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By enabling the quantification of cytokine secretion at the single-cell level, particularly interferon gamma (IFN-\u03b3), ELISPOT provides a functional readout of T cell activity with applications in both preclinical and clinical settings. This systematic review presents a structured qualitative synthesis of 78 studies investigating the use of ELISPOT in cancer immunotherapy, including cancer vaccines, oncolytic viruses, cellular therapies, immune checkpoint inhibitors, and biomarker development. Studies were selected following PRISMA guidelines from PubMed, Scopus, and Embase, focusing on both clinical and preclinical research with translational relevance. The evidence indicates that ELISPOT is widely used to validate tumor-associated antigens and neoantigens, monitor antigen-specific T cell responses during cancer immunotherapy, including immune checkpoint blockade, cancer vaccines, and adoptive cell therapies, and characterize antigen-specific T cell function. However, only a limited subset of studies establishes direct associations between ELISPOT responses and clinically meaningful outcomes. In addition, substantial variability in assay protocols and reporting criteria limits cross-study comparability and reproducibility. Overall, ELISPOT remains a valuable tool for immune monitoring in cancer research. Still, its implementation as a clinically validated biomarker requires further standardization, prospective validation, and integration with complementary analytical approaches.<\/jats:p>","DOI":"10.3390\/ijms27094056","type":"journal-article","created":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T06:50:01Z","timestamp":1777618201000},"page":"4056","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["ELISPOT as a Functional for Biomarker Study in Cancer Immunotherapy: Applications and Future Directions"],"prefix":"10.3390","volume":"27","author":[{"given":"Laura R.","family":"Fern\u00e1ndez Castro","sequence":"first","affiliation":[{"name":"GENYO, Centre for Genomics and Oncological Research, Pfizer\/University of Granada\/Andalusian Regional Government PTS, Avenida de la Ilustraci\u00f3n, 114, 18016 Granada, Spain"},{"name":"IBS Granada, Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Avenida de Madrid 15, 18012 Granada, Spain"},{"name":"Unidad de Neumolog\u00eda, Hospital Universitario Virgen de la Nieves, Avenida de las Fuerzas Armadas, 2, 18014 Granada, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matias","family":"Regiart","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica, Instituto de Qu\u00edmica San Luis (INQUISAL), Universidad Nacional de San Luis, CONICET, Ej\u00e9rcito de Los Andes 950, San Luis D5700BWS, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0563-2237","authenticated-orcid":false,"given":"Francisco Gabriel","family":"Ortega-S\u00e1nchez","sequence":"additional","affiliation":[{"name":"GENYO, Centre for Genomics and Oncological Research, Pfizer\/University of Granada\/Andalusian Regional Government PTS, Avenida de la Ilustraci\u00f3n, 114, 18016 Granada, Spain"},{"name":"IBS Granada, Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Avenida de Madrid 15, 18012 Granada, Spain"},{"name":"Unidad de Neumolog\u00eda, Hospital Universitario Virgen de la Nieves, Avenida de las Fuerzas Armadas, 2, 18014 Granada, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0991-5939","authenticated-orcid":false,"given":"Rodrigo","family":"Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"Facultad de Ingenier\u00eda, Instituto de Ciencias Aplicadas, Universidad Aut\u00f3noma de Chile, Temuco 4810101, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7069-7454","authenticated-orcid":false,"given":"Gonzalo","family":"Tortella","sequence":"additional","affiliation":[{"name":"Centro de Excelencia en Investigaci\u00f3n Biotecnol\u00f3gica Aplicada al Medio Ambiente (CIBAMA), Facultad de Ingenier\u00eda y Ciencias, Universidad de La Frontera, Av. Francisco Salazar 01145, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0385-2391","authenticated-orcid":false,"given":"Mart\u00edn A.","family":"Fern\u00e1ndez-Baldo","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica, Instituto de Qu\u00edmica San Luis (INQUISAL), Universidad Nacional de San Luis, CONICET, Ej\u00e9rcito de Los Andes 950, San Luis D5700BWS, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"ref_1","unstructured":"Ortonobes Lara, S. (2020). \u00bfQu\u00e9 Puede Salir Mal? 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