Computational Molecular Profiling of Exosomes to Monitor Hypoxia in In Vitro 3D Culture Systems for Tissue Engineering and Tumour Angiogenesis

Krupa Ann Mathew, Bernadette K Madathil


Molecular profiling is increasingly explored in biology as a diagnostic, prognostic, and discerning tool. Gene and protein expression signatures generated using bioinformatics tools and databases are of predictive significance in identifying the phenotype or clinical status of a biological sample. This predictive bioinformatics approach finds application in a wide variety of disease conditions and tissue types. Identifying the hypoxic status of three-dimensional (3D) cultures is essential due to its divergent effects on cell survival. Though hypoxia limits the viable thickness of three-dimensional tissue engineering constructs, it also promotes angiogenesis into the construct, thereby facilitating its successful integration with the host tissue. Accurate determination of hypoxia in 3D cultures also finds use in tumour angiogenesis models. Efforts to generate hypoxic and angiogenic molecular signatures using computational databases (e.g., iHypoxia, AngioDB) and tools have proved fruitful. Exosomal molecular profiling is proposed as a non-invasive and real-time extrapolative tool to determine the hypoxic status within 3D cultures. Mathematical models can be created using R packages (e.g., randomForest and rpart) and exosomal databases (e.g., ExoCarta) to generate hypoxic decision tree classifiers to determine precisely the hypoxic status of discrete 3D culture samples. Hence, bioinformatics software and databases offer potent methodologies for non-invasive, high-throughput, real-time, in vitro monitoring of cellular states in tissue engineered constructs and 3D disease models.


Molecular profiling, gene expression signature, exosomal miRNA profiling, hypoxia, angiogenesis, 3D culture, hypoxic status, tissue engineering constructs, decision tree classifiers

Full Text:



Abd Elmageed, Z.Y., Yang, Y., Thomas, R., Ranjan, M., Mondal, D., Moroz, K., Fang, Z., Rezk, B.M., Moparty, K., and Sikka, S.C. (2014). Neoplastic reprogramming of patient-derived adipose stem cells by prostate cancer cell-associated exosomes. Stem cells 32, 983-997.

Abdollahi, S. (2021). Extracellular vesicles from organoids and 3D culture systems. Biotechnology and bioengineering 118, 1029-1049.

Al-Sowayan, B., Alammari, F., and Alshareeda, A. (2020). Preparing the bone tissue regeneration ground by exosomes: from diagnosis to therapy. Molecules 25, 4205.

Altan-Bonnet, N. (2016). Extracellular vesicles are the Trojan horses of viral infection. Current opinion in microbiology 32, 77-81.

Aragonés, J., Fraisl, P., Baes, M., and Carmeliet, P. (2009). Oxygen sensors at the crossroad of metabolism. Cell metabolism 9, 11-22.

Bae, S.-H., Jeong, J.-W., Park, J.A., Kim, S.-H., Bae, M.-K., Choi, S.-J., and Kim, K.-W. (2004). Sumoylation increases HIF-1α stability and its transcriptional activity. Biochemical and biophysical research communications 324, 394-400.

Baiguera, S., and Ribatti, D. (2013). Endothelialization approaches for viable engineered tissues. Angiogenesis 16, 1-14.

Bei, H.P., Hung, P.M., Yeung, H.L., Wang, S., and Zhao, X. (2021). Bone‐a‐Petite: Engineering Exosomes towards Bone, Osteochondral, and Cartilage Repair. Small (Weinheim an der Bergstrasse, Germany) 17, 2101741.

Bellamy, W.T., Richter, L., Frutiger, Y., and Grogan, T.M. (1999). Expression of Vascular Endothelial Growth Factor and Its Receptors in Hematopoietic Malignancies1. Cancer Research 59, 728-733.

Bhattacharya, S., Calar, K., Evans, C., Petrasko, M., and de la Puente, P. (2019). Bioengineering a novel 3D in-vitro model to recreate physiological oxygen levels and tumor-immune interactions. Biorxiv, 828145.

Bister, N., Pistono, C., Huremagic, B., Jolkkonen, J., Giugno, R., and Malm, T. (2020). Hypoxia and extracellular vesicles: A review on methods, vesicular cargo and functions. Journal of Extracellular Vesicles 10, e12002.

Bland, E., Dréau, D., and Burg, K.J. (2013). Overcoming hypoxia to improve tissue-engineering approaches to regenerative medicine. Journal of tissue engineering and regenerative medicine 7, 505-514.

Bono, H., and Hirota, K. (2020). Meta-Analysis of Hypoxic Transcriptomes from Public Databases. Biomedicines 8, 10.

Bray, L.J., and Werner, C. (2018). Evaluation of three-dimensional in vitro models to study tumor angiogenesis. ACS Biomaterials Science & Engineering 4, 337-346.

Brown, D.A., MacLellan, W.R., Wu, B.M., and Beygui, R.E. (2007). Analysis of pH gradients resulting from mass transport limitations in engineered heart tissue. Annals of biomedical engineering 35, 1885-1897.

