Immune contexture defined by single cell technology for prognosis prediction and immunotherapy guidance in cancer

Albini A, Sporn MB. The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer. 2007;7(2):139–47. https://doi.org/10.1038/nrc2067. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989;49(23):6449–65. Fridman WH, Zitvogel L, Sautes-Fridman C, Kroemer G. The immune contexture in cancer prognosis and treatment. Nat Rev Clin Oncol. 2017;14(12):717–34. https://doi.org/10.1038/nrclinonc.2017.101. Taube JM, Galon J, Sholl LM, Rodig SJ, Cottrell TR, Giraldo NA, et al. Implications of the tumor immune microenvironment for staging and therapeutics. Mod Pathol. 2018;31(2):214–34. https://doi.org/10.1038/modpathol.2017.156. Littman DR. Releasing the brakes on cancer immunotherapy. Cell. 2015;162(6):1186–90. https://doi.org/10.1016/j.cell.2015.08.038. Spitzer MH, Nolan GP. Mass cytometry: single cells, many features. Cell. 2016;165(4):780–91. https://doi.org/10.1016/j.cell.2016.04.019. Newell EW, Cheng Y. Mass cytometry: blessed with the curse of dimensionality. Nat Immunol. 2016;17(8):890–5. https://doi.org/10.1038/ni.3485. Wagner A, Regev A, Yosef N. Revealing the vectors of cellular identity with single-cell genomics. Nat Biotechnol. 2016;34(11):1145–60. https://doi.org/10.1038/nbt.3711. Kargl J, Busch SE, Yang GH, Kim KH, Hanke ML, Metz HE, et al. Neutrophils dominate the immune cell composition in non-small cell lung cancer. Nat Commun. 2017;8:14381. https://doi.org/10.1038/ncomms14381. Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger K, Yatabe Y, et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society: international multidisciplinary classification of lung adenocarcinoma: executive summary. Proc Am Thorac Soc. 2011;8(5):381–5. https://doi.org/10.1513/pats.201107-042ST. Bremnes RM, Busund LT, Kilvaer TL, Andersen S, Richardsen E, Paulsen EE, et al. The role of tumor-infiltrating lymphocytes in development, progression, and prognosis of non-small cell lung cancer. J Thorac Oncol. 2016;11(6):789–800. https://doi.org/10.1016/j.jtho.2016.01.015. Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf AC, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity. 2013;39(4):782–95. https://doi.org/10.1016/j.immuni.2013.10.003. Iglesia MD, Parker JS, Hoadley KA, Serody JS, Perou CM, Vincent BG. Genomic analysis of immune cell infiltrates across 11 tumor types. J Natl Cancer Inst. 2016. https://doi.org/10.1093/jnci/djw144. Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell. 2015;160(1–2):48–61. https://doi.org/10.1016/j.cell.2014.12.033. Bremnes RM, Al-Shibli K, Donnem T, Sirera R, Al-Saad S, Andersen S, et al. The role of tumor-infiltrating immune cells and chronic inflammation at the tumor site on cancer development, progression, and prognosis: emphasis on non-small cell lung cancer. J Thorac Oncol. 2011;6(4):824–33. https://doi.org/10.1097/JTO.0b013e3182037b76. Ishibashi Y, Tanaka S, Tajima K, Yoshida T, Kuwano H. Expression of Foxp3 in non-small cell lung cancer patients is significantly higher in tumor tissues than in normal tissues, especially in tumors smaller than 30 mm. Oncol Rep. 2006;15(5):1315–9. Kataki A, Scheid P, Piet M, Marie B, Martinet N, Martinet Y, et al. Tumor infiltrating lymphocytes and macrophages have a potential dual role in lung cancer by supporting both host-defense and tumor progression. J Lab Clin Med. 2002;140(5):320–8. https://doi.org/10.1067/mlc.2002.128317. Anichini A, Tassi E, Grazia G, Mortarini R. The non-small cell lung cancer immune landscape: emerging complexity, prognostic relevance and prospective significance in the context of immunotherapy. Cancer Immunol Immunother. 