Spatial multiplexed immunofluorescence analysis reveals coordinated cellular networks associated with overall survival in metastatic osteosarcoma

Abstract

Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

References

[1.] GillJ, GorlickR. Advancing therapy for osteosarcoma. Nat. Rev. Clin. Oncol., 2021, 18: 609-624

[2.] YangC, et al. . Bone microenvironment and osteosarcoma metastasis. Int. J. Mol. Sci., 2020, 21: 6985

[3.] Fletcher, C. D., Unni, K. K., & Mertens, F. (Eds.). (2002). Pathology and genetics of tumours of soft tissue and bone (Vol. 4). Iarc.

[4.] MesserschmittPJ, GarciaRM, Abdul-KarimFW, GreenfieldEM, GettyPJ. Osteosarcoma. JAAOS - J. Am. Acad. Orthop. Surg., 2009, 17: 515-527

[5.] FoxMG, TrottaBM. Osteosarcoma: review of the various types with emphasis on recent advancements in imaging. Semin. Musculoskelet. Radio., 2013, 17: 123-136

[6.] SmrkeA, et al. . Future directions in the treatment of osteosarcoma. Cells, 2021, 10: 172

[7.] CorreI, VerrecchiaF, CrennV, RediniF, TrichetV. The osteosarcoma microenvironment: a complex but targetable ecosystem. Cells, 2020, 9: 976

[8.] GasparN, et al. . Recent advances in understanding osteosarcoma and emerging therapies. Faculty Rev., 2020, 9: 18

[9.] CasciniC, ChiodoniC. The immune landscape of osteosarcoma: implications for prognosis and treatment response. Cells, 2021, 10: 1668

[10.] MeazzaC, ScanagattaP. Metastatic osteosarcoma: a challenging multidisciplinary treatment. Expert Rev. Anticancer Ther., 2016, 16: 543-556

[11.] XiaoX, WangW, WangZ. The role of chemotherapy for metastatic, relapsed and refractory osteosarcoma. Paediatr. Drugs, 2014, 16: 503-512

[12.] HuZ, et al. . Current status and prospects of targeted therapy for osteosarcoma. Cells, 2022, 11: 3507

[13.] SupraR, AgrawalDK. Immunotherapeutic strategies in the management of osteosarcoma. J. Orthop. Sports Med., 2023, 5: 32-40

[14.] YahiroK, MatsumotoY. Immunotherapy for osteosarcoma. Hum. Vaccin Immunother., 2021, 17: 1294-1295

[15.] ParkJA, CheungNV. Promise and challenges of T cell immunotherapy for osteosarcoma. Int. J. Mol. Sci., 2023, 24: 12520

[16.] NiralaBK, YamamichiT, PetrescuDI, ShafinTN, YusteinJT. Decoding the impact of tumor microenvironment in osteosarcoma progression and metastasis. Cancers (Basel), 2023, 15: 5108

[17.] ZhuT, et al. . Immune microenvironment in osteosarcoma: components, therapeutic strategies and clinical applications. Front. Immunol., 2022, 13

[18.] TianH, et al. . Managing the immune microenvironment of osteosarcoma: the outlook for osteosarcoma treatment. Bone Res., 2023, 11: 11

[19.] GuoJ, et al. . Single-cell profiling of tumor microenvironment heterogeneity in osteosarcoma identifies a highly invasive subcluster for predicting prognosis. Front. Oncol., 2022, 12

[20.] HuangR, et al. . Combining bulk RNA-sequencing and single-cell RNA-sequencing data to reveal the immune microenvironment and metabolic pattern of osteosarcoma. Front. Genet., 2022, 13

[21.] HuangX, WangL, GuoH, ZhangW, ShaoZ. Single-cell transcriptomics reveals the regulative roles of cancer associated fibroblasts in tumor immune microenvironment of recurrent osteosarcoma. Theranostics, 2022, 12: 5877-5887

[22.] LiuY, et al. . Single-cell transcriptomics reveals the complexity of the tumor microenvironment of treatment-naive osteosarcoma. Front. Oncol., 2021, 11

[23.] LiuW, et al. . Characterizing the tumor microenvironment at the single-cell level reveals a novel immune evasion mechanism in osteosarcoma. Bone Res., 2023, 11: 4