Buchanan, C.F., Voigt, E.E., Szot, C.S., Freeman, J.W., Vlachos, P.P., and Rylander, M.N. (2014). Three-dimensional microfluidic collagen hydrogels for investigating flow-mediated tumor-endothelial signaling and vascular organization. Tissue engineering Part C, Methods 20, 64-75.

Buffa, F., Harris, A., West, C., and Miller, C. (2010). Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene. British journal of cancer 102, 428-435.

Carlsson, P.-O., Palm, F., Andersson, A., and Liss, P. (2001). Markedly decreased oxygen tension in transplanted rat pancreatic islets irrespective of the implantation site. Diabetes 50, 489-495.

Carmeliet, P. (2003). Angiogenesis in health and disease. Nature medicine 9, 653-660.

Carmeliet, P., Dor, Y., Herbert, J.-M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., and Maxwell, P. (1998). Role of HIF-1α in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394, 485-490.

Chalmin, F., Ladoire, S., Mignot, G., Vincent, J., Bruchard, M., Remy-Martin, J.-P., Boireau, W., Rouleau, A., Simon, B., and Lanneau, D. (2010). Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. The Journal of clinical investigation 120, 457-471.

Chen, P.-S., Chiu, W.-T., Hsu, P.-L., Lin, S.-C., Peng, I.C., Wang, C.-Y., and Tsai, S.-J. (2020). Pathophysiological implications of hypoxia in human diseases. Journal of Biomedical Science 27, 63.

Chen, Y.C., Lin, R.Z., Qi, H., Yang, Y., Bae, H., Melero-Martin, J.M., and Khademhosseini, A. (2012). Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels. Advanced functional materials 22, 2027-2039.

Clough, E., and Barrett, T. (2016). The Gene Expression Omnibus Database. Methods Mol Biol 1418, 93-110.

Dai, X., Ma, C., Lan, Q., and Xu, T. (2016). 3D bioprinted glioma stem cells for brain tumor model and applications of drug susceptibility. Biofabrication 8, 045005.

Deep, G., and Panigrahi, G.K. (2015). Hypoxia-induced signaling promotes prostate cancer progression: exosomes role as messenger of hypoxic response in tumor microenvironment. Critical Reviews™ in Oncogenesis 20.

Deng, Z., Lin, B., Jiang, Z., Huang, W., Li, J., Zeng, X., Wang, H., Wang, D., and Zhang, Y. (2019). Hypoxia-mimicking cobalt-doped borosilicate bioactive glass scaffolds with enhanced angiogenic and osteogenic capacity for bone regeneration. International Journal of Biological Sciences 15, 1113.

Dennis, G., Jr., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., and Lempicki, R.A. (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome biology 4, P3.

Di Paolo, A., and Bocci, G. (2007). Drug distribution in tumors: mechanisms, role in drug resistance, and methods for modification. Current oncology reports 9, 109-114.

Duddu, S., Bhattacharya, A., Chakrabarti, R., Chakravorty, N., and Shukla, P.C. (2023). Regeneration and Tissue Microenvironment. In Regenerative Medicine: Emerging Techniques to Translation Approaches, N. Chakravorty, and P.C. Shukla, eds. (Singapore: Springer Nature Singapore), pp. 1-12.

Edmondson, R., Broglie, J.J., Adcock, A.F., and Yang, L. (2014). Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay and drug development technologies 12, 207-218.

Elshabrawy, H.A., Chen, Z., Volin, M.V., Ravella, S., Virupannavar, S., and Shahrara, S. (2015). The pathogenic role of angiogenesis in rheumatoid arthritis. Angiogenesis 18, 433-448.

Eustace, A., Mani, N., Span, P.N., Irlam, J.J., Taylor, J., Betts, G.N., Denley, H., Miller, C.J., Homer, J.J., and Rojas, A.M. (2013). A 26-gene hypoxia signature predicts benefit from hypoxia-modifying therapy in laryngeal cancer but not bladder CancerGene signature predicts benefit from antihypoxia therapy. Clinical cancer research 19, 4879-4888.

Ferrara, N., and Gerber, H.P. (2001). The role of vascular endothelial growth factor in angiogenesis. Acta haematologica 106, 148-156.

Ferrara, N., and Kerbel, R.S. (2005). Angiogenesis as a therapeutic target. Nature 438, 967-974.

Folkman, J., and Hanahan, D. (1991). Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu symposia 22, 339-347.

Fong, G.-H. (2008). Mechanisms of adaptive angiogenesis to tissue hypoxia. Angiogenesis 11, 121-140.

Fong, M.Y., Zhou, W., Liu, L., Alontaga, A.Y., Chandra, M., Ashby, J., Chow, A., O’Connor, S.T.F., Li, S., and Chin, A.R. (2015). Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis. Nature cell biology 17, 183-194.

Gaudiello, E., Melly, L., Cerino, G., Boccardo, S., Jalili‐Firoozinezhad, S., Xu, L., Eckstein, F., Martin, I., Kaufmann, B.A., and Banfi, A. (2017). Scaffold Composition Determines the Angiogenic Outcome of Cell‐Based Vascular Endothelial Growth Factor Expression by Modulating Its Microenvironmental Distribution. Advanced healthcare materials 6, 1700600.

