{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T12:25:11Z","timestamp":1784291111323,"version":"3.55.0"},"reference-count":38,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The limited penetration of chemotherapeutic agents into solid tumors remains a major obstacle to effective cancer treatment and is strongly influenced by the extracellular matrix (ECM). Three\u2010dimensional (3D) tumor spheroids derived from primary tumor cells (PTCs) represent a valuable in vitro model to study how ECM composition and organization regulate drug distribution and cytotoxicity with accurate physiological relevance. By recapitulating key features of the tumor microenvironment, including endogenous collagen deposition and diffusion\u2010limited drug accessibility, these models enable mechanistic investigation of microenvironment\u2010driven drug resistance. Here, we describe a set of integrated protocols to evaluate the impact of ECM remodeling on chemotherapeutic response in primary breast tumor spheroids. Spheroids are generated from isolated primary tumor cells and subjected to controlled enzymatic degradation of collagen\u2010rich ECM using ultrapure recombinant collagenases. As a model chemotherapeutic agent, doxorubicin is employed thanks to its widespread clinical use, well\u2010characterized cytotoxic mechanism, and intrinsic fluorescence, which allows direct visualization of drug uptake and spatial distribution by confocal microscopy. In parallel, drug\u2010induced cytotoxicity is quantified using a luminescent 3D viability assay. Together, these protocols provide a reproducible and accessible platform for investigating ECM\u2010mediated barriers to drug delivery and toxicity in 3D tumor models. This framework facilitates the implementation of physiologically relevant assays to study drug efficacy and therapeutic resistance in solid tumors. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Induction of breast cancer, isolation of tumor primary cells, and generation 3D spheroids\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol<\/jats:bold>\n                    : Culture and storage of PTCs\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Evaluation of collagen expression and enzymatic degradation in primary tumor spheroids by confocal microscopy\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Enzymatic degradation of the endogenous matrix collagen in primary tumor spheroids using recombinant collagenases\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Assessment of doxorubicin uptake in spheroids by confocal microscopy\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 5<\/jats:bold>\n                    : Assessment of cell viability in doxorubicin\u2010treated spheroids using CellTiter\u2010Glo 3D assay\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70394","type":"journal-article","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T09:55:25Z","timestamp":1780307725000},"source":"Crossref","is-referenced-by-count":1,"title":["Assessment of Doxorubicin Internalization and Cytotoxicity in Primary Tumor Spheroids After Collagen Digestion"],"prefix":"10.1002","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6525-4480","authenticated-orcid":false,"given":"Alessandra","family":"Lo Cicero","sequence":"first","affiliation":[{"name":"Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF) University of Palermo  Palermo Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriele","family":"Lo Buglio","sequence":"additional","affiliation":[{"name":"Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF) University of Palermo  Palermo Italy"},{"name":"Department of Pharmacy University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3786-6812","authenticated-orcid":false,"given":"Simona","family":"Campora","sequence":"additional","affiliation":[{"name":"Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF) University of Palermo  Palermo Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7250-9808","authenticated-orcid":false,"given":"Giulio","family":"Ghersi","sequence":"additional","affiliation":[{"name":"Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF) University of Palermo  Palermo Italy"},{"name":"Abiel srl  Palermo Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,1]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jddst.2024.106033"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm3904"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2020.01488"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.transproceed.2011.09.059"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.9935"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms26157104"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers15041221"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/adtp.202400370"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers14225487"},{"key":"e_1_2_12_11_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12935\u2010025\u201003734\u2010w"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers14133132"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010020\u201079721\u20100"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12951\u2010025\u201003815\u2010y"},{"key":"e_1_2_12_15_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2019.00160"},{"key":"e_1_2_12_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392\u2010021\u201000544\u20100"},{"key":"e_1_2_12_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392\u2010021\u201000544\u20100"},{"key":"e_1_2_12_18_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13046\u2010022\u201002484\u20101"},{"key":"e_1_2_12_19_1","doi-asserted-by":"publisher","DOI":"10.5114\/aoms.2016.63743"},{"key":"e_1_2_12_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003\u2010022\u201004236\u20105"},{"key":"e_1_2_12_21_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12951\u2010023\u201001874\u20107"},{"key":"e_1_2_12_22_1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aas8998"},{"key":"e_1_2_12_23_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms252413523"},{"key":"e_1_2_12_24_1","doi-asserted-by":"publisher","DOI":"10.1111\/febs.70069"},{"key":"e_1_2_12_25_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12935\u2010025\u201004154\u20106"},{"key":"e_1_2_12_26_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471\u20102407\u201013\u201095"},{"key":"e_1_2_12_27_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12885\u2010017\u20103953\u20106"},{"key":"e_1_2_12_28_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrc1893"},{"key":"e_1_2_12_29_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers14040958"},{"key":"e_1_2_12_30_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12929\u2010024\u201000997\u20109"},{"key":"e_1_2_12_31_1","doi-asserted-by":"publisher","DOI":"10.3303\/CET1438042"},{"key":"e_1_2_12_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep28951"},{"key":"e_1_2_12_33_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.2042\u20107158.2012.01567.x"},{"key":"e_1_2_12_34_1","doi-asserted-by":"publisher","DOI":"10.3303\/CET1649011"},{"key":"e_1_2_12_35_1","doi-asserted-by":"publisher","DOI":"10.1039\/D1NR00950H"},{"key":"e_1_2_12_36_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12951\u2010024\u201002968\u20106"},{"key":"e_1_2_12_37_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12943\u2010023\u201001744\u20108"},{"key":"e_1_2_12_38_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40164\u2010025\u201000647\u20102"},{"key":"e_1_2_12_39_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41565\u2010025\u201001924\u20101"}],"container-title":["Current 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