[24.] ThomasDD, LacinskiRA, LindseyBA. Single-cell RNA-seq reveals intratumoral heterogeneity in osteosarcoma patients: a review. J. Bone Oncol., 2023, 39

[25.] ZhongC, et al. . Single-cell and bulk RNA sequencing reveals Anoikis related genes to guide prognosis and immunotherapy in osteosarcoma. Sci. Rep., 2023, 13

[26.] ZhouY, et al. . Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma. Nat. Commun., 2020, 11

[27.] EvdokimovaV, GassmannH, RadvanyiL, BurdachSEG. Current state of immunotherapy and mechanisms of immune evasion in ewing sarcoma and osteosarcoma. Cancers (Basel), 2022, 15: 272

[28.] LigonJA, et al. . Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes. J. Immunother. Cancer, 2021, 9: e001772

[29.] CilloAR, et al. . Ewing sarcoma and osteosarcoma have distinct immune signatures and intercellular communication networks. Clin. Cancer Res., 2022, 28: 4968-4982

[30.] BressanD, BattistoniG, HannonGJ. The dawn of spatial omics. Science, 2023, 381

[31.] BlackS, et al. . CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nat. Protoc., 2021, 16: 3802-3835

[32.] GoltsevY, et al. . Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell, 2018, 174: 968-981.e915

[33.] PhillipsD, et al. . Highly multiplexed phenotyping of immunoregulatory proteins in the tumor microenvironment by CODEX tissue imaging. Front. Immunol., 2021, 12

[34.] PhillipsD, et al. . Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma. Nat. Commun., 2021, 12

[35.] SchürchCM, et al. . Coordinated cellular neighborhoods orchestrate antitumoral immunity at the colorectal cancer invasive front. Cell, 2020, 183: 838

[36.] ShekarianT, et al. . Immunotherapy of glioblastoma explants induces interferon-γ responses and spatial immune cell rearrangements in tumor center, but not periphery. Sci. Adv., 2022, 8

[37.] RufB, et al. . Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin. Cell, 2023, 186: 3686-3705.e3632

[38.] MaestriE, et al. . Spatial proximity of tumor-immune interactions predicts patient outcome in hepatocellular carcinoma. Hepatology, 2023, 79: 768-779

[39.] WeedDT, et al. . The tumor immune microenvironment architecture correlates with risk of recurrence in head and neck squamous cell carcinoma. Cancer Res., 2023, 83: 3886-390

[40.] HickeyJW, et al. . T cell-mediated curation and restructuring of tumor tissue coordinates an effective immune response. Cell Rep., 2023, 42

[41.] MascharakS, et al. . Desmoplastic stromal signatures predict patient outcomes in pancreatic ductal adenocarcinoma. Cell Rep. Med., 2023, 4

[42.] MondelloP, et al. . Lack of intrafollicular memory CD4⁺ T cells is predictive of early clinical failure in newly diagnosed follicular lymphoma. Blood Cancer J., 2021, 11

[43.] EinhausJ, RochwargerA, MatternS, GaudillièreB, SchürchCM. High-multiplex tissue imaging in routine pathology-are we there yet?. Virchows Arch., 2023, 482: 801-812

[44.] MoffittJR, LundbergE, HeynH. The emerging landscape of spatial profiling technologies. Nat. Rev. Genet., 2022, 23: 741-759

[45.] WalshLA, QuailDF. Decoding the tumor microenvironment with spatial technologies. Nat. Immunol., 2023, 24: 1982-1993

[46.] GreenwaldNF, et al. . Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning. Nat. Biotechnol., 2022, 40: 555-565

[47.] HaistM, StegeH, GrabbeS, BrosM. The functional crosstalk between myeloid-derived suppressor cells and regulatory T cells within the immunosuppressive tumor microenvironment. Cancers (Basel), 2021, 13: 210

[48.] Li, Q., Huang, X. & Zhao, Y. Prediction of prognosis and immunotherapy response with a novel natural killer cell marker genes signature in osteosarcoma. Cancer Biother. Radiopharmhttps://doi.org/10.1089/cbr.2023.0103 (2023).