Gentles, A.J., Newman, A.M., Liu, C.L., Bratman, S.V., Feng, W., Kim, D., Nair, V.S., Xu, Y., Khuong, A., Hoang, C.D., et al. (2015). The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature medicine 21, 938-945.

Gézsi, A., Kovács, Á., Visnovitz, T., and Buzás, E.I. (2019). Systems biology approaches to investigating the roles of extracellular vesicles in human diseases. Experimental & molecular medicine 51, 1-11.

Greer, S.N., Metcalf, J.L., Wang, Y., and Ohh, M. (2012). The updated biology of hypoxia-inducible factor. Embo j 31, 2448-2460.

Gregson, A.L., Hoji, A., Injean, P., Poynter, S.T., Briones, C., Palchevskiy, V., Sam Weigt, S., Shino, M.Y., Derhovanessian, A., and Sayah, D. (2015). Altered exosomal RNA profiles in bronchoalveolar lavage from lung transplants with acute rejection. American journal of respiratory and critical care medicine 192, 1490-1503.

Harris, A.L. (2002). Hypoxia—a key regulatory factor in tumour growth. Nature Reviews Cancer 2, 38-47.

Harris, M.A., Clark, J., Ireland, A., Lomax, J., Ashburner, M., Foulger, R., Eilbeck, K., Lewis, S., Marshall, B., Mungall, C., et al. (2004). The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res 32, D258-261.

He, G., Peng, X., Wei, S., Yang, S., Li, X., Huang, M., Tang, S., Jin, H., Liu, J., and Zhang, S. (2022). Exosomes in the hypoxic TME: from release, uptake and biofunctions to clinical applications. Molecular Cancer 21, 1-22.

Hermawan, A., and Putri, H. (2022). Bioinformatics analysis reveals the potential target of rosiglitazone as an antiangiogenic agent for breast cancer therapy. BMC Genomic Data 23, 1-17.

Hildebrandt, A., Kirchner, B., Nolte-'t Hoen, E.N.M., and Pfaffl, M.W. (2021). miREV: An Online Database and Tool to Uncover Potential Reference RNAs and Biomarkers in Small-RNA Sequencing Data Sets from Extracellular Vesicles Enriched Samples. Journal of Molecular Biology 433, 167070.

Hiraoka, Y., Yamada, K., Kawasaki, Y., Hirose, H., Matsumoto, K., Ishikawa, K., and Yasumizu, Y. (2019). Ikra: RNAseq pipeline centered on Salmon. Zenodo https://doi org/10 5281.

Hong, P., Yang, H., Wu, Y., Li, K., and Tang, Z. (2019). The functions and clinical application potential of exosomes derived from adipose mesenchymal stem cells: a comprehensive review. Stem cell research & therapy 10, 1-12.

Hsieh, Y.-K., Chen, S.-C., Huang, W.-L., Hsu, K.-P., Gorday, K.A.V., Wang, T., and Wang, J. (2017). Direct micromachining of microfluidic channels on biodegradable materials using laser ablation. Polymers 9, 242.

Hsu, Y., Hung, J., Chang, W., Lin, Y., Pan, Y., Tsai, P., Wu, C., and Kuo, P. (2017). Hypoxic lung cancer-secreted exosomal miR-23a increased angiogenesis and vascular permeability by targeting prolyl hydroxylase and tight junction protein ZO-1. Oncogene 36, 4929-4942.

Hua, J., Stevenson, W., Dohlman, T.H., Inomata, T., Tahvildari, M., Calcagno, N., Pirmadjid, N., Sadrai, Z., Chauhan, S.K., and Dana, R. (2016). Graft Site Microenvironment Determines Dendritic Cell Trafficking Through the CCR7-CCL19/21 Axis. Invest Ophthalmol Vis Sci 57, 1457-1467.

Huang, S.H., Lo, Y.S., Luo, Y.C., Chuang, Y.H., Lee, J.Y., and Yang, J.M. (2022). CoMI: consensus mutual information for tissue-specific gene signatures. BMC bioinformatics 22, 624.

Indira Chandran, V., Welinder, C., Gonçalves de Oliveira, K., Cerezo-Magaña, M., Månsson, A.-S., Johansson, M.C., Marko-Varga, G., and Belting, M. (2019). Global extracellular vesicle proteomic signature defines U87-MG glioma cell hypoxic status with potential implications for non-invasive diagnostics. Journal of Neuro-oncology 144, 477-488.

Jafari, R., Rahbarghazi, R., Ahmadi, M., Hassanpour, M., and Rezaie, J. (2020). Hypoxic exosomes orchestrate tumorigenesis: molecular mechanisms and therapeutic implications. Journal of Translational Medicine 18, 474.

Janvier, R., Sourla, A., Koutsilieris, M., and Doillon, C.J. (1997). Stromal fibroblasts are required for PC-3 human prostate cancer cells to produce capillary-like formation of endothelial cells in a three-dimensional co-culture system. Anticancer research 17, 1551-1557.

