腫瘤會出現缺氧是因為其不正常的細胞生長和血管給給不足而導致供氧不足，這樣的缺氧環境直接導致了化療抗藥性的產生，也間接導致了血管新生及蛋白質組/基因組的變化。因此本研究的目的是透過帶氧微氣泡來恢復缺氧環境及提升化療藥物的吸收效率，進而改善化療的治療效果。在體外實驗測試中，帶氧微氣泡在高能量聚焦式超音波 (High-intensity focused ultrasound, HIFU) 的作用下會破裂而提升環境的氧分壓。為了證明超音波搭配帶氧微氣泡在體外模型中改善化療的治療效果，本研究建立了套缺氧的細胞模型。本研究以Image-iT作為判斷缺氧程度的指示劑，在經過4個小時的培養後，細胞出現抗藥性。體外實驗根據降低缺氧程度及細胞活力的相關性來調整OMB的最佳劑量，在這個劑量下，本研究證明了超音波搭配帶氧微氣泡的組別降低了70%的缺氧程度並增加20%的細胞通透性。若是搭配阿黴素 (Dox) 化療藥物一同使用時，超音波搭配帶氧微氣泡的組別更能增加14±5%的阿黴素藥物攝取量。在細胞活力的表現上，相較於只有使用阿黴素藥物的組別高達88±5%，超音波搭配帶氧微氣泡的組別細胞活力降低至45±10%，足見其發展潛力。

Hypoxia develops in solid tumors because of insufficient oxygen delivery caused by exponential cellular growth and poor vascular supply. Chemotherapeutic resistance occurs in hypoxic region as a direct result of a shortage of oxygen in the tumor, as well as indirectly a response to angiogenesis and changes of proteome/genome. In this study, we propose a hypothesis of promoting chemotherapy by using oxygen microbubbles (OMB) to recover hypoxic condition and enhance drug uptake. Under the effect of high intensity focused ultrasound (HIFU), OMB are disrupted and directly boost the oxygen pressure in vitro. To illustrate the performance of OMB+US (oxygen microbubbles and high intensity focused ultrasound) in modulating chemotherapy outcomes in vitro, a hypoxic TRAMP cell model was created. The hypoxic degree was indicated by hypoxic reagent Image-iT, the drug resistance occurred after 4 hours hypoxic incubation. To cope with the in vitro created hypoxia, an optimal dose of OMB was determined depending on the correlation of hypoxic level reduction and cell viability. With this dose, OMB+US was proved to decay approximately 70% hypoxic degree, increase about 20% cell permeability. In combination with Doxorubicin (Dox), Dox+OMB+US enhances Dox uptake 14±5%, results in decrease cell viability down to 45±10%, while the cell viability is 88±5% in Dox only and 72±6% in Dox+CMB+US in hypoxic condition.

References
[1] M. Wang et al., “Role of tumor microenvironment in tumorigenesis,” J. Cancer, vol. 8, no. 5, pp. 761–773, 2017, doi: 10.7150/jca.17648.
[2] C. Roma-Rodrigues, R. Mendes, P. V. Baptista, and A. R. Fernandes, “Targeting tumor microenvironment for cancer therapy,” Int. J. Mol. Sci., vol. 20, no. 4, 2019, doi: 10.3390/ijms20040840.
[3] A. L. Harris, “Hypoxia - A key regulatory factor in tumour growth,” Nat. Rev. Cancer, vol. 2, no. 1, pp. 38–47, 2002, doi: 10.1038/nrc704.
[4] H. Wang et al., “Nanoparticles-mediated reoxygenation strategy relieves tumor hypoxia for enhanced cancer therapy,” J. Control. Release, vol. 319, pp. 25–45, 2020, doi: 10.1016/j.jconrel.2019.12.028.
[5] G. L. Semenza, “Targeting HIF-1 for cancer therapy,” Nat. Rev. Cancer, vol. 3, no. 10, pp. 721–732, 2003, doi: 10.1038/nrc1187.
[6] B. Muz, P. de la Puente, F. Azab, and A. K. Azab, “The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy,” Hypoxia, p. 83, 2015, doi: 10.2147/hp.s93413.
[7] E. Henke, R. Nandigama, and S. Ergün, “Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy,” Front. Mol. Biosci., vol. 6, no. January, pp. 1–24, 2020, doi: 10.3389/fmolb.2019.00160.
[8] C. H. Heldin, K. Rubin, K. Pietras, and A. Östman, “High interstitial fluid pressure - An obstacle in cancer therapy,” Nat. Rev. Cancer, vol. 4, no. 10, pp. 806–813, 2004, doi: 10.1038/nrc1456.
[9] S. K. Golombek et al., “Tumor targeting via EPR: Strategies to enhance patient responses,” Adv. Drug Deliv. Rev., vol. 130, pp. 17–38, 2018, doi: 10.1016/j.addr.2018.07.007.
