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. 2023 Nov 4;13(1):19079.
doi: 10.1038/s41598-023-46305-7.

Novel PD-L1- and collagen-expressing patient-derived cell line of undifferentiated pleomorphic sarcoma (JBT19) as a model for cancer immunotherapy

Pavla Taborska  1 , Pavol Lukac  2   3 , Dmitry Stakheev  1   2 , Lenka Rajsiglova  2   3 , Katerina Kalkusova  1 , Karolina Strnadova  4   5 , Lukas Lacina  4   5   6 , Barbora Dvorankova  4   5 , Jiri Novotny  7 , Michal Kolar  7 , Milena Vrana  8 , Hana Cechova  8 , Sarka Ransdorfova  9 , Marie Valerianova  9 , Karel Smetana Jr  4   5 , Luca Vannucci  2 , Daniel Smrz  10   11
Affiliations

Novel PD-L1- and collagen-expressing patient-derived cell line of undifferentiated pleomorphic sarcoma (JBT19) as a model for cancer immunotherapy

Pavla Taborska et al. Sci Rep. .

Abstract

Soft tissue sarcomas are aggressive mesenchymal-origin malignancies. Undifferentiated pleomorphic sarcoma (UPS) belongs to the aggressive, high-grade, and least characterized sarcoma subtype, affecting multiple tissues and metastasizing to many organs. The treatment of localized UPS includes surgery in combination with radiation therapy. Metastatic forms are treated with chemotherapy. Immunotherapy is a promising treatment modality for many cancers. However, the development of immunotherapy for UPS is limited due to its heterogeneity, antigenic landscape variation, lower infiltration with immune cells, and a limited number of established patient-derived UPS cell lines for preclinical research. In this study, we established and characterized a novel patient-derived UPS cell line, JBT19. The JBT19 cells express PD-L1 and collagen, a ligand of the immune checkpoint molecule LAIR-1. JBT19 cells can form spheroids in vitro and solid tumors in immunodeficient nude mice. We found JBT19 cells induce expansion of JBT19-reactive autologous and allogeneic NK, T, and NKT-like cells, and the reactivity of the expanded cells was associated with cytotoxic impact on JBT19 cells. The PD-1 and LAIR-1 ligand-expressing JBT19 cells show ex vivo immunogenicity and effective in vivo xenoengraftment properties that can offer a unique resource in the preclinical research developing novel immunotherapeutic interventions in the treatment of UPS.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of JBT19 cells. (a) JBT19 growth curve. (b) Brightfield microscopy of adherent JBT19 cells. (c) Confocal microscopy of JBT19 cells intracellularly stained with collagen- or PD-L1-specific antibody. (d) Immunohistochemistry of JBT19 cells stained with nestin- (top left panel), FAP- (top right panel), vimentin- (bottom left panel), or desmin- (bottom right panel) specific antibody. In (b) and ( d), the bars represent 100 μm. In (c), the bar represents 50 μm.
Figure 2
Figure 2
JBT19 phenotypic characterization. (a) The gating strategy of flow cytometry data. (b) Intracellular staining with Ki-67, collagen-, αSMA-, nestin-, or vimentin-specific antibody. (c) The evaluation of the mean fluorescence intensity (MFI) staining in b. (d) Extracellular staining with FAP-, CD44-, HLA-ABC, CD95(Fas)-, CD47-, or PD-L1-specific antibody. (e) The evaluation of the mean fluorescence intensity (MFI) staining in d. (f) Extracellular staining with CD31-, PD-1-, CD133-, CD30-, TRAIL-, HLA-DP, DQ, DR-, CD34-, CD117-, Gal-9-, DR3-, or CD95L(FasL)-specific antibody. In (b), (d), and (f), representative histograms are shown, and Ctrl for individual fluorochromes means staining with the vehicle alone. In (c) and (e), mean + SEM and statistical significances of differences between the group of unstained and stained samples are indicated (*p < 0.05; n = 6 (FAP, CD44), n = 5 (Ki-67, Collagen, αSMA, nestin, CD47, PD-L1), n = 4 (vimentin, HLA-ABC, CD95); paired two-tailed Student’s t-test).
Figure 3
Figure 3
Short tandem repeats, HLA genotype, and karyotype characterization of JBT19 cells. (a) JBT19 short tandem repeats (STR). (b) HLA genotype of JBT19 cells and peripheral blood cells of the patient. (c) Karyotype analysis by probe 24XCyte mFISH Probe (MetaSystems). One metaphase representing a composite karyotype (in this cell some abnormalities are absent, some non-clonal abnormalities are present).
Figure 4
Figure 4
Transcriptome signatures of JBT19 cells. (a) The single-cell transcriptome profiling of 2D-cultured JBT19 cells shows the homogeneity of the JBT19 cell population. The cells do not show any significant stratification, with the exception of a weakly distinguishable subpopulation marked by the hexagon in the bottom left part of the t-distributed stochastic neighborhood embedding (tSNE) plot. These cells express less the marker of proliferation MKI67 (left), tend to be in the G1 phase of the cell cycle (center), and have blurry cell annotation (right, iPS induced pluripotent cells, MSC mesenchymal stem cells, NA not annotated). (b) When combined in a pseudo bulk sample, the JBT19 cells display the expression profile that matches the undifferentiated pleomorphic sarcoma or myxofibrosarcoma samples from the TCGA SARC study. These sarcoma subtypes are hardly distinguishable by molecular markers. Each point represents one clinical sarcoma sample; MPNST stands for malignant peripheral nerve sheath tumor.
Figure 5
Figure 5
JBT19 spheroids and characteristics of JBT19 tumors developed in nude mice. (a,b) Representative images of a 4-day (a) and 14-day (b) spheroid originated by seeding of 0.273 × 106 JBT19 cells/agarose 81-microwell dish at day 0. (c) Nude mice after s.c. inoculation of JBT19 cells (1 × 106). Representative images of the development of a localized JBT19 tumor (red arrow) in a nude mouse 14 (top left panel), 24 (top right panel), 42 (bottom left panel), and 60 (bottom right panel) days after the inoculation. (d) Growth of JBT19 tumors after s.c. inoculation of JBT19 cells (1 × 106) in nude mice (n = 6). (e) Collagen distribution and organization in 24-day-old JBT19 tumor sections imaged by two-photon confocal microscopy in SHG mode. (f) Representative visualization of collagen structures (left panel) and PD-L1 (right panel) in 24-day JBT19 tumor sections using immunohistochemistry staining with specific antibodies and one-photon confocal microscopy. In (f), collagen is visualized in red, PD-L1 is visualized in green; nuclei are stained with Hoechst 33,258 (blue) or Draq5 (red). The bars in (a,b) and (e,f) are 100 μm.
Figure 6
Figure 6
Characterization of K-562- or JBT19-primed and expanded healthy donors' and patient's lymphocytes (a) Proliferation, viability, and lymphocyte population frequencies of the K-562-primed and expanded healthy donors' (HD) and patient's (Pac) lymphocytes determined using the gating strategy in Fig. 7a. (b) Same tests like in (a) but using JBT19-primed and expanded HD and Pac lymphocytes. Bars represent the mean of values and SEM determined in each group (HD, n = 4 donors; Pac, n = 2 preparations).
Figure 7
Figure 7
Reactivity of K-562-primed and expanded healthy donors' and the patient's lymphocytes to K-562 and JBT19 cells. (a) The gating strategy of flow cytometry data. (b) The frequencies of K-562- or JBT19-reactive TNFα+- (left panels), IFNγ+- (middle panels), and TNFα+/IFNγ+- (right panels) producing K-562-primed and expanded healthy donors' (HD) or patient's (Pac) lymphocyte populations after stimulation with K-562 (K-562 stim) or JBT19 (JBT19 stim) cells. Bars represent the mean of values and SEM determined in each group (HD, n = 4 donors; Pac, n = 2 preparations).
Figure 8
Figure 8
Reactivity of JBT19-primed and expanded healthy donors' and the patient's lymphocytes to K-562 and JBT19 cells. Cytotoxic impact of K-562- or JBT19-primed and expanded HD and patient's lymphocytes on 2D-cultured TurboGFP-JBT19 cells. (a) The flow cytometry data were gated as in Fig. 7a. Shown are frequencies of K-562- or JBT19-reactive TNFα+- (left panels), IFNγ+- (middle panels), and TNFα+/IFNγ+- (right panels) producing JBT19-primed and expanded healthy donors' (HD) or patient's (Pac) lymphocyte populations after stimulation with K-562 (K-562 stim) or JBT19 (JBT19 stim) cells. Bars represent the mean of values and SEM determined in each group (HD, n = 4 donors; Pac, n = 2 preparations). (b) Adherent TurboGFP-JBT19 cells (left panel) were cocultured with expanded HD and patient's lymphocytes, and the cytotoxic impact of the coculture on TurboGFP-JBT19 cells was evaluated via the TurboGFP fluorescence in the cell culture (right panel). Bars represent the mean of values and SEM determined in each group (HD, n = 4 donors; Pac, n = 2 preparations).
Figure 9
Figure 9
LAIR-1 expression in JBT19-primed and expanded healthy donors' lymphocytes after stimulation with JBT19 cells. (a) The flow cytometry data were gated as in Fig. 7a. Shown are representative images of JBT19-stimulated cells. (b) The frequencies of LAIR-1- (left bars) and LAIR-1+ (right bars) JBT19-primed and expanded healthy donors' (HD) NK cells, CD4+ T cells, CD8+ T cells, and CD8+ NKT-like cells. (c) The frequencies of JBT19-reactive and TNFα-producing LAIR-1- (left bars) or LAIR-1+ (right bars) JBT19-primed and expanded healthy donors' (HD) NK cells, CD4+ T cells, CD8+ T cells, and CD8+ NKT-like cells after stimulation with JBT19 cells. Bars in (a) and (b) represent the mean of values and SEM determined in each group, and statistical significances of differences between the group of LAIR-1− and LAIR-1+ populations are indicated (*p < 0.05; HD, n = 4; paired two-tailed Student's t-test).
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References

