{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T03:22:02Z","timestamp":1782789722716,"version":"3.54.5"},"reference-count":37,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2020,1,31]],"date-time":"2020-01-31T00:00:00Z","timestamp":1580428800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["No.81874331"],"award-info":[{"award-number":["No.81874331"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The tumor microenvironment has been recently recognized as a critical contributor to cancer progression and anticancer therapy-resistance. Cyclophosphamide (CTX) is a cytotoxic agent commonly used in clinics for the treatment of cancer. Previous reports demonstrated that CTX given at low continuous doses, known as metronomic schedule, mainly targets endothelial cells and circulating Tregs with unknown mechanisms. Here, we investigated the antitumor activity of two different metronomic schedules of CTX along with their corresponding MTD regimen and further explored their effect on immune function and tumor microenvironment. Toxicity evaluation was monitored by overall survival rate, weight loss, and histopathological analysis. A nude mouse model of Lewis lung cancer was established to assess the anti-metastatic effects of CTX in vivo. CD4+, CD8+, and CD4+CD25+FoxP3 T cells were selected by flow cytometry analysis. Low and continuous administration of CTX was able to restore immune function via increase of CD4+\/CD8+ T cells and depletion of T regulatory cells, not only in circulatory and splenic compartments, but also at the tumor site. Low-dose CTX also reduced myofibroblasts, accompanied with an increased level of E-cadherin and low N-cadherin, both in the primary tumor and lung through the TGF-\u03b2 pathway by the downregulated expression of TGF-\u03b2 receptor 2. Our data may indicate that several other molecular mechanisms of CTX for tumor may be involved in metronomic chemotherapy, besides targeting angiogenesis and regulatory T cells.<\/jats:p>","DOI":"10.3390\/ijms21030957","type":"journal-article","created":{"date-parts":[[2020,1,31]],"date-time":"2020-01-31T11:55:56Z","timestamp":1580471756000},"page":"957","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":35,"title":["Low Dose Cyclophosphamide Modulates Tumor Microenvironment by TGF-\u03b2 Signaling Pathway"],"prefix":"10.3390","volume":"21","author":[{"given":"Hui","family":"Zhong","sequence":"first","affiliation":[{"name":"State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China"},{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifan","family":"Lai","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China"},{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China"},{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdelkader","family":"Daoud","sequence":"additional","affiliation":[{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingyuan","family":"Feng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China"},{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China"},{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Shang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China"},{"name":"School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"394","DOI":"10.3322\/caac.21492","article-title":"Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"68","author":"Bray","year":"2018","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1126\/science.1241328","article-title":"Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA","volume":"343","author":"Nathanson","year":"2014","journal-title":"Science"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s11302-012-9349-9","article-title":"Adenosine as an endogenous immunoregulator in cancer pathogenesis: Where to go?","volume":"9","author":"Kumar","year":"2013","journal-title":"Purinergic Signal"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e25962","DOI":"10.4161\/onci.25962","article-title":"Enhancing immunotherapy using chemotherapy and radiation to modify the tumor microenvironment","volume":"2","author":"Kershaw","year":"2013","journal-title":"Oncoimmunology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.imlet.2014.03.001","article-title":"Mechanisms of tumor escape from immune system: Role of mesenchymal stromal cells","volume":"159","author":"Poggi","year":"2014","journal-title":"Immunol. Lett."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1038\/nrc1877","article-title":"Fibroblasts in cancer","volume":"6","author":"Kalluri","year":"2006","journal-title":"Nat. Rev. Cancer"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1038\/emboj.2010.351","article-title":"TGF-\u03b2 regulates isoform switching of FGF receptors and epithelial-mesenchymal transition","volume":"30","author":"Shirakihara","year":"2011","journal-title":"EMBO J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1007\/s00262-001-0237-3","article-title":"Th2\/Th1 switch induced by a single low dose of cyclophosphamide in a rat metastatic lymphoma model","volume":"50","author":"Matar","year":"2002","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_9","first-page":"1878","article-title":"Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer","volume":"60","author":"Browder","year":"2000","journal-title":"Cancer Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"R15","DOI":"10.1172\/JCI8829","article-title":"Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity","volume":"105","author":"Hicklin","year":"2000","journal-title":"J. Clin. Invest."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1172\/JCI9872","article-title":"Less is more, regularly: Metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice","volume":"105","author":"Hanahan","year":"2000","journal-title":"J. Clin. Invest."