[10] T. P. Padera, B. R. Stoll, J. B. Tooredman, D. Capen, E. Di Tomaso, and R. K. Jain, “Cancer cells compress intratumour vessels,” Nature, vol. 427, no. 6976, p. 695, 2004, doi: 10.1038/427695a.
[11] S. M. Kim, P. H. Faix, and J. E. Schnitzer, “Overcoming key biological barriers to cancer drug delivery and efficacy,” J. Control. Release, vol. 267, pp. 15–30, 2017, doi: 10.1016/j.jconrel.2017.09.016.
[12] J.-P. Cosse and C. Michiels, “Tumour Hypoxia Affects the Responsiveness of Cancer Cells to Chemotherapy and Promotes Cancer Progression,” Anticancer. Agents Med. Chem., vol. 8, no. 7, pp. 790–797, 2012, doi: 10.2174/187152008785914798.
[13] D. J. McKenna, R. Errington, and K. Pors, “Current challenges and opportunities in treating hypoxic prostate tumors,” J. Cancer Metastasis Treat., vol. 4, no. 3, p. 11, 2018, doi: 10.20517/2394-4722.2017.54.
[14] M. Reprogramming and L. Cancer, “Hypoxia, Metabolic Reprogramming, and Drug Resistance in Liver Cancer,” pp. 1–18, 2021.
[15] T. Hompland, C. S. Fjeldbo, and H. Lyng, “Tumor hypoxia as a barrier in cancer therapy: Why levels matter,” Cancers (Basel)., vol. 13, no. 3, pp. 1–23, 2021, doi: 10.3390/cancers13030499.
[16] B. Movsas et al., “Hypoxic regions exist in human prostate carcinoma,” Urology, vol. 53, no. 1, pp. 11–18, 1999, doi: 10.1016/S0090-4295(98)00500-7.
[17] P. Vaupel, M. Höckel, and A. Mayer, “Detection and characterization of tumor hypoxia using pO2 histography.,” Antioxid. Redox Signal., vol. 9, no. 8, pp. 1221–1235, Aug. 2007, doi: 10.1089/ars.2007.1628.
[18] Q. T. Le et al., “An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers,” Clin. Cancer Res., vol. 12, no. 5, pp. 1507–1514, 2006, doi: 10.1158/1078-0432.CCR-05-2049.
[19] A. C. Koong et al., “Pancreatic tumors show high levels of hypoxia,” Int. J. Radiat. Oncol. Biol. Phys., vol. 48, no. 4, pp. 919–922, 2000, doi: 10.1016/S0360-3016(00)00803-8.
[20] J. Mattern, F. Kallinowski, C. Herfarth, and M. Volm, “Association of resistance related protein expression with poor vascularization and low levels of oxygen in human rectal cancer,” vol. 23, pp. 20–23, 1996.
[21] A. M. Shannon, D. J. Bouchier-Hayes, C. M. Condron, and D. Toomey, “Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies,” Cancer Treat. Rev., vol. 29, no. 4, pp. 297–307, 2003, doi: 10.1016/S0305-7372(03)00003-3.
[22] M. V. Sales Amaral et al., “Establishment of drug-resistant cell lines as a model in experimental oncology: A review,” Anticancer Res., vol. 39, no. 12, pp. 6443–6455, 2019, doi: 10.21873/anticanres.13858.
[23] T. W. Loo and D. M. Clarke, “Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux,” J. Membr. Biol., vol. 206, no. 3, pp. 173–185, 2005, doi: 10.1007/s00232-005-0792-1.
[24] N. Rohwer and T. Cramer, “Hypoxia-mediated drug resistance: Novel insights on the functional interaction of HIFs and cell death pathways,” Drug Resist. Updat., vol. 14, no. 3, pp. 191–201, 2011, doi: 10.1016/j.drup.2011.03.001.
[25] J. G. Park et al., “MDR1 gene expression: Its effect on drug resistance to doxorubicin in human hepatocellular carcinoma cell lines,” J. Natl. Cancer Inst., vol. 86, no. 9, pp. 700–705, 1994, doi: 10.1093/jnci/86.9.700.
[26] P. Fortini, C. Ferretti, and E. Dogliotti, “The response to DNA damage during differentiation: Pathways and consequences,” Mutat. Res. - Fundam. Mol. Mech. Mutagen., vol. 743–744, pp. 160–168, 2013, doi: 10.1016/j.mrfmmm.2013.03.004.
[27] L. Li et al., “Targeting DNA damage response in the radio(Chemo)therapy of non-small cell lung cancer,” Int. J. Mol. Sci., vol. 17, no. 6, 2016, doi: 10.3390/ijms17060839.
[28] Y. Xia, L. Jiang, and T. Zhong, “The role of HiF-1α in chemo-/radioresistant tumors,” OncoTargets and Therapy. pp. 11–3003, 2018.
