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Senescent Alveolospheres: A Preliminary 3D Model for Exploring Epithelial Senescence and Pro-Fibrotic Signaling.

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Title: Senescent Alveolospheres: A Preliminary 3D Model for Exploring Epithelial Senescence and Pro-Fibrotic Signaling.
Authors: Longhin A; Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy., Gatta V; Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy., Teti G; Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy., Falconi M; Department of Medical and Surgical Sciences (DIMEC), University of Bologna, 40126 Bologna, Italy.
Source: International journal of molecular sciences [Int J Mol Sci] 2026 Jul 10; Vol. 27 (14). Date of Electronic Publication: 2026 Jul 10.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
Imprint Name(s): Original Publication: Basel, Switzerland : MDPI, [2000-
MeSH Terms: Idiopathic Pulmonary Fibrosis*/pathology , Idiopathic Pulmonary Fibrosis*/metabolism , Alveolar Epithelial Cells*/metabolism , Alveolar Epithelial Cells*/pathology , Spheroids, Cellular*/metabolism , Pulmonary Alveoli*/metabolism , Pulmonary Alveoli*/pathology , Cellular Senescence* , Signal Transduction*, Doxorubicin/pharmacology ; Epithelial Cells/metabolism ; Humans ; Cell Survival
Abstract: Idiopathic pulmonary fibrosis (IPF) is one of the most severe forms of idiopathic interstitial pneumonia. Increasing evidence indicates that the gradual accumulation of senescent fibroblasts and alveolar epithelial cells contributes significantly to IPF pathogenesis, suggesting senescence as a potentially targetable process. Recurrent injury to the alveolar epithelium promotes senescence in epithelial cells, impairing their regenerative capacity and thereby predisposing the tissue to fibrotic degeneration. Although epithelial senescence is strongly implicated in the initiation and progression of lung fibrosis, the mechanisms through which it drives IPF remain challenging, partially due to the lack of physiologically relevant in vitro models capable of recapitulating lung architecture under both normal and pathological conditions. The objective of the present study was to develop a reproducible alveolosphere model in healthy and senescent conditions as a preliminary approach to investigate epithelial features that may be relevant to aspects of the IPF microenvironment. An alveolosphere system was generated by culturing alveolar epithelial cells with or without basement membrane components in combination with alveolar/epithelial optimized medium. Cultures were maintained for 3, 6, and 8 days, and cell viability together with morphological assessment confirmed the absence of cytotoxicity. The expression of keratin 8/18 and AQP5 was consistent with the maintenance of epithelial and alveolar-associated features. Cellular senescence was induced by exposing alveolospheres to doxorubicin for 24 h. Subsequent analyses of viability, along with the expression of senescent and pro-fibrotic markers, inflammatory mediators, and tissue remodeling factors, such as MMPs, were carried out in senescent 3D structures. The results demonstrated robust cell viability at all time points, supported by morphological observations. Marker expression suggested preservation of key epithelial characteristics, while senescence-inducing conditions were associated with an increase in senescence-associated, pro-fibrotic, inflammatory, and matrix-modulating markers. Collectively, these findings describe the preliminary establishment of a cost-effective and reproducible alveolosphere platform that may represent a useful starting point for studying epithelial senescence and its potential association with pro-fibrotic signaling relevant to aspects of IPF pathogenesis. Furthermore, this model may provide a basis for the preliminary evaluation of senotherapeutic compounds aimed at delaying or preventing the onset of cellular senescence.