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.drudis.2012.07.015","article-title":"Low-dose metronomic chemotherapy: From past experience to new paradigms in the treatment of cancer","volume":"18","author":"Loven","year":"2013","journal-title":"Drug Discov. Today"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1007\/s00262-009-0797-1","article-title":"Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma","volume":"59","author":"Tongu","year":"2010","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1007\/s00262-012-1343-0","article-title":"Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo","volume":"62","author":"Tongu","year":"2013","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1060","DOI":"10.1016\/S0959-8049(00)00044-7","article-title":"Mechanism of antimetastatic immunopotentiation by low-dose cyclophosphamide","volume":"36","author":"Matar","year":"2000","journal-title":"Eur. J. Cancer"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2703","DOI":"10.1016\/j.lfs.2005.05.038","article-title":"Failure in peripheral immuno-surveillance due to thymic atrophy: Importance of thymocyte maturation and apoptosis in adult tumor-bearer","volume":"77","author":"Mandal","year":"2005","journal-title":"Life Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1038\/cmi.2010.11","article-title":"Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts","volume":"7","author":"Bhattacharyya","year":"2010","journal-title":"Cell. Mol. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1158\/0008-5472.CAN-12-2225","article-title":"Dose-dense chemotherapy improves mechanisms of antitumor immune response","volume":"73","author":"Chang","year":"2013","journal-title":"Cancer Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1737","DOI":"10.1038\/mt.2011.113","article-title":"Immunological effects of low-dose cyclophosphamide in cancer patients treated with oncolytic adenovirus","volume":"19","author":"Cerullo","year":"2011","journal-title":"Mol. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"209","DOI":"10.4110\/in.2009.9.6.209","article-title":"The role of regulatory T cells in cancer","volume":"9","author":"Ha","year":"2009","journal-title":"Immune Netw."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1002\/eji.200324181","article-title":"CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative","volume":"34","author":"Ghiringhelli","year":"2004","journal-title":"Eur. J. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2862","DOI":"10.1182\/blood-2004-06-2410","article-title":"Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide","volume":"105","author":"Lutsiak","year":"2005","journal-title":"Blood"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1007\/s00262-006-0225-8","article-title":"Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients","volume":"56","author":"Ghiringhelli","year":"2007","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1138","DOI":"10.1172\/JCI63836","article-title":"IQGAP1 suppresses T\u03b2RII-mediated myofibroblastic activation and metastatic growth in liver","volume":"123","author":"Liu","year":"2013","journal-title":"J. Clin. Invest."},{"key":"ref_25","first-page":"1950","article-title":"Inhibition of growth of primary and metastatic lewis lung carcinoma cells by the phosphodiesterase inhibitor isobutylmethylxanthine","volume":"40","author":"Janik","year":"1980","journal-title":"Cancer Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"940","DOI":"10.1593\/neo.08456","article-title":"Myofibroblasts in pulmonary and brain metastases of alveolar soft-part sarcoma: A novel target for treatment?","volume":"10","author":"Genin","year":"2008","journal-title":"Neoplasia"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7003","DOI":"10.1158\/1078-0432.CCR-07-1263","article-title":"A switch from E-cadherin to N-cadherin expression indicates epithelial to mesenchymal transition and is of strong and independent importance for the progress of prostate cancer","volume":"13","author":"Gravdal","year":"2007","journal-title":"Clin. Cancer Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"7","DOI":"10.3747\/co.v16i2.420","article-title":"Metronomic chemotherapy: Changing the paradigm that more is better","volume":"16","author":"Scharovsky","year":"2009","journal-title":"Curr. Oncol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1182","DOI":"10.1056\/NEJM197111182852108","article-title":"Tumor angiogenesis: Therapeutic implications","volume":"285","author":"Folkman","year":"1971","journal-title":"N. Engl. J. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1002\/bies.950130106","article-title":"Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents","volume":"13","author":"Kerbel","year":"1991","journal-title":"Bioessays"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3387","DOI":"10.1016\/j.ejca.2013.06.038","article-title":"Low-dose metronomic chemotherapy: A systematic literature analysis","volume":"49","author":"Lien","year":"2013","journal-title":"Eur. J. Cancer"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1064","DOI":"10.1007\/s00262-005-0092-8","article-title":"CD4(+)CD25 high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers","volume":"55","author":"Kono","year":"2006","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Motoyoshi, Y., Kaminoda, K., Saitoh, O., Hamasaki, K., Nakao, K., Ishii, N., Nagayama, Y., and Eguchi, K. (2006). Different mechanisms for anti-tumor effects of low- and high-dose cyclophosphamide. Oncol. Rep.","DOI":"10.3892\/or.16.1.141"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1007\/s00262-011-1106-3","article-title":"Metronomic cyclophosphamide treatment in metastasized breast cancer patients: Immunological effects and clinical outcome","volume":"61","author":"Ge","year":"2012","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1513\/pats.200701-020AW","article-title":"Regulatory T cells, transforming growth factor-beta, and immune suppression","volume":"4","author":"Wan","year":"2007","journal-title":"Proc. Am. Thorac. Soc."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1093\/jmcb\/mjr033","article-title":"TGF-\u03b2: The sword, the wand, and the shield of FOXP3(+) regulatory T cells","volume":"4","author":"Tran","year":"2012","journal-title":"J. Mol. Cell Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1038","DOI":"10.4161\/onci.20684","article-title":"Single low-dose cyclophosphamide combined with interleukin-12 gene therapy is superior to a metronomic schedule in inducing immunity against colorectal carcinoma in mice","volume":"1","author":"Malvicini","year":"2012","journal-title":"Oncoimmunology"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/3\/957\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T08:53:32Z","timestamp":1760172812000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/3\/957"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,31]]},"references-count":37,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2020,2]]}},"alternative-id":["ijms21030957"],"URL":"https:\/\/doi.org\/10.3390\/ijms21030957","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,1,31]]}}}