Jing, X., Yang, F., Shao, C., Wei, K., Xie, M., Shen, H., and Shu, Y. (2019). Role of hypoxia in cancer therapy by regulating the tumor microenvironment. Molecular Cancer 18, 157.

Jung, F., Palmer, L.A., Zhou, N., and Johns, R.A. (2000). Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circulation research 86, 319-325.

Kairuz, E., Upton, Z., Dawson, R.A., and Malda, J. (2007). Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents. Wound repair and regeneration 15, 266-274.

Kalluri, R. (2016). The biology and function of exosomes in cancer. The Journal of clinical investigation 126, 1208-1215.

Kalluri, R., and LeBleu, V.S. (2020). The biology, function, and biomedical applications of exosomes. Science 367.

Kalra, H., Simpson, R.J., Ji, H., Aikawa, E., Altevogt, P., Askenase, P., Bond, V.C., Borràs, F.E., Breakefield, X., and Budnik, V. (2012). Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation. PLoS biology 10, e1001450.

Kanehisa, M., and Goto, S. (2000). KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28, 27-30.

Karsch-Mizrachi, I., Takagi, T., and Cochrane, G. (2018). The international nucleotide sequence database collaboration. Nucleic Acids Res 46, D48-d51.

Keerthikumar, S., Chisanga, D., Ariyaratne, D., Al Saffar, H., Anand, S., Zhao, K., Samuel, M., Pathan, M., Jois, M., and Chilamkurti, N. (2016). ExoCarta: a web-based compendium of exosomal cargo. Journal of molecular biology 428, 688-692.

Keith, B., and Simon, M.C. (2007). Hypoxia-inducible factors, stem cells, and cancer. Cell 129, 465-472.

Kershaw, N.J., and Babon, J.J. (2015). VHL: Cullin-g the hypoxic response. Structure 23, 435-436.

Khalyfa, A., Zhang, C., Khalyfa, A.A., Foster, G.E., Beaudin, A.E., Andrade, J., Hanly, P.J., Poulin, M.J., and Gozal, D. (2016). Effect on intermittent hypoxia on plasma exosomal micro RNA signature and endothelial function in healthy adults. Sleep 39, 2077-2090.

Khattak, S.F., Chin, K.s., Bhatia, S.R., and Roberts, S.C. (2007). Enhancing oxygen tension and cellular function in alginate cell encapsulation devices through the use of perfluorocarbons. Biotechnology and bioengineering 96, 156-166.

Khurana, P., Sugadev, R., Jain, J., and Singh, S.B. (2013). HypoxiaDB: a database of hypoxia-regulated proteins. Database : the journal of biological databases and curation 2013, bat074.

Kim, D.-K., Lee, J., Kim, S.R., Choi, D.-S., Yoon, Y.J., Kim, J.H., Go, G., Nhung, D., Hong, K., and Jang, S.C. (2015). EVpedia: a community web portal for extracellular vesicles research. Bioinformatics 31, 933-939.

Kim, J.-w., Tchernyshyov, I., Semenza, G.L., and Dang, C.V. (2006). HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell metabolism 3, 177-185.

Kim, M.Y., Shin, H., Moon, H.W., Park, Y.H., Park, J., and Lee, J.Y. (2021). Urinary exosomal microRNA profiling in intermediate-risk prostate cancer. Scientific Reports 11, 7355.

King, H.W., Michael, M.Z., and Gleadle, J.M. (2012). Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer 12, 421.

Knitsch, R., AlWahsh, M., Raschke, H., Lambert, J., and Hergenröder, R. (2021). In Vitro Spatio-Temporal NMR Metabolomics of Living 3D Cell Models. Analytical Chemistry 93, 13485-13494.

Koenig, T., Menze, B.H., Kirchner, M., Monigatti, F., Parker, K.C., Patterson, T., Steen, J.J., Hamprecht, F.A., and Steen, H. (2008). Robust Prediction of the MASCOT Score for an Improved Quality Assessment in Mass Spectrometric Proteomics. Journal of Proteome Research 7, 3708-3717.

Kondo, J., Endo, H., Okuyama, H., Ishikawa, O., Iishi, H., Tsujii, M., Ohue, M., and Inoue, M. (2011). Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc Natl Acad Sci U S A 108, 6235-6240.

Kore, R.A., and Abraham, E.C. (2014). Inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, upregulated in glioblastoma multiforme, raise the levels of CRYAB in exosomes secreted by U373 glioma cells. Biochemical and biophysical research communications 453, 326-331.

Kore, R.A., Edmondson, J.L., Jenkins, S.V., Jamshidi-Parsian, A., Dings, R.P.M., Reyna, N.S., and Griffin, R.J. (2018). Hypoxia-derived exosomes induce putative altered pathways in biosynthesis and ion regulatory channels in glioblastoma cells. Biochemistry and Biophysics Reports 14, 104-113.

Krämer, A., Green, J., Pollard, J., Jr, and Tugendreich, S. (2013). Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30, 523-530.