References: J Cancer Res Clin Oncol. 2010 May;136(5):717-36. (PMID: 19898866)
Cell. 2023 Jan 19;186(2):243-278. (PMID: 36599349)
Int J Mol Sci. 2021 Mar 04;22(5):. (PMID: 33806395)
BMC Pulm Med. 2020 Mar 14;20(1):64. (PMID: 32171287)
Acta Biomater. 2025 Sep 15;204:277-292. (PMID: 40782923)
Front Mol Biosci. 2025 Apr 24;12:1564176. (PMID: 40343260)
Br J Hosp Med (Lond). 2026 Feb 09;87(2):50844. (PMID: 41762087)
Biomolecules. 2024 Jan 16;14(1):. (PMID: 38254715)
Clin Exp Med. 2025 Sep 1;25(1):312. (PMID: 40888966)
Pulm Ther. 2026 Mar;12(1):161-180. (PMID: 41733797)
Eur J Pharmacol. 2025 Jun 15;997:177461. (PMID: 40049575)
Trends Cell Biol. 2018 Jun;28(6):436-453. (PMID: 29477613)
Int J Mol Sci. 2025 Jun 04;26(11):. (PMID: 40508199)
Medicina (Kaunas). 2019 Mar 28;55(4):. (PMID: 30925805)
Eur Respir J. 2026 Jun 18;67(6):. (PMID: 41819537)
Biochem Genet. 2026 Mar 25;:. (PMID: 41880115)
Int J Mol Med. 2021 Jul;48(1):. (PMID: 34013369)
Am J Respir Cell Mol Biol. 1998 Apr;18(4):554-61. (PMID: 9533944)
Respir Res. 2017 Jun 19;18(1):122. (PMID: 28629363)
Exp Ther Med. 2023 Feb 15;25(4):145. (PMID: 36911379)
Pharmaceuticals (Basel). 2025 May 28;18(6):. (PMID: 40573209)
Int J Mol Sci. 2020 Apr 30;21(9):. (PMID: 32366033)
Int J Mol Sci. 2023 Feb 23;24(5):. (PMID: 36901843)
Exp Cell Res. 1998 Sep 15;243(2):359-66. (PMID: 9743595)
Front Pharmacol. 2022 Nov 15;13:1059434. (PMID: 36457712)
Trends Pharmacol Sci. 2026 Apr;47(4):403-422. (PMID: 41826159)
Nat Rev Mol Cell Biol. 2024 Dec;25(12):958-978. (PMID: 38654098)
Respir Res. 2023 Apr 7;24(1):101. (PMID: 37029417)
Int J Biochem Cell Biol. 2013 Nov;45(11):2568-73. (PMID: 23988571)
Compr Physiol. 2025 Aug;15(4):e70041. (PMID: 40820551)
Front Med (Lausanne). 2025 Sep 08;12:1658001. (PMID: 40988754)
Geriatr Gerontol Int. 2024 Mar;24 Suppl 1:60-66. (PMID: 37604771)
Can Respir J. 2025 Jul 15;2025:3183241. (PMID: 40697404)
Exp Cell Res. 2003 Jan 15;282(2):90-100. (PMID: 12531695)
Biochim Biophys Acta. 1979 May 25;573(2):276-95. (PMID: 444551)
Biomedicines. 2026 Jan 11;14(1):. (PMID: 41595688)
Stem Cells. 2024 Jun 14;42(6):491-498. (PMID: 38526067)
Int J Mol Sci. 2023 Nov 16;24(22):. (PMID: 38003600)
Cell Biol Int. 2020 Feb;44(2):477-487. (PMID: 31631444)
Tuberc Respir Dis (Seoul). 2024 Jan;87(1):52-64. (PMID: 37993994)
Grant Information: 2023.0274 Cassa di Risparmio in Bologna
Contributed Indexing: Keywords: alveolospheres; cellular senescence; idiopathic pulmonary fibrosis; inflammation; tissue remodeling related to epithelial–mesenchymal transition
Substance Nomenclature: 80168379AG (Doxorubicin)
Entry Date(s): Date Created: 20260728 Date Completed: 20260729 Latest Revision: 20260813
Update Code: 20260814
PubMed Central ID: PMC13409869
DOI: 10.3390/ijms27146171
PMID: 42511519
Database: MEDLINE
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  Data: Senescent Alveolospheres: A Preliminary 3D Model for Exploring Epithelial Senescence and Pro-Fibrotic Signaling.
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  Data: <searchLink fieldCode="AU" term="%22Longhin+A%22">Longhin A</searchLink>; Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy.<br /><searchLink fieldCode="AU" term="%22Gatta+V%22">Gatta V</searchLink>; Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy.<br /><searchLink fieldCode="AU" term="%22Teti+G%22">Teti G</searchLink>; Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy.<br /><searchLink fieldCode="AU" term="%22Falconi+M%22">Falconi M</searchLink>; Department of Medical and Surgical Sciences (DIMEC), University of Bologna, 40126 Bologna, Italy.