2018;67(6):1011–22. https://doi.org/10.1007/s00262-018-2147-7. Lavin Y, Kobayashi S, Leader A, Amir ED, Elefant N, Bigenwald C, et al. Innate immune landscape in early lung adenocarcinoma by paired single-cell analyses. Cell. 2017;169(4):750.e17–765.e17. https://doi.org/10.1016/j.cell.2017.04.014. Engblom C, Pfirschke C, Pittet MJ. The role of myeloid cells in cancer therapies. Nat Rev Cancer. 2016;16(7):447–62. https://doi.org/10.1038/nrc.2016.54. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol. 2012;12(4):253–68. https://doi.org/10.1038/nri3175. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol. 2013;31:563–604. https://doi.org/10.1146/annurev-immunol-020711-074950. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell. 2014;26(6):938. https://doi.org/10.1016/j.ccell.2014.11.010. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M, et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 2008;322(5904):1097–100. https://doi.org/10.1126/science.1164206. Salmon H, Idoyaga J, Rahman A, Leboeuf M, Remark R, Jordan S, et al. Expansion and activation of CD103(+) dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition. Immunity. 2016;44(4):924–38. https://doi.org/10.1016/j.immuni.2016.03.012. Sanchez-Paulete AR, Cueto FJ, Martinez-Lopez M, Labiano S, Morales-Kastresana A, Rodriguez-Ruiz ME, et al. Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires BATF3-dependent dendritic cells. Cancer Discov. 2016;6(1):71–9. https://doi.org/10.1158/2159-8290.CD-15-0510. Becht E, Goc J, Germain C, Giraldo NA, Dieu-Nosjean MC, Sautes-Fridman C, et al. Shaping of an effective immune microenvironment to and by cancer cells. Cancer Immunol Immunother. 2014;63(10):991–7. https://doi.org/10.1007/s00262-014-1590-3. Wendel M, Galani IE, Suri-Payer E, Cerwenka A. Natural killer cell accumulation in tumors is dependent on IFN-gamma and CXCR28 ligands. Cancer Res. 2008;68(20):8437–45. https://doi.org/10.1158/0008-5472.CAN-08-1440. Geukes Foppen MH, Donia M, Svane IM, Haanen JB. Tumor-infiltrating lymphocytes for the treatment of metastatic cancer. Mol Oncol. 2015;9(10):1918–35. https://doi.org/10.1016/j.molonc.2015.10.018. Man YG, Stojadinovic A, Mason J, Avital I, Bilchik A, Bruecher B, et al. Tumor-infiltrating immune cells promoting tumor invasion and metastasis: existing theories. J Cancer. 2013;4(1):84–95. https://doi.org/10.7150/jca.5482. Hendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. Assessing tumor-infiltrating lymphocytes in solid tumors: a practical review for pathologists and proposal for a standardized method from the international immuno-oncology biomarkers working group: Part 2: TILs in melanoma, gastrointestinal tract carcinomas, non-small cell lung carcinoma and mesothelioma, endometrial and ovarian carcinomas, squamous cell carcinoma of the head and neck, genitourinary carcinomas, and primary brain tumors. Adv Anat Pathol. 2017;24(6):311–35. https://doi.org/10.1097/PAP.0000000000000161. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science. 2011;331(6024):1565–70. https://doi.org/10.1126/science.1203486. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12(4):298–306. https://doi.org/10.1038/nrc3245. Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer. Curr Opin Immunol. 