[29] H.-K. L. Abdol-Hossein Rezaeian, Yu-Hui Wang, “DNA damage players are linked to HIF-1α hypoxia signaling,” Cell Cycle, vol. 16, no. No.8, pp. 725–726, 2017.
[30] R. Wirthner, S. Wrann, K. Balamurugan, R. H. Wenger, and D. P. Stiehl, “Impaired DNA double-strand break repair contributes to chemoresistance in HIF-1α-deficient mouse embryonic fibroblasts,” Carcinogenesis, vol. 29, no. 12, pp. 2306–2316, 2008, doi: 10.1093/carcin/bgn231.
[31] X. Jing et al., “Role of hypoxia in cancer therapy by regulating the tumor microenvironment,” Mol. Cancer, vol. 18, no. 1, pp. 1–15, 2019, doi: 10.1186/s12943-019-1089-9.
[32] C. Bayer and P. Vaupel, “Acute versus chronic hypoxia in tumors : Controversial data concerning time frames and biological consequences,” Strahlentherapie und Onkol., vol. 188, no. 7, pp. 616–627, 2012, doi: 10.1007/s00066-012-0085-4.
[33] M. S. Khan et al., “Engineering oxygen nanobubbles for the effective reversal of hypoxia,” Artif. Cells, Nanomedicine Biotechnol., vol. 46, no. sup3, pp. S318–S327, 2018, doi: 10.1080/21691401.2018.1492420.
[34] M. S. Khan et al., “Anti-tumor drug-loaded oxygen nanobubbles for the degradation of HIF-1α and the upregulation of reactive oxygen species in tumor cells,” Cancers (Basel)., vol. 11, no. 10, pp. 19–23, 2019, doi: 10.3390/cancers11101464.
[35] C. C. Huang et al., “An Implantable Depot That Can Generate Oxygen in Situ for Overcoming Hypoxia-Induced Resistance to Anticancer Drugs in Chemotherapy,” J. Am. Chem. Soc., vol. 138, no. 16, pp. 5222–5225, 2016, doi: 10.1021/jacs.6b01784.
[36] A. M. Poff, N. Ward, T. N. Seyfried, P. Arnold, and D. P. D’Agostino, “Non-toxic metabolic management of metastatic cancer in VM mice: Novel combination of ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy,” PLoS One, vol. 10, no. 6, pp. 1–21, 2015, doi: 10.1371/journal.pone.0127407.
[37] S. Y. Chen, K. Tsuneyama, M. H. Yen, J. T. Lee, J. L. Chen, and S. M. Huang, “Hyperbaric oxygen suppressed tumor progression through the improvement of tumor hypoxia and induction of tumor apoptosis in A549-cell-transferred lung cancer,” Sci. Rep., vol. 11, no. 1, pp. 1–15, 2021, doi: 10.1038/s41598-021-91454-2.
[38] M. S. Iyikesici, “Feasibility study of metabolically supported chemotherapy with weekly carboplatin/paclitaxel combined with ketogenic diet, hyperthermia and hyperbaric oxygen therapy in metastatic non-small cell lung cancer,” Int. J. Hyperth., vol. 36, no. 1, pp. 446–455, 2019, doi: 10.1080/02656736.2019.1589584.
[39] P. M. Petre et al., “Hyperbaric oxygen as a chemotherapy adjuvant in the treatment of metastatic lung tumors in a rat model,” J. Thorac. Cardiovasc. Surg., vol. 125, no. 1, pp. 85–95, 2003, doi: 10.1067/mtc.2003.90.
[40] T. Beppu, K. Kamada, Y. Yoshida, H. Arai, K. Ogasawara, and A. Ogawa, “Change of oxygen pressure in glioblastoma tissue under various conditions,” J. Neurooncol., vol. 58, no. 1, pp. 47–52, 2002, doi: 10.1023/A:1015832726054.
[41] A. Becker, T. Kuhnt, H. Liedtke, A. Krivokuca, M. Bloching, and J. Dunst, “Oxygenation measurements in head and neck cancers during hyperbaric oxygenation,” Strahlentherapie und Onkol., vol. 178, no. 2, pp. 105–108, 2002, doi: 10.1007/s00066-002-0892-0.
[42] T. C. Kian, N. B. Hampson, D. G. Bostwick, R. L. Vessella, and J. M. Corman, “Hyperbaric oxygen does not accelerate latent in vivo prostate cancer: Implications for the treatment of radiation-induced haemorrhagic cystitis,” BJU Int., vol. 94, no. 9, pp. 1275–1278, 2004, doi: 10.1111/j.1464-410X.2004.05156.x.