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  Data: <searchLink fieldCode="MM" term="%22Idiopathic+Pulmonary+Fibrosis%22">Idiopathic Pulmonary Fibrosis*</searchLink>/<searchLink fieldCode="MM" term="%22Idiopathic+Pulmonary+Fibrosis+pathology%22">pathology</searchLink> <br /><searchLink fieldCode="MM" term="%22Idiopathic+Pulmonary+Fibrosis%22">Idiopathic Pulmonary Fibrosis*</searchLink>/<searchLink fieldCode="MM" term="%22Idiopathic+Pulmonary+Fibrosis+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Alveolar+Epithelial+Cells%22">Alveolar Epithelial Cells*</searchLink>/<searchLink fieldCode="MM" term="%22Alveolar+Epithelial+Cells+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Alveolar+Epithelial+Cells%22">Alveolar Epithelial Cells*</searchLink>/<searchLink fieldCode="MM" term="%22Alveolar+Epithelial+Cells+pathology%22">pathology</searchLink> <br /><searchLink fieldCode="MM" term="%22Spheroids%2C+Cellular%22">Spheroids, Cellular*</searchLink>/<searchLink fieldCode="MM" term="%22Spheroids%2C+Cellular+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Pulmonary+Alveoli%22">Pulmonary Alveoli*</searchLink>/<searchLink fieldCode="MM" term="%22Pulmonary+Alveoli+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Pulmonary+Alveoli%22">Pulmonary Alveoli*</searchLink>/<searchLink fieldCode="MM" term="%22Pulmonary+Alveoli+pathology%22">pathology</searchLink> <br /><searchLink fieldCode="MM" term="%22Cellular+Senescence%22">Cellular Senescence*</searchLink> <br /><searchLink fieldCode="MM" term="%22Signal+Transduction%22">Signal Transduction*</searchLink><br /><searchLink fieldCode="MH" term="%22Doxorubicin%22">Doxorubicin</searchLink>/<searchLink fieldCode="MH" term="%22Doxorubicin+pharmacology%22">pharmacology</searchLink> ; <searchLink fieldCode="MH" term="%22Epithelial+Cells%22">Epithelial Cells</searchLink>/<searchLink fieldCode="MH" term="%22Epithelial+Cells+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Cell+Survival%22">Cell Survival</searchLink>
– Name: Abstract
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  Data: Idiopathic pulmonary fibrosis (IPF) is one of the most severe forms of idiopathic interstitial pneumonia. Increasing evidence indicates that the gradual accumulation of senescent fibroblasts and alveolar epithelial cells contributes significantly to IPF pathogenesis, suggesting senescence as a potentially targetable process. Recurrent injury to the alveolar epithelium promotes senescence in epithelial cells, impairing their regenerative capacity and thereby predisposing the tissue to fibrotic degeneration. Although epithelial senescence is strongly implicated in the initiation and progression of lung fibrosis, the mechanisms through which it drives IPF remain challenging, partially due to the lack of physiologically relevant in vitro models capable of recapitulating lung architecture under both normal and pathological conditions. The objective of the present study was to develop a reproducible alveolosphere model in healthy and senescent conditions as a preliminary approach to investigate epithelial features that may be relevant to aspects of the IPF microenvironment. An alveolosphere system was generated by culturing alveolar epithelial cells with or without basement membrane components in combination with alveolar/epithelial optimized medium. Cultures were maintained for 3, 6, and 8 days, and cell viability together with morphological assessment confirmed the absence of cytotoxicity. The expression of keratin 8/18 and AQP5 was consistent with the maintenance of epithelial and alveolar-associated features. Cellular senescence was induced by exposing alveolospheres to doxorubicin for 24 h. Subsequent analyses of viability, along with the expression of senescent and pro-fibrotic markers, inflammatory mediators, and tissue remodeling factors, such as MMPs, were carried out in senescent 3D structures. The results demonstrated robust cell viability at all time points, supported by morphological observations. Marker expression suggested preservation of key epithelial characteristics, while senescence-inducing conditions were associated with an increase in senescence-associated, pro-fibrotic, inflammatory, and matrix-modulating markers. Collectively, these findings describe the preliminary establishment of a cost-effective and reproducible alveolosphere platform that may represent a useful starting point for studying epithelial senescence and its potential association with pro-fibrotic signaling relevant to aspects of IPF pathogenesis. Furthermore, this model may provide a basis for the preliminary evaluation of senotherapeutic compounds aimed at delaying or preventing the onset of cellular senescence.