Kucharzewska, P., Christianson, H.C., Welch, J.E., Svensson, K.J., Fredlund, E., Ringnér, M., Mörgelin, M., Bourseau-Guilmain, E., Bengzon, J., and Belting, M. (2013). Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proceedings of the National Academy of Sciences 110, 7312-7317.

Law, A.M., Rodriguez de la Fuente, L., Grundy, T.J., Fang, G., Valdes-Mora, F., and Gallego-Ortega, D. (2021). Advancements in 3D cell culture systems for personalizing anti-cancer therapies. Front Oncol 11, 782766.

Lei, D., Yang, Y., Liu, Z., Yang, B., Gong, W., Chen, S., Wang, S., Sun, L., Song, B., and Xuan, H. (2019). 3D printing of biomimetic vasculature for tissue regeneration. Materials Horizons 6, 1197-1206.

Lesman, A., Koffler, J., Atlas, R., Blinder, Y.J., Kam, Z., and Levenberg, S. (2011). Engineering vessel-like networks within multicellular fibrin-based constructs. Biomaterials 32, 7856-7869.

Lewis, M.C., MacArthur, B.D., Malda, J., Pettet, G., and Please, C.P. (2005). Heterogeneous proliferation within engineered cartilaginous tissue: the role of oxygen tension. Biotechnology and bioengineering 91, 607-615.

Li, L., Li, C., Wang, S., Wang, Z., Jiang, J., Wang, W., Li, X., Chen, J., Liu, K., and Li, C. (2016). Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic PhenotypeExosomal miR-21 Mediates Hypoxia-Induced Cell Invasiveness. Cancer research 76, 1770-1780.

Li, L., Mu, J., Zhang, Y., Zhang, C., Ma, T., Chen, L., Huang, T., Wu, J., Cao, J., and Feng, S. (2022a). Stimulation by exosomes from hypoxia preconditioned human umbilical vein endothelial cells facilitates mesenchymal stem cells angiogenic function for spinal cord repair. ACS nano 16, 10811-10823.

Li, P., Liu, Y., Wang, H., He, Y., Wang, X., He, Y., Lv, F., Chen, H., Pang, X., Liu, M., et al. (2014). PubAngioGen: a database and knowledge for angiogenesis and related diseases. Nucleic Acids Research 43, D963-D967.

Li, X.-y., Ma, W.-N., Su, L.-x., Shen, Y., Zhang, L., Shao, Y., Wang, D., Wang, Z., Wen, M.-Z., and Yang, X.-t. (2022b). Association of Angiogenesis Gene Expression With Cancer Prognosis and Immunotherapy Efficacy. Frontiers in cell and developmental biology 10, 19.

Li, Z., Guo, X., and Guan, J. (2012). An oxygen release system to augment cardiac progenitor cell survival and differentiation under hypoxic condition. Biomaterials 33, 5914-5923.

Liaw, A., and Wiener, M. (2002). Classification and regression by randomForest. R news 2, 18-22.

Liu, T., Zhang, Q., Zhang, J., Li, C., Miao, Y.-R., Lei, Q., Li, Q., and Guo, A.-Y. (2018). EVmiRNA: a database of miRNA profiling in extracellular vesicles. Nucleic Acids Research 47, D89-D93.

Liu, X., Wang, J., Wang, P., Zhong, L., Wang, S., Feng, Q., Wei, X., and Zhou, L. (2022a). Hypoxia-pretreated mesenchymal stem cell-derived exosomes-loaded low-temperature extrusion 3D-printed implants for neuroregeneration after traumatic brain injury in beagle dogs. Frontiers in Bioengineering and Biotechnology, 1828.

Liu, Y., Zhou, G., and Cao, Y. (2017). Recent Progress in Cartilage Tissue Engineering—Our Experience and Future Directions. Engineering 3, 28-35.

Liu, Z.X., Wang, P., Zhang, Q., Li, S., Zhang, Y., Guo, Y., Jia, C., Shao, T., Li, L., Cheng, H., et al. (2022b). iHypoxia: An Integrative Database of Protein Expression Dynamics in Response to Hypoxia in Animals. Genomics, proteomics & bioinformatics.

Llorente, A., Skotland, T., Sylvänne, T., Kauhanen, D., Róg, T., Orłowski, A., Vattulainen, I., Ekroos, K., and Sandvig, K. (2013). Molecular lipidomics of exosomes released by PC-3 prostate cancer cells. Biochim Biophys Acta 1831, 1302-1309.

Lu, X., Yan, C.H., Yuan, M., Wei, Y., Hu, G., and Kang, Y. (2010). In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer. Cancer Res 70, 3905-3914.

Luo, Y.-C., Huang, S.-H., Pathak, N., Chuang, Y.-H., and Yang, J.-M. (2021). An integrated systematic approach for investigating microcurrent electrical nerve stimulation (MENS) efficacy in STZ-induced diabetes mellitus. Life Sciences 279, 119650.

Lv, D., Hu, Z., Lu, L., Lu, H., and Xu, X. (2017). Three-dimensional cell culture: A powerful tool in tumor research and drug discovery. Oncology letters 14, 6999-7010.

