Secretome profiling of heterotypic spheroids suggests a role of fibroblasts in HIF-1 pathway modulation and colorectal cancer photodynamic resistance
Previous analyses of the tumor microenvironment (TME) have resulted in a concept that tumor progression may depend on interactions between cancer cells and its surrounding stroma. An important aspect of these interactions is the ability of cancer cells to modulate stroma behavior, and vice versa, through the action of a variety of soluble mediators. Here, we aimed to identify soluble factors present in the TME of colorectal cancer cells that may affect relevant pathways through secretome profiling.
To partially recapitulate the TME and its architecture, we co-cultured colorectal cancer cells (SW480, TC) with stromal fibroblasts (MRC-5, F) as 3D-spheroids. Subsequent characterization of both homotypic (TC) and heterotypic (TC + F) spheroid secretomes was performed using label-free liquid chromatography-mass spectrometry (LC-MS).
Through bioinformatic analysis using the NCI-Pathway Interaction Database (NCI-PID) we found that the HIF-1 signaling pathway was most highly enriched among the proteins whose secretion was enhanced in the heterotypic spheroids. Previously, we found that HIF-1 may be associated with resistance of colorectal cancer cells to photodynamic therapy (PDT), an antitumor therapy that combines photosensitizing agents, O2 and light to create a harmful photochemical reaction. Here, we found that the presence of fibroblasts considerably diminished the sensitivity of colorectal cancer cells to photodynamic activity. Although the biological significance of the HIF-1 pathway of secretomes was decreased after photosensitization, this decrease was partially reversed in heterotypic 3D-spheroids. HIF-1 pathway modulation by both PDT and stromal fibroblasts was confirmed through expression assessment of the HIF-target VEGF, as well as through HIF transcriptional activity assessment.
Collectively, our results delineate a potential mechanism by which stromal fibroblasts may enhance colorectal cancer cell survival and photodynamic treatment resistance via HIF-1 pathway modulation.
KeywordsTumor microenvironment Photodynamic therapy Secretome Spheroids Cancer associated fibroblasts Colorectal cancer cells
Automatic gain control
Fetal bovine serum
Green fluorescent protein
Glucose transporter 1
High energy collision induced dissociation
Hypoxia inducible factor-1
Liquid chromatography–Mass spectrometry
Aminolevulinic acid methyl ester
National Cancer Institute–Pathway Interaction Database
Plasminogen activator inhibitor 1
Phosphate buffer saline
Transferrin receptor protein 1
Vascular endothelial growth factor
This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (PICT), Secretaría de Ciencia y Técnica (SECyT), Universidad Nacional de Río Cuarto, and a Christian Boulin Fellowship (EMBL). VAR and NBRV are members of the Scientific Researcher Career at CONICET. MJL holds a fellowship from CONICET.
Compliance with ethical standards
Conflict of interest
- 9.N. Rumie Vittar, M. Lamberti, M. Pansa, R. Vera, M. Rodriguez, I. Cogno, L. Milla Sanabria, V. Rivarola, Ecological photodynamic therapy: New trend to disrupt the intricate networks within tumor ecosystem. Biochim. Biophys. Acta 1835, 86 (2013)Google Scholar
- 26.M. Ashburner, C. Ball, J. Blake, D. Botstein, H. Butler, J. Cherry, A. Davis, K. Dolinski, S. Dwight, J. Eppig, M. Harris, D. Hill, L. Issel-Tarver, A. Kasarskis, S. Lewis, J. Matese, J. Richardson, M. Ringwald, G. Rubin, G. Sherlock, Gene ontology: Tool for the unification of biology. The gene ontology consortium. Nat. Genet. 25, 25 (2000)CrossRefGoogle Scholar
- 31.C. Koumenis, L. Coussens, A. Giaccia, E. Hammond, Tumor Microenvironment: Study Protocols, vol. 899 (Springer, 2016)Google Scholar
- 37.V. Carroll, M. Ashcroft, Role of hypoxia-inducible factor (HIF)-1alpha versus HIF-2alpha in the regulation of HIF target genes in response to hypoxia, insulin-like growth factor-I, or loss of von Hippel-Lindau function: Implications for targeting the HIF pathway. Cancer Res. 66, 6264 (2006)CrossRefGoogle Scholar
- 41.A. Amann, M. Zwierzina, G. Gamerith, M. Bitsche, J. Huber, G. Vogel, M. Blumer, S. Koeck, E. Pechriggl, J. Kelm, W. Hilbe, H. Zwierzina, Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells. PLoS One 9, e92511 (2014)CrossRefGoogle Scholar
- 48.S. Jung, H. Song, S. Park, S. Chung, Y. Kim, Pyruvate promotes tumor angiogenesis through HIF-1-dependent PAI-1 expression. Int. J. Oncol. 38, 571 (2011)Google Scholar
- 49.Y. Ahn, M. Chua, J.J. Whitlock, Y. Shin, W. Song, Y. Kim, C. Eom, W. An, Rodent-specific hypoxia response elements enhance PAI-1 expression through HIF-1 or HIF-2 in mouse hepatoma cells. Int. J. Oncol. 37, 1627 (2010)Google Scholar
- 50.S. Peng, G. Xue, L. Gong, C. Fang, J. Chen, C. Yuan, Z. Chen, L. Yao, B. Furie, M. Huang, A long-acting PAI-1 inhibitor reduce thrombus formation. Thromb. Haemost. 117, 1338 (2017)Google Scholar
- 52.C. Isogai, W. Laug, H. Shimada, P. Declerck, M. Stins, D. Durden, A. Erdreich-Epstein, Y. DeClerck, Plasminogen activator inhibitor-1 promotes angiogenesis by stimulating endothelial cell migration toward fibronectin. Cancer Res. 61, 5587 (2001)Google Scholar
- 53.R. Gozzelino, P. Arosio, Iron homeostasis in health and disease. Int. J. Mol. Sci. 17, (2016)Google Scholar
- 58.A. Calzolari, C. Raggi, S. Deaglio, N. Sposi, M. Stafsnes, K. Fecchi, I. Parolini, F. Malavasi, C. Peschle, M. Sargiacomo, U. Testa, TfR2 localizes in lipid raft domains and is released in exosomes to activate signal transduction along the MAPK pathway. J. Cell Sci. Pt 21, 4486 (119AD)Google Scholar
- 61.R. Han, F. Wang, P. Zhang, X. Zhou, Y. Li, miR-383 inhibits ovarian cancer cell proliferation, invasion and aerobic glycolysis by targeting LDHA. Neoplasma 64 (2017)Google Scholar
- 65.G. Semenza, B. Jiang, S. Leung, R. Passantino, J. Concordet, P. Maire, A. Giallongo, Hypoxia response elements in the aldolase a, enolase 1, and lactate dehydrogenase a gene promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem. 27, 32529 (1996)CrossRefGoogle Scholar
- 73.N. Yoneda-Kato, A. Look, M. Kirstein, M. Valentine, S. Raimondi, K. Cohen, A. Carroll, S. Morris, The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1. Oncogene 12, 265 (1996)Google Scholar
- 76.N. Feuerstein, J. Mond, Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells. J. Immunol. 139, 1818 (1987)Google Scholar