was denoted to represent a promising upcoming strategy for pediatric sarcoma patients. We here investigated a combinatorial approach employing oncolytic measles vaccine virotherapeutics (MeV) together with activated human NK cells (or PBMCs).

Methods: The human sarcoma cell lines A673 and HT1080 were used to evaluate the efficacy of this combinatorial treatment modality. Oncolysis was determined by measuring real-time cell proliferation using the xCELLigence RTCA SP system. Furthermore, expression of receptors on NK cells and the respective ligands on A673 cells was analyzed by flow cytometry. To measure the protein release of activated NK cells a LEGENDplex™ assay was performed.

Results: Monotherapy with MeV led to a time- and dose-dependent oncolytic reduction of A673 and HT1080 sarcoma tumor cell masses. Concurrently, such MeV infections did not change the expression of NK cell ligands MICA/B, ULBP1, 2, and 3, CD112, and CD155. As shown by real-time proliferation assays, infections of A673 and HT1080 sarcoma cells with MeV followed by co-culture with activated NK cells or PBMCs led to enhanced sarcoma cell destruction when compared to the respective monotherapies. In parallel, this dual therapy resulted in an increased release of granzymes, perforin, and granulysin from NK cells. In contrast, expression of activation and ontogenesis receptors on NK cells was not found to be altered after co-culture with MeV-infected A673 sarcoma cells.

Conclusions: Taken together, the combined treatment strategy comprising oncolytic MeV and activated NK cells resulted in enhanced oncolysis of A673 and HT1080 cells when compared to the respective monotherapies. In parallel, we observed an increased release of NK cell activation markers upon co-culture with MeV-infected A673 human sarcoma cells. These results support the onset of clinical trials combining oncolytic virotherapy with NK cell based immunotherapies.