Lydic, T.A., Townsend, S., Adda, C.G., Collins, C., Mathivanan, S., and Reid, G.E. (2015). Rapid and comprehensive 'shotgun' lipidome profiling of colorectal cancer cell derived exosomes. Methods (San Diego, Calif) 87, 83-95.

Maeda, K., Chung, Y.S., Ogawa, Y., Takatsuka, S., Kang, S.M., Ogawa, M., Sawada, T., and Sowa, M. (1996). Prognostic value of vascular endothelial growth factor expression in gastric carcinoma. Cancer 77, 858-863.

Mahon, P.C., Hirota, K., and Semenza, G.L. (2001). FIH-1: a novel protein that interacts with HIF-1α and VHL to mediate repression of HIF-1 transcriptional activity. Genes & development 15, 2675-2686.

Malda, J., Klein, T.J., and Upton, Z. (2007). The Roles of Hypoxia in the In Vitro Engineering of Tissues. Tissue Engineering 13, 2153-2162.

McQuilling, J.P., Sittadjody, S., Pendergraft, S., Farney, A.C., and Opara, E.C. (2017). Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas. Biomaterials science 5, 2437-2447.

Menon, R., Dixon, C.L., Sheller-Miller, S., Fortunato, S.J., Saade, G.R., Palma, C., Lai, A., Guanzon, D., and Salomon, C. (2019). Quantitative proteomics by SWATH-MS of maternal plasma exosomes determine pathways associated with term and preterm birth. Endocrinology 160, 639-650.

Minchinton, A.I., and Tannock, I.F. (2006). Drug penetration in solid tumours. Nature reviews Cancer 6, 583-592.

Müller, R., Weirick, T., John, D., Militello, G., Chen, W., Dimmeler, S., and Uchida, S. (2016). ANGIOGENES: knowledge database for protein-coding and noncoding RNA genes in endothelial cells. Scientific Reports 6, 32475.

Muschler, G.F., Nakamoto, C., and Griffith, L.G. (2004). Engineering principles of clinical cell-based tissue engineering. JBJS 86, 1541-1558.

Muz, B., de la Puente, P., Azab, F., and Azab, A.K. (2015). The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia 3, 83-92.

Nesvizhskii, A.I., Keller, A., Kolker, E., and Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Analytical chemistry 75, 4646-4658.

Niklińska, W., Burzykowski, T., Chyczewski, L., and Nikliński, J. (2001). Expression of vascular endothelial growth factor (VEGF) in non-small cell lung cancer (NSCLC): association with p53 gene mutation and prognosis. Lung cancer (Amsterdam, Netherlands) 34 Suppl 2, S59-64.

Noerholm, M., Balaj, L., Limperg, T., Salehi, A., Zhu, L.D., Hochberg, F.H., Breakefield, X.O., Carter, B.S., and Skog, J. (2012). RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC cancer 12, 1-11.

Orsburn, B.C. (2021). Proteome Discoverer-A Community Enhanced Data Processing Suite for Protein Informatics. Proteomes 9.

Papantoniou, I., Sonnaert, M., Lambrechts, T., Aerts, J.-M., Geris, L., Luyten, F.P., and Schrooten, J. (2014). Analysis of gene expression signatures for osteogenic 3D perfusion-bioreactor cell cultures based on a multifactorial DoE approach. Processes 2, 639-657.

Patel, T.H., Kimura, H., Weiss, C.R., Semenza, G.L., and Hofmann, L.V. (2005). Constitutively active HIF-1α improves perfusion and arterial remodeling in an endovascular model of limb ischemia. Cardiovascular research 68, 144-154.

Perez-Amodio, S., Tra, W.M., Rakhorst, H.A., Hovius, S.E., and van Neck, J.W. (2011). Hypoxia preconditioning of tissue-engineered mucosa enhances its angiogenic capacity in vitro. Tissue Engineering Part A 17, 1583-1593.

Peters, E.B. (2018). Endothelial progenitor cells for the vascularization of engineered tissues. Tissue Engineering Part B: Reviews 24, 1-24.

Pruitt, K.D., Tatusova, T., and Maglott, D.R. (2007). NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic acids research 35, D61-D65.

Puente-Santamaría, L., Sanchez-Gonzalez, L., Ramos-Ruiz, R., and Del Peso, L. (2022). Hypoxia classifier for transcriptome datasets. BMC bioinformatics 23, 204.

Rashid, I., Nagpure, N.S., Srivastava, P., Kumar, R., Pathak, A.K., Singh, M., and Kushwaha, B. (2017). HRGFish: A database of hypoxia responsive genes in fishes. Scientific Reports 7, 42346.

Rauniyar, N. (2015). Parallel reaction monitoring: a targeted experiment performed using high resolution and high mass accuracy mass spectrometry. International journal of molecular sciences 16, 28566-28581.

Ravi, M., Paramesh, V., Kaviya, S.R., Anuradha, E., and Solomon, F.D.P. (2015). 3D Cell Culture Systems: Advantages and Applications. Journal of Cellular Physiology 230, 16-26.

