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Herein, we report a bio\u2010orthogonal click\u2010chemistry\u2010based strategy for engineering mechanically robust and functionally durable macrophage spheroids without exogenous polarizing stimuli. Macrophage membranes were functionalized via cholesterol\u2010mediated hydrophobic insertion of azide and dibenzocyclooctyne (DBCO) groups, enabling strain\u2010promoted alkyne\u2013azide cycloaddition (SPAAC)\u2010mediated intercellular coupling. Combined with micropatterned nanofiber scaffolds, this approach generates compact, spatially confined spheroids from inherently low\u2010adhesion macrophages. The engineered spheroids exhibit spontaneous M1\u2010skewed polarization, associated with increased hypoxia\u2010inducible factor\u20101\u03b1 (HIF\u20101\u03b1) signaling and elevated inducible nitric oxide synthase (iNOS) expression, pro\u2010inflammatory cytokine secretion, and suppressed IL\u201010 production. In paracrine co\u2010culture assays, click\u2010assembled spheroids display time\u2010dependent, contact\u2010independent antitumor activity against melanoma cells. Proteomic profiling revealed activation of innate immune signaling, cytokine\u2010associated pathways, and redox\u2010related metabolic programs, consistent with sustained pro\u2010inflammatory functionality. Collectively, this work demonstrates that bio\u2010orthogonal surface engineering and three\u2010dimensional spheroid architecture can stabilize macrophage phenotype and function, providing a versatile proof\u2010of\u2010concept platform for engineering immune spheroids with durable M1\u2010skewed functionality in vitro. While these findings remain to be validated in vivo, they highlight the potential of this approach for cancer immunoengineering applications.<\/jats:p>","DOI":"10.1002\/smll.75002","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T07:53:09Z","timestamp":1785743589000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Bio\u2010Orthogonally Click\u2010Assembled Macrophage Spheroids on Micropatterned Scaffolds for Sustained M1 Polarization and Antitumor Function"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-9883-6753","authenticated-orcid":false,"given":"Taehyeon","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering Yonsei University  Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hye Jin","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering Yonsei University  Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juhwan","family":"Choi","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering Yonsei University  Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6443-0902","authenticated-orcid":false,"given":"Hyun Jong","family":"Lee","sequence":"additional","affiliation":[{"name":"School of Chemical Biological and Battery Engineering Gachon University  Seongnam\u2010Si Gyeonggi\u2010Do Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5191-2531","authenticated-orcid":false,"given":"Won\u2010Gun","family":"Koh","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering Yonsei University  Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,3]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pdpdt.2024.103975"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1152\/physiol.00036.2016"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41536\u2010022\u201000266\u2010z"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s44385\u2010025\u201000041\u2010x"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/smll.202101931"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1159\/000541416"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13206\u2010022\u201000086\u20109"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/biot.201700417"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010017\u201000475\u2010x"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392\u2010023\u201001452\u20101"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12195\u2010024\u201000824\u2010z"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.canlet.2025.217772"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13045\u2010024\u201001559\u20100"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms22136995"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.lfs.2023.121857"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejphar.2020.173090"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.lfs.2025.123601"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2024.116930"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1042\/BST20221008"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2017.12.006"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2020.08.002"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.3892\/ijo.2024.5705"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep12599"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood\u20102011\u201010\u2010384289"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1039\/d3sc04597h"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202304040"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio0605\u201013"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200900942"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.4c16669"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bioadv.2022.213059"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1002\/adma.202304172"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.nanolett.3c02889"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.actbio.2018.05.034"},{"key":"e_1_2_9_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mtbio.2024.101061"},{"key":"e_1_2_9_36_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms241310763"},{"key":"e_1_2_9_37_1","doi-asserted-by":"publisher","DOI":"10.1177\/20417314221085645"},{"key":"e_1_2_9_38_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2004.07.040"},{"key":"e_1_2_9_39_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsbiomaterials.8b00894"},{"key":"e_1_2_9_40_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bioactmat.2025.06.024"},{"key":"e_1_2_9_41_1","doi-asserted-by":"publisher","DOI":"10.1039\/c1lc20186g"},{"key":"e_1_2_9_42_1","doi-asserted-by":"publisher","DOI":"10.1002\/adma.202107192"},{"key":"e_1_2_9_43_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsomega.2c06052"},{"key":"e_1_2_9_44_1","doi-asserted-by":"publisher","DOI":"10.1002\/jlb.3a0520\u2010338r"},{"key":"e_1_2_9_45_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2025.1638102"},{"key":"e_1_2_9_46_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12672\u2010025\u201003258\u20109"},{"key":"e_1_2_9_47_1","doi-asserted-by":"publisher","DOI":"10.3892\/mmr.2018.8834"},{"key":"e_1_2_9_48_1","doi-asserted-by":"publisher","DOI":"10.12703\/p6\u201013"},{"key":"e_1_2_9_49_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.15.1.323"},{"key":"e_1_2_9_50_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.60646"},{"key":"e_1_2_9_51_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.ircmb.2017.07.007"},{"key":"e_1_2_9_52_1","doi-asserted-by":"publisher","DOI":"10.1098\/rsif.2015.0262"},{"key":"e_1_2_9_53_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ctrv.2017.11.007"},{"key":"e_1_2_9_54_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.cdd.4400476"},{"key":"e_1_2_9_55_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2018.02520"}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