[49.] RazmaraAM, et al. . Natural killer and T cell infiltration in canine osteosarcoma: clinical implications and translational relevance. Front. Vet. Sci., 2021, 8

[50.] OmerN, NichollsW, RueggB, Souza-Fonseca-GuimaraesF, RossiGR. Enhancing natural killer cell targeting of pediatric sarcoma. Front. Immunol., 2021, 12

[51.] TulliusBP, SettyBA, LeeDA. Natural killer cell immunotherapy for osteosarcoma. Adv. Exp. Med. Biol., 2020, 1257: 141-154

[52.] YangX, ZhangW, XuP. NK cell and macrophages confer prognosis and reflect immune status in osteosarcoma. J. Cell Biochem., 2019, 120: 8792-8797

[53.] FernándezL, et al. . Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D-NKG2DL dependent manner. Cancer Lett., 2015, 368: 54-63

[54.] TarekN, LeeDA. Natural killer cells for osteosarcoma. Adv. Exp. Med. Biol., 2014, 804: 341-353

[55.] GumaSR, et al. . Natural killer cell therapy and aerosol interleukin-2 for the treatment of osteosarcoma lung metastasis. Pediatr. Blood Cancer, 2014, 61: 618-626

[56.] ZhengW, XiaoH, LiuH, ZhouY. Expression of programmed death 1 is correlated with progression of osteosarcoma. APMIS, 2015, 123: 102-107

[57.] SunCY, et al. . T cell exhaustion drives osteosarcoma pathogenesis. Ann. Transl. Med., 2021, 9: 1447

[58.] LussierDM, et al. . Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions. J. Immunother., 2015, 38: 96-106

[59.] HuangX, et al. . Prognostic value of programmed cell death 1 ligand-1 (PD-L1) or PD-1 expression in patients with osteosarcoma: a meta-analysis. J. Cancer, 2018, 9: 2525-2531

[60.] TawbiHA, et al. . Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol., 2017, 18: 1493-1501

[61.] ScheinbergT, et al. . PD-1 blockade using pembrolizumab in adolescent and young adult patients with advanced bone and soft tissue sarcoma. Cancer Rep. (Hoboken), 2021, 4

[62.] DavisKL, et al. . Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): a multicentre, open-label, single-arm, phase 1-2 trial. Lancet Oncol., 2020, 21: 541-550

[63.] GeoergerB, et al. . Atezolizumab for children and young adults with previously treated solid tumours, non-Hodgkin lymphoma, and Hodgkin lymphoma (iMATRIX): a multicentre phase 1-2 study. Lancet Oncol., 2020, 21: 134-144

[64.] Le CesneA, et al. . Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study. Eur. J. Cancer, 2019, 119: 151-157

[65.] ZhangZ, TanX, JiangZ, WangH, YuanH. Immune checkpoint inhibitors in osteosarcoma: a hopeful and challenging future. Front. Pharm., 2022, 13

[66.] GrothC, et al. . Immunosuppression mediated by myeloid-derived suppressor cells (MDSCs) during tumour progression. Br. J. Cancer, 2019, 120: 16-25

[67.] UmanskyV, BlattnerC, GebhardtC, UtikalJ. The role of myeloid-derived suppressor cells (MDSC) in cancer progression. Vaccines (Basel), 2016, 4: 36

[68.] LawAMK, Valdes-MoraF, Gallego-OrtegaD. Myeloid-derived suppressor cells as a therapeutic target for cancer. Cells, 2020, 9: 561

[69.] LiT, et al. . Targeting MDSC for immune-checkpoint blockade in cancer immunotherapy: current progress and new prospects. Clin. Med. Insights Oncol., 2021, 15

[70.] WeberR, et al. . Myeloid-derived suppressor cells hinder the anti-cancer activity of immune checkpoint inhibitors. Front. Immunol., 2018, 9: 1310

[71.] PetrovaV, et al. . Immunosuppressive capacity of circulating MDSC predicts response to immune checkpoint inhibitors in melanoma patients. Front. Immunol., 2023, 14

[72.] HouA, HouK, HuangQ, LeiY, ChenW. Targeting myeloid-derived suppressor cell, a promising strategy to overcome resistance to immune checkpoint inhibitors. Front. Immunol., 2020, 11: 783