2013;25(2):261–7. https://doi.org/10.1016/j.coi.2013.03.004. Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C, et al. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J Pathol. 2014;232(2):199–209. https://doi.org/10.1002/path.4287. Yan X, Jiao SC, Zhang GQ, Guan Y, Wang JL. Tumor-associated immune factors are associated with recurrence and metastasis in non-small cell lung cancer. Cancer Gene Ther. 2017;24(2):57–63. https://doi.org/10.1038/cgt.2016.40. Al-Shibli KI, Donnem T, Al-Saad S, Persson M, Bremnes RM, Busund LT. Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer. Clin Cancer Res. 2008;14(16):5220–7. https://doi.org/10.1158/1078-0432.CCR-08-0133. Lin Y, Liu L, Zhang T, Liu J. Functional investigation of Fas ligand expressions in human non-small cell lung cancer cells and its clinical implications. Ann Thorac Surg. 2013;95(2):412–8. https://doi.org/10.1016/j.athoracsur.2012.08.012. Rutkowski J, Cyman M, Slebioda T, Bemben K, Rutkowska A, Gruchala M, et al. Evaluation of peripheral blood T lymphocyte surface activation markers and transcription factors in patients with early stage non-small cell lung cancer. Cell Immunol. 2017;322:26–33. https://doi.org/10.1016/j.cellimm.2017.09.007. Liu H, Zhang T, Ye J, Li H, Huang J, Li X, et al. Tumor-infiltrating lymphocytes predict response to chemotherapy in patients with advance non-small cell lung cancer. Cancer Immunol Immunother. 2012;61(10):1849–56. https://doi.org/10.1007/s00262-012-1231-7. Masucci GV, Cesano A, Hawtin R, Janetzki S, Zhang J, Kirsch I, et al. Validation of biomarkers to predict response to immunotherapy in cancer: volume I—pre-analytical and analytical validation. J Immunother Cancer. 2016;4:76. https://doi.org/10.1186/s40425-016-0178-1. Brody R, Zhang Y, Ballas M, Siddiqui MK, Gupta P, Barker C, et al. PD-L1 expression in advanced NSCLC: insights into risk stratification and treatment selection from a systematic literature review. Lung Cancer. 2017;112:200–15. https://doi.org/10.1016/j.lungcan.2017.08.005. Doroshow DB, Sanmamed MF, Hastings K, Politi K, Rimm DL, Chen L, et al. Immunotherapy in non-small cell lung cancer: facts and hopes. Clin Cancer Res. 2019. https://doi.org/10.1158/1078-0432.ccr-18-1538. Wang VE, Urisman A, Albacker L, Ali S, Miller V, Aggarwal R, et al. Checkpoint inhibitor is active against large cell neuroendocrine carcinoma with high tumor mutation burden. J Immunother Cancer. 2017;5(1):75. https://doi.org/10.1186/s40425-017-0281-y. Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, et al. Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362(6411):eaar3593. https://doi.org/10.1126/science.aar3593. Fruh M, Peters S. Genomic features of response to combination immunotherapy in lung cancer. Cancer Cell. 2018;33(5):791–3. https://doi.org/10.1016/j.ccell.2018.04.005. Rolfo C, Caglevic C, Santarpia M, Araujo A, Giovannetti E, Gallardo CD, et al. Immunotherapy in NSCLC: a promising and revolutionary weapon. Adv Exp Med Biol. 2017;995:97–125. https://doi.org/10.1007/978-3-319-53156-4_5. Zheng C, Zheng L, Yoo JK, Guo H, Zhang Y, Guo X, et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell. 2017;169(7):1342.e16–1356.e16. https://doi.org/10.1016/j.cell.2017.05.035. Jiang Y, Li Y, Zhu B. T-cell exhaustion in the tumor microenvironment. Cell Death Dis. 2015;6:e1792. https://doi.org/10.1038/cddis.2015.162. Xiao X, Lao XM, Chen MM, Liu RX, Wei Y, Ouyang FZ, et al. PD-1hi identifies a novel regulatory B-cell population in human hepatoma that promotes disease progression. Cancer Discov. 