– Name: Ref
  Label: References
  Group: RefInfo
  Data: J Cancer Res Clin Oncol. 2010 May;136(5):717-36. (PMID: <searchLink fieldCode="PM" term="%2219898866%22">19898866)</searchLink><br />Cell. 2023 Jan 19;186(2):243-278. (PMID: <searchLink fieldCode="PM" term="%2236599349%22">36599349)</searchLink><br />Int J Mol Sci. 2021 Mar 04;22(5):. (PMID: <searchLink fieldCode="PM" term="%2233806395%22">33806395)</searchLink><br />BMC Pulm Med. 2020 Mar 14;20(1):64. (PMID: <searchLink fieldCode="PM" term="%2232171287%22">32171287)</searchLink><br />Acta Biomater. 2025 Sep 15;204:277-292. (PMID: <searchLink fieldCode="PM" term="%2240782923%22">40782923)</searchLink><br />Front Mol Biosci. 2025 Apr 24;12:1564176. (PMID: <searchLink fieldCode="PM" term="%2240343260%22">40343260)</searchLink><br />Br J Hosp Med (Lond). 2026 Feb 09;87(2):50844. (PMID: <searchLink fieldCode="PM" term="%2241762087%22">41762087)</searchLink><br />Biomolecules. 2024 Jan 16;14(1):. (PMID: <searchLink fieldCode="PM" term="%2238254715%22">38254715)</searchLink><br />Clin Exp Med. 2025 Sep 1;25(1):312. (PMID: <searchLink fieldCode="PM" term="%2240888966%22">40888966)</searchLink><br />Pulm Ther. 2026 Mar;12(1):161-180. (PMID: <searchLink fieldCode="PM" term="%2241733797%22">41733797)</searchLink><br />Eur J Pharmacol. 2025 Jun 15;997:177461. (PMID: <searchLink fieldCode="PM" term="%2240049575%22">40049575)</searchLink><br />Trends Cell Biol. 2018 Jun;28(6):436-453. (PMID: <searchLink fieldCode="PM" term="%2229477613%22">29477613)</searchLink><br />Int J Mol Sci. 2025 Jun 04;26(11):. (PMID: <searchLink fieldCode="PM" term="%2240508199%22">40508199)</searchLink><br />Medicina (Kaunas). 2019 Mar 28;55(4):. (PMID: <searchLink fieldCode="PM" term="%2230925805%22">30925805)</searchLink><br />Eur Respir J. 2026 Jun 18;67(6):. (PMID: <searchLink fieldCode="PM" term="%2241819537%22">41819537)</searchLink><br />Biochem Genet. 2026 Mar 25;:. (PMID: <searchLink fieldCode="PM" term="%2241880115%22">41880115)</searchLink><br />Int J Mol Med. 2021 Jul;48(1):. (PMID: <searchLink fieldCode="PM" term="%2234013369%22">34013369)</searchLink><br />Am J Respir Cell Mol Biol. 1998 Apr;18(4):554-61. (PMID: <searchLink fieldCode="PM" term="%229533944%22">9533944)</searchLink><br />Respir Res. 2017 Jun 19;18(1):122. (PMID: <searchLink fieldCode="PM" term="%2228629363%22">28629363)</searchLink><br />Exp Ther Med. 2023 Feb 15;25(4):145. (PMID: <searchLink fieldCode="PM" term="%2236911379%22">36911379)</searchLink><br />Pharmaceuticals (Basel). 2025 May 28;18(6):. (PMID: <searchLink fieldCode="PM" term="%2240573209%22">40573209)</searchLink><br />Int J Mol Sci. 2020 Apr 30;21(9):. (PMID: <searchLink fieldCode="PM" term="%2232366033%22">32366033)</searchLink><br />Int J Mol Sci. 2023 Feb 23;24(5):. (PMID: <searchLink fieldCode="PM" term="%2236901843%22">36901843)</searchLink><br />Exp Cell Res. 1998 Sep 15;243(2):359-66. (PMID: <searchLink fieldCode="PM" term="%229743595%22">9743595)</searchLink><br />Front Pharmacol. 