Rocca-Serra, P., Brazma, A., Parkinson, H., Sarkans, U., Shojatalab, M., Contrino, S., Vilo, J., Abeygunawardena, N., Mukherjee, G., Holloway, E., et al. (2003). ArrayExpress: a public database of gene expression data at EBI. Comptes Rendus Biologies 326, 1075-1078.

Rocha, S., Carvalho, J., Oliveira, P., Voglstaetter, M., Schvartz, D., Thomsen, A.R., Walter, N., Khanduri, R., Sanchez, J.C., and Keller, A. (2019). 3D cellular architecture affects microRNA and protein cargo of extracellular vesicles. Advanced science 6, 1800948.

Rouwkema, J., Boer, J.D., and Blitterswijk, C.A.V. (2006). Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct. Tissue engineering 12, 2685-2693.

Saravanan, P.B., Vasu, S., Yoshimatsu, G., Darden, C.M., Wang, X., Gu, J., Lawrence, M.C., and Naziruddin, B. (2019). Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress. Diabetologia 62, 1901-1914.

Searle, B.C. (2010). Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomics 10, 1265-1269.

Seigneuric, R., Starmans, M.H., Fung, G., Krishnapuram, B., Nuyten, D.S., van Erk, A., Magagnin, M.G., Rouschop, K.M., Krishnan, S., and Rao, R.B. (2007). Impact of supervised gene signatures of early hypoxia on patient survival. Radiotherapy and Oncology 83, 374-382.

Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome research 13, 2498-2504.

Sharma, K., Sharma, S., and Kanwar, J.R. (2023). MicroRNA Signatures of Tumor Hypoxia. In Hypoxia in Cancer: Significance and Impact on Cancer Therapy (Springer), pp. 139-159.

Sohn, T.K., Moon, E.J., Lee, S.K., Cho, H.G., and Kim, K.W. (2002). AngioDB: database of angiogenesis and angiogenesis-related molecules. Nucleic Acids Res 30, 369-371.

Stocum, D.L. (2012). Chapter 10 - Regenerative Medicine of Epidermal Structures. In Regenerative Biology and Medicine (Second Edition), D.L. Stocum, ed. (San Diego: Academic Press), pp. 261-284.

Sun, Y. (2016). Tumor microenvironment and cancer therapy resistance. Cancer Lett 380, 205-215.

Szvicsek, Z., Oszvald, Á., Szabó, L., Sándor, G.O., Kelemen, A., Soós, A.Á., Pálóczi, K., Harsányi, L., Tölgyes, T., and Dede, K. (2019). Extracellular vesicle release from intestinal organoids is modulated by Apc mutation and other colorectal cancer progression factors. Cellular and Molecular Life Sciences 76, 2463-2476.

Tauro, B.J., Greening, D.W., Mathias, R.A., Mathivanan, S., Ji, H., and Simpson, R.J. (2013). Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids. Molecular & Cellular Proteomics 12, 587-598.

Taylor, D.D., and Gercel-Taylor, C. (2008). MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecologic oncology 110, 13-21.

Therneau, T., Atkinson, B., and Ripley, B. (2015). rpart: Recursive partitioning and regression trees. R package version 4, 1-9.

Thippabhotla, S., Zhong, C., and He, M. (2019). 3D cell culture stimulates the secretion of in vivo like extracellular vesicles. Scientific reports 9, 13012.

To, K.K., and Cho, W.C. (2022). Exosome secretion from hypoxic cancer cells reshapes the tumor microenvironment and mediates drug resistance. Cancer Drug Resistance 5, 577.

Uhlén, M., Fagerberg, L., Hallström, B.M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, Å., Kampf, C., Sjöstedt, E., Asplund, A., et al. (2015). Proteomics. Tissue-based map of the human proteome. Science 347, 1260419.

van Duinen, V., Stam, W., Mulder, E., Famili, F., Reijerkerk, A., Vulto, P., Hankemeier, T., and van Zonneveld, A.J. (2020). Robust and scalable angiogenesis assay of perfused 3D human iPSC-derived endothelium for anti-angiogenic drug screening. International journal of molecular sciences 21, 4804.

Vaupel, P., Mayer, A., and Höckel, M. (2004). Tumor hypoxia and malignant progression. Methods in enzymology 381, 335-354.

Volkmer, E., Drosse, I., Otto, S., Stangelmayer, A., Stengele, M., Kallukalam, B.C., Mutschler, W., and Schieker, M. (2008). Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone. Tissue Engineering Part A 14, 1331-1340.

Volkmer, E., Otto, S., Polzer, H., Saller, M., Trappendreher, D., Zagar, D., Hamisch, S., Ziegler, G., Wilhelmi, A., and Mutschler, W. (2012). Overcoming hypoxia in 3D culture systems for tissue engineering of bone in vitro using an automated, oxygen-triggered feedback loop. Journal of Materials Science: Materials in Medicine 23, 2793-2801.