2016;6(5):546–59. https://doi.org/10.1158/2159-8290.CD-15-1408. Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric cancer. Lancet. 2016;388(10060):2654–64. https://doi.org/10.1016/S0140-6736(16)30354-3. Wang B, Xu D, Yu X, Ding T, Rao H, Zhan Y, et al. Association of intra-tumoral infiltrating macrophages and regulatory T cells is an independent prognostic factor in gastric cancer after radical resection. Ann Surg Oncol. 2011;18(9):2585–93. https://doi.org/10.1245/s10434-011-1609-3. Feichtenbeiner A, Haas M, Buttner M, Grabenbauer GG, Fietkau R, Distel LV. Critical role of spatial interaction between CD8(+) and Foxp3(+) cells in human gastric cancer: the distance matters. Cancer Immunol Immunother. 2014;63(2):111–9. https://doi.org/10.1007/s00262-013-1491-x. Okita Y, Ohira M, Tanaka H, Tokumoto M, Go Y, Sakurai K, et al. Alteration of CD4 T cell subsets in metastatic lymph nodes of human gastric cancer. Oncol Rep. 2015;34(2):639–47. https://doi.org/10.3892/or.2015.4064. Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, et al. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol. 2007;25(18):2586–93. https://doi.org/10.1200/JCO.2006.09.4565. Greten TF, Sangro B. Targets for immunotherapy of liver cancer. J Hepatol. 2017. https://doi.org/10.1016/j.jhep.2017.09.007. Finger EC, Giaccia AJ. Hypoxia, inflammation, and the tumor microenvironment in metastatic disease. Cancer Metastasis Rev. 2010;29(2):285–93. https://doi.org/10.1007/s10555-010-9224-5. Jimenez-Sanchez A, Memon D, Pourpe S, Veeraraghavan H, Li Y, Vargas HA, et al. Heterogeneous tumor-immune microenvironments among differentially growing metastases in an ovarian cancer patient. Cell. 2017;170(5):927.e20–938.e20. https://doi.org/10.1016/j.cell.2017.07.025. Azizi E, Carr AJ, Plitas G, Cornish AE, Konopacki C, Prabhakaran S, et al. Single-cell map of diverse immune phenotypes in the breast tumor microenvironment. Cell. 2018;174(5):1293.e36–1308.e36. https://doi.org/10.1016/j.cell.2018.05.060. Liu PS, Wang H, Li X, Chao T, Teav T, Christen S, et al. alpha-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol. 2017;18(9):985–94. https://doi.org/10.1038/ni.3796. Mantovani A, Locati M. Tumor-associated macrophages as a paradigm of macrophage plasticity, diversity, and polarization: lessons and open questions. Arterioscler Thromb Vasc Biol. 2013;33(7):1478–83. https://doi.org/10.1161/ATVBAHA.113.300168. Denkert C, Loibl S, Noske A, Roller M, Muller BM, Komor M, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol. 2010;28(1):105–13. https://doi.org/10.1200/JCO.2009.23.7370. Ali HR, Provenzano E, Dawson SJ, Blows FM, Liu B, Shah M, et al. Association between CD8+ T-cell infiltration and breast cancer survival in 12,439 patients. Ann Oncol. 2014;25(8):1536–43. https://doi.org/10.1093/annonc/mdu191. Aaltomaa S, Lipponen P, Eskelinen M, Kosma VM, Marin S, Alhava E, et al. Lymphocyte infiltrates as a prognostic variable in female breast cancer. Eur J Cancer. 1992;28A(4–5):859–64. Baitsch L, Baumgaertner P, Devevre E, Raghav SK, Legat A, Barba L, et al. Exhaustion of tumor-specific CD8(+) T cells in metastases from melanoma patients. J Clin Invest. 2011;121(6):2350–60. https://doi.org/10.1172/JCI46102. Lee PP, Yee C, Savage PA, Fong L, Brockstedt D, Weber JS, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999;5(6):677–85. https://doi.org/10.1038/9525. Liu F, Lang R, Zhao J, Zhang X, Pringle GA, Fan Y, et al. CD8(+) cytotoxic T cell and FOXP3(+) regulatory T cell infiltration in relation to breast cancer survival and molecular subtypes. Breast Cancer Res Treat. 