2022 Nov 15;13:1059434. (PMID: <searchLink fieldCode="PM" term="%2236457712%22">36457712)</searchLink><br />Trends Pharmacol Sci. 2026 Apr;47(4):403-422. (PMID: <searchLink fieldCode="PM" term="%2241826159%22">41826159)</searchLink><br />Nat Rev Mol Cell Biol. 2024 Dec;25(12):958-978. (PMID: <searchLink fieldCode="PM" term="%2238654098%22">38654098)</searchLink><br />Respir Res. 2023 Apr 7;24(1):101. (PMID: <searchLink fieldCode="PM" term="%2237029417%22">37029417)</searchLink><br />Int J Biochem Cell Biol. 2013 Nov;45(11):2568-73. (PMID: <searchLink fieldCode="PM" term="%2223988571%22">23988571)</searchLink><br />Compr Physiol. 2025 Aug;15(4):e70041. (PMID: <searchLink fieldCode="PM" term="%2240820551%22">40820551)</searchLink><br />Front Med (Lausanne). 2025 Sep 08;12:1658001. (PMID: <searchLink fieldCode="PM" term="%2240988754%22">40988754)</searchLink><br />Geriatr Gerontol Int. 2024 Mar;24 Suppl 1:60-66. (PMID: <searchLink fieldCode="PM" term="%2237604771%22">37604771)</searchLink><br />Can Respir J. 2025 Jul 15;2025:3183241. (PMID: <searchLink fieldCode="PM" term="%2240697404%22">40697404)</searchLink><br />Exp Cell Res. 2003 Jan 15;282(2):90-100. (PMID: <searchLink fieldCode="PM" term="%2212531695%22">12531695)</searchLink><br />Biochim Biophys Acta. 1979 May 25;573(2):276-95. (PMID: <searchLink fieldCode="PM" term="%22444551%22">444551)</searchLink><br />Biomedicines. 2026 Jan 11;14(1):. (PMID: <searchLink fieldCode="PM" term="%2241595688%22">41595688)</searchLink><br />Stem Cells. 2024 Jun 14;42(6):491-498. (PMID: <searchLink fieldCode="PM" term="%2238526067%22">38526067)</searchLink><br />Int J Mol Sci. 2023 Nov 16;24(22):. (PMID: <searchLink fieldCode="PM" term="%2238003600%22">38003600)</searchLink><br />Cell Biol Int. 2020 Feb;44(2):477-487. (PMID: <searchLink fieldCode="PM" term="%2231631444%22">31631444)</searchLink><br />Tuberc Respir Dis (Seoul). 2024 Jan;87(1):52-64. (PMID: <searchLink fieldCode="PM" term="%2237993994%22">37993994)</searchLink>
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  Data: <i>Keywords: </i>alveolospheres; cellular senescence; idiopathic pulmonary fibrosis; inflammation; tissue remodeling related to epithelial–mesenchymal transition
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        Text: English
    Subjects:
      – SubjectFull: Doxorubicin pharmacology
        Type: general
      – SubjectFull: Epithelial Cells metabolism
        Type: general
      – SubjectFull: Humans
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      – SubjectFull: Idiopathic Pulmonary Fibrosis pathology
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      – SubjectFull: Idiopathic Pulmonary Fibrosis metabolism
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      – SubjectFull: Spheroids, Cellular metabolism
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      – SubjectFull: Pulmonary Alveoli metabolism
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      – SubjectFull: Pulmonary Alveoli pathology
        Type: general
      – SubjectFull: Cellular Senescence
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      – TitleFull: Senescent Alveolospheres: A Preliminary 3D Model for Exploring Epithelial Senescence and Pro-Fibrotic Signaling.
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