[73.] ShiX, et al. . Specific inhibition of PI3Kδ/γ enhances the efficacy of anti-PD1 against osteosarcoma cancer. J. Bone Oncol., 2019, 16

[74.] JiangK, et al. . SDF-1/CXCR4 axis facilitates myeloid-derived suppressor cells accumulation in osteosarcoma microenvironment and blunts the response to anti-PD-1 therapy. Int Immunopharmacol., 2019, 75

[75.] GuanY, et al. . Inhibition of IL-18-mediated myeloid derived suppressor cell accumulation enhances anti-PD1 efficacy against osteosarcoma cancer. J. Bone Oncol., 2017, 9: 59-64

[76.] YuCP, et al. . The clinicopathological and prognostic significance of ido1 expression in human solid tumors: evidence from a systematic review and meta-analysis. Cell Physiol. Biochem., 2018, 49: 134-143

[77.] WangS, WuJ, ShenH, WangJ. The prognostic value of IDO expression in solid tumors: a systematic review and meta-analysis. BMC Cancer, 2020, 20

[78.] LiF, ZhangR, LiS, LiuJ. IDO1: an important immunotherapy target in cancer treatment. Int. Immunopharmacol., 2017, 47: 70-77

[79.] FujiwaraY, et al. . Indoleamine 2,3-dioxygenase (IDO) inhibitors and cancer immunotherapy. Cancer Treat. Rev., 2022, 110

[80.] ZhaiL, et al. . Immunosuppressive IDO in cancer: mechanisms of action, animal models, and targeting strategies. Front. Immunol., 2020, 11: 1185

[81.] LiuM, et al. . Targeting the IDO1 pathway in cancer: from bench to bedside. J. Hematol. Oncol., 2018, 11: 100

[82.] FarooqA, ZulfiqarB, AsgharK. Indoleamine 2,3-Dioxygenase: a novel immunotherapeutic target for osteosarcoma. J. Cancer Allied Spec., 2023, 9: 501

[83.] FanQ, et al. . Nanoengineering a metal-organic framework for osteosarcoma chemo-immunotherapy by modulating indoleamine-2,3-dioxygenase and myeloid-derived suppressor cells. J. Exp. Clin. Cancer Res., 2022, 41: 162

[84.] XiangD, et al. . Combination of IDO inhibitors and platinum(IV) prodrugs reverses low immune responses to enhance cancer chemotherapy and immunotherapy for osteosarcoma. Mater. Today Bio., 2023, 20

[85.] Barbosa, P. M., Matheus. Investigating the Role of Indoleamine 2,3-Dioxygenase 1 and Transforming Growth Factor Beta 1 in Rebound Immune Suppression Following Chemotherapy in Osteosarcoma M.S. thesis, University of Illinois at Urbana-Champaign (2023).

[86.] KellyCM, et al. . A phase II study of epacadostat and pembrolizumab in patients with advanced sarcoma. Clin. Cancer Res., 2023, 29: 2043-2051

[87.] Le NaourJ, GalluzziL, ZitvogelL, KroemerG, VacchelliE. Trial watch: IDO inhibitors in cancer therapy. Oncoimmunology, 2020, 9

[88.] FanL, RuJ, LiuT, MaC. Identification of a novel prognostic gene signature from the immune cell infiltration landscape of osteosarcoma. Front. Cell Dev. Biol., 2021, 9

[89.] LiuR, HuY, LiuT, WangY. Profiles of immune cell infiltration and immune-related genes in the tumor microenvironment of osteosarcoma cancer. BMC Cancer, 2021, 21

[90.] HeL, YangH, HuangJ. The tumor immune microenvironment and immune-related signature predict the chemotherapy response in patients with osteosarcoma. BMC Cancer, 2021, 21

[91.] YuY, et al. . Development of a prognostic gene signature based on an immunogenomic infiltration analysis of osteosarcoma. J. Cell Mol. Med., 2020, 24: 11230-11242

[92.] ZhangC, et al. . Profiles of immune cell infiltration and immune-related genes in the tumor microenvironment of osteosarcoma. Aging (Albany NY), 2020, 12: 3486-3501

[93.] YangH, ZhaoL, ZhangY, LiFF. A comprehensive analysis of immune infiltration in the tumor microenvironment of osteosarcoma. Cancer Med., 2021, 10: 5696-5711