2011;130(2):645–55. https://doi.org/10.1007/s10549-011-1647-3. West NR, Kost SE, Martin SD, Milne K, Deleeuw RJ, Nelson BH, et al. Tumour-infiltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor-negative breast cancer. Br J Cancer. 2013;108(1):155–62. https://doi.org/10.1038/bjc.2012.524. Ott PA, Bang YJ, Piha-Paul SA, Razak ARA, Bennouna J, Soria JC, et al. T-Cell-Inflamed gene-expression profile, programmed death ligand 1 expression, and tumor mutational burden predict efficacy in patients treated with pembrolizumab across 20 cancers: KEYNOTE-028. J Clin Oncol. 2019;37(4):318–27. https://doi.org/10.1200/JCO.2018.78.2276. Heimes AS, Schmidt M. Atezolizumab for the treatment of triple-negative breast cancer. Expert Opin Investig Drugs. 2019;28(1):1–5. https://doi.org/10.1080/13543784.2019.1552255. Zhou X, Li B, Zhang Y, Gu Y, Chen B, Shi T, et al. A relative ordering-based predictor for tamoxifen-treated estrogen receptor-positive breast cancer patients: multi-laboratory cohort validation. Breast Cancer Res Treat. 2013;142(3):505–14. https://doi.org/10.1007/s10549-013-2767-8. Chevrier S, Levine JH, Zanotelli VRT, Silina K, Schulz D, Bacac M, et al. An immune atlas of clear cell renal cell carcinoma. Cell. 2017;169(4):736.e18–749.e18. https://doi.org/10.1016/j.cell.2017.04.016. Shin DS, Ribas A. The evolution of checkpoint blockade as a cancer therapy: what’s here, what’s next? Curr Opin Immunol. 2015;33:23–35. https://doi.org/10.1016/j.coi.2015.01.006. Maus MV, June CH. Making better chimeric antigen receptors for adoptive T-cell therapy. Clin Cancer Res. 2016;22(8):1875–84. https://doi.org/10.1158/1078-0432.CCR-15-1433. Komohara Y, Niino D, Saito Y, Ohnishi K, Horlad H, Ohshima K, et al. Clinical significance of CD163+ tumor-associated macrophages in patients with adult T-cell leukemia/lymphoma. Cancer Sci. 2013;104(7):945–51. https://doi.org/10.1111/cas.12167. Behnes CL, Bremmer F, Hemmerlein B, Strauss A, Strobel P, Radzun HJ. Tumor-associated macrophages are involved in tumor progression in papillary renal cell carcinoma. Virchows Arch. 2014;464(2):191–6. https://doi.org/10.1007/s00428-013-1523-0. Dannenmann SR, Thielicke J, Stockli M, Matter C, von Boehmer L, Cecconi V, et al. Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma. Oncoimmunology. 2013;2(3):e23562. https://doi.org/10.4161/onci.23562. Daurkin I, Eruslanov E, Stoffs T, Perrin GQ, Algood C, Gilbert SM, et al. Tumor-associated macrophages mediate immunosuppression in the renal cancer microenvironment by activating the 15-lipoxygenase-2 pathway. Cancer Res. 2011;71(20):6400–9. https://doi.org/10.1158/0008-5472.CAN-11-1261. Steidl C, Connors JM, Gascoyne RD. Molecular pathogenesis of Hodgkin’s lymphoma: increasing evidence of the importance of the microenvironment. J Clin Oncol. 2011;29(14):1812–26. https://doi.org/10.1200/JCO.2010.32.8401. Roemer MGM, Redd RA, Cader FZ, Pak CJ, Abdelrahman S, Ouyang J, et al. Major histocompatibility complex class II and programmed death ligand 1 expression predict outcome after programmed death 1 blockade in classic hodgkin lymphoma. J Clin Oncol. 2018;36(10):942–50. https://doi.org/10.1200/JCO.2017.77.3994. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568–71. https://doi.org/10.1038/nature13954. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature. 