    1. Gamboa AC, Gronchi A, Cardona K. Soft-tissue sarcoma in adults: An update on the current state of histiotype-specific management in an era of personalized medicine. CA Cancer J. Clin. 2020;70:200–229. doi: 10.3322/caac.21605. - DOI - PubMed
    1. Jo VY, Fletcher CD. WHO classification of soft tissue tumours: An update based on the 2013 (4th) edition. Pathology. 2014;46:95–104. doi: 10.1097/PAT.0000000000000050. - DOI - PubMed
    1. O'Brien JE, Stout AP. Malignant fibrous xanthomas. Cancer. 1964;17:1445–1455. doi: 10.1002/1097-0142(196411)17:11<1445::aid-cncr2820171112>3.0.co;2-g. - DOI - PubMed
    1. Choi JH, Ro JY. The 2020 WHO classification of tumors of soft tissue: Selected changes and new entities. Adv. Anatom. Pathol. 2021;28:44–58. doi: 10.1097/PAP.0000000000000284. - DOI - PubMed
    1. Widemann BC, Italiano A. Biology and management of undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and malignant peripheral nerve sheath tumors: State of the art and perspectives. J. Clin. Oncol. 2018;36:160–167. doi: 10.1200/JCO.2017.75.3467. - DOI - PMC - PubMed

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