[94.] Sautès-FridmanC, et al. . Tertiary lymphoid structures in cancers: prognostic value, regulation, and manipulation for therapeutic intervention. Front. Immunol., 2016, 7: 407

[95.] DomblidesC, et al. . Tumor-associated tertiary lymphoid structures: from basic and clinical knowledge to therapeutic manipulation. Front. Immunol., 2021, 12

[96.] SchumacherTN, ThommenDS. Tertiary lymphoid structures in cancer. Science, 2022, 375

[97.] KangW, et al. . Tertiary lymphoid structures in cancer: the double-edged sword role in antitumor immunity and potential therapeutic induction strategies. Front. Immunol., 2021, 12

[98.] ZouJ, et al. . Tertiary lymphoid structures: a potential biomarker for anti-cancer therapy. Cancers (Basel), 2022, 14: 5968

[99.] MartinetL, et al. . Human solid tumors contain high endothelial venules: association with T- and B-lymphocyte infiltration and favorable prognosis in breast cancer. Cancer Res., 2011, 71: 5678-5687

[100.] GermainC, et al. . Presence of B cells in tertiary lymphoid structures is associated with a protective immunity in patients with lung cancer. Am. J. Respir. Crit. Care Med., 2014, 189: 832-844

[101.] GocJ, et al. . Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8⁺ T cells. Cancer Res., 2014, 74: 705-715

[102.] KroegerDR, MilneK, NelsonBH. Tumor-infiltrating plasma cells are associated with tertiary lymphoid structures, cytolytic T-cell responses, and superior prognosis in ovarian cancer. Clin. Cancer Res., 2016, 22: 3005-3015

[103.] VanherseckeL, et al. . Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression. Nat. Cancer, 2021, 2: 794-802

[104.] CottrellTR, et al. . Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC). Ann. Oncol., 2018, 29: 1853-1860

[105.] TangJ, et al. . B cells and tertiary lymphoid structures influence survival in lung cancer patients with resectable tumors. Cancers (Basel), 2020, 12: 2644

[106.] TrübM, ZippeliusA. Tertiary lymphoid structures as a predictive biomarker of response to cancer immunotherapies. Front. Immunol., 2021, 12

[107.] ZhangQ, WuS. Tertiary lymphoid structures are critical for cancer prognosis and therapeutic response. Front. Immunol., 2022, 13

[108.] WakasuS, et al. . Preventive effect of tertiary lymphoid structures on lymph node metastasis of lung adenocarcinoma. Cancer Immunol. Immunother., 2023, 72: 1823-1834

[109.] VaghjianiRG, SkitzkiJJ. Tertiary lymphoid structures as mediators of immunotherapy response. Cancers (Basel), 2022, 14: 3748

[110.] BrunetM, et al. . Mature tertiary lymphoid structure is a specific biomarker of cancer immunotherapy and does not predict outcome to chemotherapy in non-small-cell lung cancer. Ann. Oncol., 2022, 33: 1084-1085

[111.] QinM, et al. . Tertiary lymphoid structures are associated with favorable survival outcomes in patients with endometrial cancer. Cancer Immunol. Immunother., 2022, 71: 1431-1442

[112.] LiR, et al. . The 12-Ck score: global measurement of tertiary lymphoid structures. Front. Immunol., 2021, 12

[113.] LiH, et al. . Peritumoral tertiary lymphoid structures correlate with protective immunity and improved prognosis in patients with hepatocellular carcinoma. Front. Immunol., 2021, 12

[114.] JacquelotN, TellierJ, NuttSL, BelzGT. Tertiary lymphoid structures and B lymphocytes in cancer prognosis and response to immunotherapies. Oncoimmunology, 2021, 10

[115.] ItalianoA, et al. . Pembrolizumab in soft-tissue sarcomas with tertiary lymphoid structures: a phase 2 PEMBROSARC trial cohort. Nat. Med., 2022, 28: 1199-1206

[116.] PetitprezF, et al. . B cells are associated with survival and immunotherapy response in sarcoma. Nature, 2020, 577: 556-560

[117.] HendersonT, et al. . Alterations in cancer stem-cell marker CD44 expression predict oncologic outcome in soft-tissue sarcomas. J. Surg. Res., 2018, 223: 207-214

[118.] MaQ, et al. . The clinical value of CXCR4, HER2 and CD44 in human osteosarcoma: a pilot study. Oncol. Lett., 2012, 3: 797-801

[119.] Kim, H. S. et al. Expression of CD44 isoforms correlates with the metastatic potential of osteosarcoma. Clin. Orthop. Relat. Res. 184–190, https://doi.org/10.1097/00003086-200203000-00028 (2002).