2016;537(7620):417–21. https://doi.org/10.1038/nature19330. Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, et al. Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017;355(6332):1423–7. https://doi.org/10.1126/science.aaf0683. Juneja VR, McGuire KA, Manguso RT, LaFleur MW, Collins N, Haining WN, et al. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med. 2017;214(4):895–904. https://doi.org/10.1084/jem.20160801. Reichel J, Chadburn A, Rubinstein PG, Giulino-Roth L, Tam W, Liu Y, et al. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood. 2015;125(7):1061–72. https://doi.org/10.1182/blood-2014-11-610436. Cader FZ, Schackmann RCJ, Hu X, Wienand K, Redd R, Chapuy B, et al. Mass cytometry of Hodgkin lymphoma reveals a CD4(+) regulatory T-cell-rich and exhausted T-effector microenvironment. Blood. 2018;132(8):825–36. https://doi.org/10.1182/blood-2018-04-843714. Tsirigotis P, Savani BN, Nagler A. Programmed death-1 immune checkpoint blockade in the treatment of hematological malignancies. Ann Med. 2016;48(6):428–39. https://doi.org/10.1080/07853890.2016.1186827. Schadendorf D, van Akkooi ACJ, Berking C, Griewank KG, Gutzmer R, Hauschild A, et al. Melanoma. Lancet. 2018;392(10151):971–84. https://doi.org/10.1016/S0140-6736(18)31559-9. Li H, van der Leun AM, Yofe I, Lubling Y, Gelbard-Solodkin D, van Akkooi ACJ, et al. Dysfunctional CD8 T cells form a proliferative, dynamically regulated compartment within human melanoma. Cell. 2018. https://doi.org/10.1016/j.cell.2018.11.043. Hashimoto M, Kamphorst AO, Im SJ, Kissick HT, Pillai RN, Ramalingam SS, et al. CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annu Rev Med. 2018;69:301–18. https://doi.org/10.1146/annurev-med-012017-043208. Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell. 2019;176(1–2):404. https://doi.org/10.1016/j.cell.2018.12.034. Landhuis E. Single-cell approaches to immune profiling. Nature. 2018;557(7706):595–7. https://doi.org/10.1038/d41586-018-05214-w. Bandura DR, Baranov VI, Ornatsky OI, Antonov A, Kinach R, Lou X, et al. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem. 2009;81(16):6813–22. https://doi.org/10.1021/ac901049w. Ntranos V, Yi L, Melsted P, Pachter L. A discriminative learning approach to differential expression analysis for single-cell RNA-seq. Nat Methods. 2019;16(2):163–6. https://doi.org/10.1038/s41592-018-0303-9. Haque A, Engel J, Teichmann SA, Lonnberg T. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications. Genome Med. 2017;9(1):75. https://doi.org/10.1186/s13073-017-0467-4. Kiselev VY, Andrews TS, Hemberg M. Challenges in unsupervised clustering of single-cell RNA-seq data. Nat Rev Genet. 2019. https://doi.org/10.1038/s41576-018-0088-9. Restrepo-Perez L, Joo C, Dekker C. Paving the way to single-molecule protein sequencing. Nat Nanotechnol. 2018;13(9):786–96. https://doi.org/10.1038/s41565-018-0236-6. Doerr A. Single-cell proteomics. Nat Methods. 2019;16(1):20. https://doi.org/10.1038/s41592-018-0273-y. Thul PJ, Akesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, et al. A subcellular map of the human proteome. Science. 2017;356(6340):eaal3321. https://doi.org/10.1126/science.aal3321. Ho YJ, Anaparthy N, Molik D, Mathew G, Aicher T, Patel A, et al. Single-cell RNA-seq analysis identifies markers of resistance to targeted BRAF inhibitors in melanoma cell populations. Genome Res. 2018;28(9):1353–63. https://doi.org/10.1101/gr.234062.117. Gerber T, Willscher E, Loeffler-Wirth H, Hopp L, Schadendorf D, Schartl M, et al. Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq. Oncotarget. 2017;8(1):846–62. https://doi.org/10.18632/oncotarget.13666. Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016;352(6282):189–96. https://doi.org/10.1126/science.aad0501. Su Y, Wei W, Robert L, Xue M, Tsoi J, Garcia-Diaz A, et al. Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance. Proc Natl Acad Sci USA. 2017;114(52):13679–84. https://doi.org/10.1073/pnas.1712064115. Povinelli BJ, Rodriguez-Meira A, Mead AJ. Single cell analysis of normal and leukemic hematopoiesis. Mol Aspects Med. 2018;59:85–94. https://doi.org/10.1016/j.mam.2017.08.006. Hou Y, Song L, Zhu P, Zhang B, Tao Y, Xu X, et al. Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell. 2012;148(5):873–85. https://doi.org/10.1016/j.cell.2012.02.028. Zeng Z, Konopleva M, Andreeff M. Single-cell mass cytometry of acute myeloid leukemia and leukemia stem/progenitor cells. Methods Mol Biol. 2017;1633:75–86. https://doi.org/10.1007/978-1-4939-7142-8_5. Kuboki Y, Fischer CG, Beleva Guthrie V, Huang W, Yu J, Chianchiano P, et al. Single-cell sequencing defines genetic heterogeneity in pancreatic cancer precursor lesions. J Pathol. 2018. https://doi.org/10.1002/path.5194. Bernard V, Semaan A, Huang J, San Lucas FA, Mulu FC, Stephens BM, et al. Single cell transcriptomics of pancreatic cancer precursors demonstrates epithelial and microenvironmental heterogeneity as an early event in neoplastic progression. Clin Cancer Res. 2018. https://doi.org/10.1158/1078-0432.ccr-18-1955. Santegoets SJ, van Ham VJ, Ehsan I, Charoentong P, Duurland CL, van Unen V, et al. The anatomical location shapes the immune infiltrate in tumors of same etiology and affects survival. Clin Cancer Res. 2018. https://doi.org/10.1158/1078-0432.ccr-18-1749. Yang D, Zhang W, Liu Y, Liang J, Zhang T, Bai Y, et al. Single-cell whole-genome sequencing identifies human papillomavirus integration in cervical tumour cells prior to and following radiotherapy. Oncol Lett. 2018;15(6):9633–40. https://doi.org/10.3892/ol.2018.8567. Wang J, Roy B. Single-cell RNA-seq reveals lincRNA expression differences in Hela-S3 cells. Biotechnol Lett. 2017;39(3):359–66. https://doi.org/10.1007/s10529-016-2260-7. Cai YD, Zhang S, Zhang YH, Pan X, Feng K, Chen L, et al. Identification of the gene expression rules that define the subtypes in glioma. J Clin Med. 2018;7(10):350. https://doi.org/10.3390/jcm7100350. Yuan J, Levitin HM, Frattini V, Bush EC, Boyett DM, Samanamud J, et al. Single-cell transcriptome analysis of lineage diversity in high-grade glioma. Genome Med. 2018;10(1):57. https://doi.org/10.1186/s13073-018-0567-9. Johnson E, Dickerson KL, Connolly ID, Hayden Gephart M. Single-cell RNA-sequencing in glioma. Curr Oncol Rep. 2018;20(5):42. https://doi.org/10.1007/s11912-018-0673-2. Filbin MG, Tirosh I, Hovestadt V, Shaw ML, Escalante LE, Mathewson ND, et al. Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq. Science. 2018;360(6386):331–5. https://doi.org/10.1126/science.aao4750. Jin W, Tang Q, Wan M, Cui K, Zhang Y, Ren G, et al. Genome-wide detection of DNase I hypersensitive sites in single cells and FFPE tissue samples. Nature. 2015;528(7580):142–6. https://doi.org/10.1038/nature15740. Lobastova L, Kraus D, Glassmann A, Khan D, Steinhauser C, Wolff C, et al. Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates. Cell Oncol (Dordr). 2017;40(1):63–76. https://doi.org/10.1007/s13402-016-0305-5.