Keywords: Immunotherapy; Measles vaccine virus; NK cells; Pediatric sarcoma; Virotherapy.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Viability of A673 (a) and HT1080 (b) sarcoma cell lines after infection with measles vaccine virus MeV-GFP. A673 (a) and HT1080 (b) cells were infected with MeV-GFP at multiplicities of infection (MOIs) of 0.1, 1, and 10, or MOCK-infected. At 24, 48, 72, and 96 h post infection (hpi) the remaining tumor cell mass was determined by SRB viability assay. MeV-GFP-mediated oncolysis is calculated relative to the MOCK-infected control. The mean ± SD of three independent experiments performed in quadruplicates is shown. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant
Fig. 2
Fig. 2
Expression of NK cell ligands on mock- vs. MeV-GFP-infected A673 sarcoma cells. A673 cells were mock-infected (left panels) or infected with MeV-GFP at MOI 0.5 (right panels). At 48 hpi expression levels of NK cell ligands MICA/MICB (a), ULBP1 (b), ULBP2 (c), ULBP3 (d), CD112 (e), and CD155 (f) were determined by flow cytometry. One representative of three independent experiments is shown. MeV, measles vaccine virus; GFP, green fluorescent protein
Fig. 3
Fig. 3
MeV-mediated induction of programmed death ligand 1 (PD-L1) expression on A673 cells. A673 cells were mock-infected (left panel) or infected with MeV-GFP (MOI 0.5) (right panel). At 48 hpi expression of PD-L1 was determined by flow cytometry. One representative of three independent experiments is shown
Fig. 4
Fig. 4
Characterization of NK cell receptors after co-culture of NKAES with MeV-infected A673 sarcoma cells. NKAES: activated and expanded NK cells; NKAES d2: on d2 without co-culture; NKAES+A673 d2: two days co-culture (E:T ratio = 2.5:1) with uninfected A673 sarcoma cells; NKAES+A673* d2: two days co-culture (E:T ratio = 2.5:1) with MeV-GFP infected A673 sarcoma cells (MOI 1). Samples were subjected to flow cytometric quantification of the proportion of cells expressing the given receptors. Bars represent the mean percentage of the respective CD56+ CD3- NK cell subsets, error bars display SD. Note, that the CD56 receptor is not included in this diagram as all cells were gated on CD56 prior to subset analysis. Results represent data from 5 different donors. * p < 0.05
Fig. 5
Fig. 5
Real-time analysis of MeV-GFP-mediated oncolysis of A673 sarcoma cells after co-treatment with PBMC/NKAES isolated from a healthy donor. At 21 h after seeding, A673 cells were infected with MeV (MOI 0.5) (right panels) or mock-infected (left panels; base line controls). At 51 hpi, (a) PBMC, (b) PBMC stimulated with IL-2, or (c) NKAES from a healthy donor were added at an E:T ratio of 2.5:1. Triton X-100 was added as a negative control inducing maximum lysis of tumor cells. Real-time cell proliferation was monitored using the xCELLigence RTCA SP system. Measured electrode impedance is expressed as Cell Index. One representative of three independent experiments performed in triplicates using different donors is shown
Fig. 6
Fig. 6
Statistical analysis of MeV-GFP-mediated oncolysis of A673 sarcoma cells after co-treatment with PBMC/NK cells isolated from a healthy donor. Analysis was performed as described in Fig. 5. Real-time cell proliferation is depicted as Cell Index after addition of PBMCs (a), PBMC stimulated with IL-2 (b), and NK cells (c) at 107 hpi. * p < 0.05; ** p < 0.01, *** p < 0.001, n.s. not significant
Fig. 7
Fig. 7
Real-time analysis of MeV-GFP-mediated oncolysis of HT1080 sarcoma cells after co-treatment with PBMC/NKAES isolated from a healthy donor. At 24 h after seeding, HT1080 cells were infected with MeV (MOI 5) (A, lower panel) or mock-infected (a, upper panel; base line controls). At 23 hpi, NK cells from a healthy donor were added at E:T ratios of 1:1, 2.5:1 and 5:1. Triton X-100 was added as a negative control inducing maximum lysis of tumor cells. Real-time cell proliferation was monitored using the xCELLigence RTCA SP system until 72 hpi. Measured electrode impedance is expressed as Cell Index. One representative of two independent experiments performed in quadruplicates using different donors is shown. (b) Statistical analysis of the same experiment. * p < 0.05; ** p < 0.01, *** p < 0.001, n.s. not significant
Fig. 8
Fig. 8
Quantification of protein release from NK cells after co-culture with MeV-infected A673 sarcoma cells. A673 sarcoma cells were infected with MeV-GFP (MOI 1) or mock-infected. At 24 hpi, NK cells from healthy donors were added at an E:T ratio of 2.5:1. Then, 24 h later, supernatants were collected and protein contents were determined by flow cytometry using LEGENDplex™ assay. A673 sarcoma cells without addition of NK cells were used as controls. The mean ± SD resulting from three different donors is shown. * p < 0.05; ** p < 0.01, *** p < 0.001, n.s. not significant

Similar articles

See all similar articles

References

    1. Russell SJ, Peng KW. Viruses as anticancer drugs. Trends Pharmacol Sci. 2007;28(7):326–333. doi: 10.1016/j.tips.2007.05.005. - DOI - PMC - PubMed
    1. Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol. 2012;30(7):658–670. doi: 10.1038/nbt.2287. - DOI - PMC - PubMed
    1. Hamid O, Hoffner B, Gasal E, Hong J, Carvajal RD. Oncolytic immunotherapy: unlocking the potential of viruses to help target cancer. Cancer immunology, immunotherapy : CII. 2017;66(10):1249–1264. doi: 10.1007/s00262-017-2025-8. - DOI - PMC - PubMed
    1. Lichty BD, Breitbach CJ, Stojdl DF, Bell JC. Going viral with cancer immunotherapy. Nat Rev Cancer. 2014;14(8):559–567. doi: 10.1038/nrc3770. - DOI - PubMed
    1. Russell SJ, Peng KW. Oncolytic Virotherapy: a contest between apples and oranges. Molecular therapy : the journal of the American Society of Gene Therapy. 2017;25(5):1107–1116. doi: 10.1016/j.ymthe.2017.03.026. - DOI - PMC - PubMed

LinkOut - more resources

Feedback