[120.] GvozdenovicA, et al. . CD44 enhances tumor formation and lung metastasis in experimental osteosarcoma and is an additional predictor for poor patient outcome. J. Bone Min. Res., 2013, 28: 838-847

[121.] MayrL, et al. . CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model. Oncotarget, 2017, 8: 114095-114108

[122.] Gerardo-RamírezM, et al. . CD44 contributes to the regulation of MDR1 protein and doxorubicin chemoresistance in osteosarcoma. Int. J. Mol. Sci., 2022, 23: 8616

[123.] SpaethEL, et al. . Mesenchymal CD44 expression contributes to the acquisition of an activated fibroblast phenotype via TWIST activation in the tumor microenvironment. Cancer Res., 2013, 73: 5347-5359

[124.] BaroneF, et al. . Stromal fibroblasts in tertiary lymphoid structures: a novel target in chronic inflammation. Front. Immunol., 2016, 7: 477

[125.] NayarS, et al. . Immunofibroblasts are pivotal drivers of tertiary lymphoid structure formation and local pathology. Proc. Natl. Acad. Sci. USA, 2019, 116: 13490-13497

[126.] RodriguezAB, et al. . Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts. Cell Rep., 2021, 36

[127.] ClarkRA, AlonR, SpringerTA. CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma. J. Cell Biol., 1996, 134: 1075-1087

[128.] KincadePW, et al. . CD44 and other cell interaction molecules contributing to B lymphopoiesis. Curr. Top. Microbiol. Immunol., 1993, 184: 215-222

[129.] Casanova, J. M. et al. Tumor-infiltration lymphocytes and cancer markers in osteosarcoma: influence on patient survival. Cancers (Basel)13, 6075 (2021).

[130.] ZhuG, et al. . Induction of tertiary lymphoid structures with antitumor function by a lymph node-derived stromal cell line. Front. Immunol., 2018, 9: 1609

[131.] JohnsonP, RuffellB. CD44 and its role in inflammation and inflammatory diseases. Inflamm. Allergy Drug Targets, 2009, 8: 208-220

[132.] GaggeroS, WittK, CarlstenM, MitraS. Cytokines orchestrating the natural killer-myeloid cell crosstalk in the tumor microenvironment: implications for natural killer cell-based cancer immunotherapy. Front. Immunol., 2020, 11

[133.] ZhouY, ChengL, LiuL, LiX. NK cells are never alone: crosstalk and communication in tumour microenvironments. Mol. Cancer, 2023, 22

[134.] JoshiS, SharabiA. Targeting myeloid-derived suppressor cells to enhance natural killer cell-based immunotherapy. Pharm. Ther., 2022, 235

[135.] ZalfaC, PaustS. Natural killer cell interactions with myeloid derived suppressor cells in the tumor microenvironment and implications for cancer immunotherapy. Front. Immunol., 2021, 12

[136.] SarkarT, DharS, SaG. Tumor-infiltrating T-regulatory cells adapt to altered metabolism to promote tumor-immune escape. Curr. Res. Immunol., 2021, 2: 132-141

[137.] MurrayS, LundqvistA. Targeting the tumor microenvironment to improve natural killer cell-based immunotherapies: on being in the right place at the right time, with resilience. Hum. Vaccin Immunother., 2016, 12: 607-611

[138.] KitamuraT. Tumour-associated macrophages as a potential target to improve natural killer cell-based immunotherapies. Essays Biochem., 2023, 67: 1003-1014

[139.] KisseberthWC, LeeDA. Adoptive natural killer cell immunotherapy for canine osteosarcoma. Front. Vet. Sci., 2021, 8