Walsh, J.C., Lebedev, A., Aten, E., Madsen, K., Marciano, L., and Kolb, H.C. (2014). The clinical importance of assessing tumor hypoxia: relationship of tumor hypoxia to prognosis and therapeutic opportunities. Antioxid Redox Signal 21, 1516-1554.

Wang, Q., Gan, H., Chen, C., Sun, Y., Chen, J., Xu, M., Weng, W., Cao, L., Xu, Q., and Wang, J. (2017). Identification and validation of a 44-gene expression signature for the classification of renal cell carcinomas. Journal of Experimental & Clinical Cancer Research 36, 176.

Wang, X., Sun, L., Maffini, M.V., Soto, A., Sonnenschein, C., and Kaplan, D.L. (2010). A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function. Biomaterials 31, 3920-3929.

Wei, W., Ao, Q., Wang, X., Cao, Y., Liu, Y., Zheng, S.G., and Tian, X. (2020). Mesenchymal Stem Cell-Derived Exosomes: A Promising Biological Tool in Nanomedicine. Front Pharmacol 11, 590470.

Weinstein, J.N., Collisson, E.A., Mills, G.B., Shaw, K.R., Ozenberger, B.A., Ellrott, K., Shmulevich, I., Sander, C., and Stuart, J.M. (2013). The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet 45, 1113-1120.

Wen, S.W., Lima, L.G., Lobb, R.J., Norris, E.L., Hastie, M.L., Krumeich, S., and Möller, A. (2019). Breast cancer‐derived exosomes reflect the cell‐of‐origin phenotype. Proteomics 19, 1800180.

Xie, Y., Su, X., Wen, Y., Zheng, C., and Li, M. (2022). Artificial Intelligent Label-Free SERS Profiling of Serum Exosomes for Breast Cancer Diagnosis and Postoperative Assessment. Nano Letters 22, 7910-7918.

Yao, Y., Jiang, Y., Song, J., Wang, R., Li, Z., Yang, L., Wu, W., Zhang, L., and Peng, Q. (2022). Exosomes as Potential Functional Nanomaterials for Tissue Engineering. Advanced healthcare materials, 2201989.

Yu, J.R., Navarro, J., Coburn, J.C., Mahadik, B., Molnar, J., Holmes IV, J.H., Nam, A.J., and Fisher, J.P. (2019). Current and future perspectives on skin tissue engineering: Key features of biomedical research, translational assessment, and clinical application. Advanced healthcare materials 8, 1801471.

Yuan, N., Ge, Z., Ji, W., and Li, J. (2021). Exosomes Secreted from Hypoxia-Preconditioned Mesenchymal Stem Cells Prevent Steroid-Induced Osteonecrosis of the Femoral Head by Promoting Angiogenesis in Rats. BioMed Research International 2021, 6655225.

Zhang, H., Deng, T., Liu, R., Bai, M., Zhou, L., Wang, X., Li, S., Wang, X., Yang, H., and Li, J. (2017). Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis. Nature communications 8, 15016.

Zhang, X., Sai, B., Wang, F., Wang, L., Wang, Y., Zheng, L., Li, G., Tang, J., and Xiang, J. (2019). Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT. Molecular cancer 18, 1-15.

Zhao, J.-L., Tan, B., Chen, G., Che, X.-M., Du, Z.-Y., Yuan, Q., Yu, J., Sun, Y.-R., Li, X.-M., Hu, J., et al. (2020). Hypoxia-Induced Glioma-Derived Exosomal miRNA-199a-3p Promotes Ischemic Injury of Peritumoral Neurons by Inhibiting the mTOR Pathway. Oxidative Medicine and Cellular Longevity 2020, 5609637.

Zheng, S., Zhang, Z., Ding, N., Sun, J., Lin, Y., Chen, J., Zhong, J., Shao, L., Lin, Z., and Xue, M. (2021). Identification of the angiogenesis related genes for predicting prognosis of patients with gastric cancer. BMC gastroenterology 21, 1-11.

Zheng, X., Wang, X., Ma, Z., Sunkari, V.G., Botusan, I., Takeda, T., Björklund, A., Inoue, M., Catrina, S., and Brismar, K. (2012). Acute hypoxia induces apoptosis of pancreatic β-cell by activation of the unfolded protein response and upregulation of CHOP. Cell death & disease 3, e322-e322.

Zhou, Y., Zhou, B., Pache, L., Chang, M., Khodabakhshi, A.H., Tanaseichuk, O., Benner, C., and Chanda, S.K. (2019). Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature Communications 10, 1523.

Zimna, A., and Kurpisz, M. (2015). Hypoxia-inducible factor-1 in physiological and pathophysiological angiogenesis: applications and therapies. BioMed research international 2015.

Zou, D., He, J., Zhang, K., Dai, J., Zhang, W., Wang, S., Zhou, J., Huang, Y., Zhang, Z., and Jiang, X. (2012). The bone-forming effects of HIF-1α-transduced BMSCs promote osseointegration with dental implant in canine mandible. PLoS One 7, e32355.


  • There are currently no refbacks.

Informatics Studies:  ISSN: 2583-8994 (Online), 2320-530X (Print)