[140.] WangZ, LiB, WangS, ChenT, YeZ. Innate immune cells: a potential and promising cell population for treating osteosarcoma. Front. Immunol., 2019, 10: 1114

[141.] QuamineAE, OlsenMR, ChoMM, CapitiniCM. Approaches to enhance natural killer cell-based immunotherapy for pediatric solid tumors. Cancers (Basel), 2021, 13: 2796

[142.] CanterRJ, et al. . Radiotherapy enhances natural killer cell cytotoxicity and localization in pre-clinical canine sarcomas and first-in-dog clinical trial. J. Immunother. Cancer, 2017, 5: 98

[143.] BarekeH, et al. . Prospects and advances in adoptive natural killer cell therapy for unmet therapeutic needs in pediatric bone sarcomas. Int. J. Mol. Sci., 2023, 24: 8324

[144.] LachotaM, et al. . Prospects for NK cell therapy of sarcoma. Cancers (Basel), 2020, 12: 3719

[145.] RebhunRB, et al. . Inhaled recombinant human IL-15 in dogs with naturally occurring pulmonary metastases from osteosarcoma or melanoma: a phase 1 study of clinical activity and correlates of response. J. Immunother Cancer, 2022, 10: e004493

[146.] ZhouY, et al. . Interleukin 15 in cell-based cancer immunotherapy. Int. J. Mol. Sci., 2022, 23: 7311

[147.] CruzSM, et al. . Intratumoral NKp46⁺ natural killer cells are spatially distanced from T and MHC-I⁺ cells with prognostic implications in soft tissue sarcoma. Front. Immunol., 2023, 14

[148.] VäänänenHK, Laitala-LeinonenT. Osteoclast lineage and function. Arch. Biochem. Biophys., 2008, 473: 132-138

[149.] HouJM, LinJL, WenJP, JinL, TangFQ. Immunohistochemical identification of osteoclasts and multinucleated macrophages. Cell Immunol., 2014, 292: 53-56

[150.] MaggianiF, ForsythR, HogendoornPC, KrenacsT, AthanasouNA. The immunophenotype of osteoclasts and macrophage polykaryons. J. Clin. Pathol., 2011, 64: 701-705

[151.] LeaderM, CollinsM, PatelJ, HenryK. Vimentin: an evaluation of its role as a tumour marker. Histopathology, 1987, 11: 63-72

[152.] Al-KhanAA, et al. . Immunohistochemical validation of spontaneously arising canine osteosarcoma as a model for human osteosarcoma. J. Comp. Pathol., 2017, 157: 256-265

[153.] BruneJC, et al. . Mesenchymal stromal cells from primary osteosarcoma are non-malignant and strikingly similar to their bone marrow counterparts. Int. J. Cancer, 2011, 129: 319-330

[154.] JiangL, LiuJ, WeiQ, WangY. KPNA2 expression is a potential marker for differential diagnosis between osteosarcomas and other malignant bone tumor mimics. Diagn. Pathol., 2020, 15: 135

[155.] SalasS, et al. . Ezrin and alpha-smooth muscle actin are immunohistochemical prognostic markers in conventional osteosarcomas. Virchows Arch., 2007, 451: 999-1007

[156.] SalasS, et al. . Ezrin immunohistochemical expression in cartilaginous tumours: a useful tool for differential diagnosis between chondroblastic osteosarcoma and chondrosarcoma. Virchows Arch., 2009, 454: 81-87

[157.] BakhshiS, RadhakrishnanV. Prognostic markers in osteosarcoma. Expert Rev. Anticancer Ther., 2010, 10: 271-287

[158.] HanC, LiuT, YinR. Biomarkers for cancer-associated fibroblasts. Biomark. Res., 2020, 8

[159.] AoyamaS, NakagawaR, MuléJJ, MaillouxAW. Inducible tertiary lymphoid structures: promise and challenges for translating a new class of immunotherapy. Front. Immunol., 2021, 12

[160.] YoonTJ, HaMY, LeeWB, LeeYW. Probabilistic characterization of the Widom delta in supercritical region. J. Chem. Phys., 2018, 149

Funding

U.S. Department of Health & Human Services | National Institutes of Health (NIH)(P30CA047904); Pittsburgh Cure Sarcoma West Virginia University School of Medicine, Department of Orthopaedics