[43] H. Tang, Y. Sun, C. Xu, T. Zhou, X. Gao, and L. Wang, “Effects of Hyperbaric Oxygen Therapy on Tumor Growth in Murine Model of PC-3 Prostate Cancer Cell Line,” Urology, vol. 73, no. 1, pp. 205–208, 2009, doi: 10.1016/j.urology.2008.04.057.
[44] I. Moen and L. E. B. Stuhr, “Hyperbaric oxygen therapy and cancer - A review,” Target. Oncol., vol. 7, no. 4, pp. 233–242, 2012, doi: 10.1007/s11523-012-0233-x.
[45] S. Yildiz, H. Ay, and T. Qyrdedi, “Central nervous system oxygen toxicity during routine hyperbaric oxygen therapy.,” Undersea & hyperbaric medicine : journal of the Undersea and Hyperbaric Medical Society, Inc, vol. 31, no. 2. pp. 189–190, 2004.
[46] I. Grover and T. Neuman, “Hyperbaric Oxygen Therapy,” Encycl. Respir. Med. Four-Volume Set, vol. 317, no. 0831, pp. 292–296, 2006, doi: 10.1016/B0-12-370879-6/00180-0.
[47] Y. J. Ho, C. C. Huang, C. H. Fan, H. L. Liu, and C. K. Yeh, “Ultrasonic technologies in imaging and drug delivery,” Cell. Mol. Life Sci., vol. 78, no. 17–18, pp. 6119–6141, 2021, doi: 10.1007/s00018-021-03904-9.
[48] D. Cosgrove and C. Harvey, “Clinical uses of microbubbles in diagnosis and treatment,” Med. Biol. Eng. Comput., vol. 47, no. 8, pp. 813–826, 2009, doi: 10.1007/s11517-009-0434-3.
[49] J. R. Lindner, “Microbubbles in medical imaging: Current applications and future directions,” Nat. Rev. Drug Discov., vol. 3, no. 6, pp. 527–532, 2004, doi: 10.1038/nrd1417.
[50] Y. J. Ho et al., “Normalization of tumor vasculature by oxygen microbubbles with ultrasound,” Theranostics, vol. 9, no. 24, pp. 7370–7383, 2019, doi: 10.7150/thno.37750.
[51] Y. J. Ho et al., “Superhydrophobic drug-loaded mesoporous silica nanoparticles capped with β-cyclodextrin for ultrasound image-guided combined antivascular and chemo-sonodynamic therapy,” Biomaterials, vol. 232, no. 101, 2020, doi: 10.1016/j.biomaterials.2019.119723.
[52] E. Stride, “Physical Principles of Microbubbles for ultrasound imaging and therapy,” Cerebrovasc. Dis., vol. 27, no. suppl 2, pp. 1–13, 2009, doi: 10.1159/000203122.
[53] Y. J. Ho, T. C. Wang, C. H. Fan, and C. K. Yeh, “Spatially Uniform Tumor Treatment and Drug Penetration by Regulating Ultrasound with Microbubbles,” ACS Appl. Mater. Interfaces, vol. 10, no. 21, pp. 17784–17791, 2018, doi: 10.1021/acsami.8b05508.
[54] M. L. Fabiilli, K. J. Haworth, N. H. Fakhri, O. D. Kripfgans, P. L. Carson, and J. B. Fowlkes, “The role of inertial cavitation in acoustic droplet vaporization,” IEEE Trans. Ultrason. Ferroelectr. Freq. Control, vol. 56, no. 5, pp. 1006–1017, 2009, doi: 10.1109/TUFFC.2009.1132.
[55] D. Qin, Q. Zou, S. Lei, W. Wang, and Z. Li, “Cavitation dynamics and inertial cavitation threshold of lipid coated microbubbles in viscoelastic media with bubble–bubble interactions,” Micromachines, vol. 12, no. 9, 2021, doi: 10.3390/mi12091125.
[56] Y. J. Ho, T. C. Wang, C. H. Fan, and C. K. Yeh, “Current progress in antivascular tumor therapy,” Drug Discov. Today, vol. 22, no. 10, pp. 1503–1515, 2017, doi: 10.1016/j.drudis.2017.06.001.
[57] Y. J. Ho, J. P. Li, C. H. Fan, H. L. Liu, and C. K. Yeh, “Ultrasound in tumor immunotherapy: Current status and future developments,” J. Control. Release, vol. 323, no. February, pp. 12–23, 2020, doi: 10.1016/j.jconrel.2020.04.023.
[58] E. Osei and A. Al-Asady, “A review of ultrasound-mediated microbubbles technology for cancer therapy: A vehicle for chemotherapeutic drug delivery,” J. Radiother. Pract., vol. 19, no. 3, pp. 291–298, 2020, doi: 10.1017/S1460396919000633.
[59] and K. W. F. A. Shengping Qin, Charles F Caskey, “Ultrasound contrast microbubbles in imaging and therapy physical principles and engineering,” Phys Med Biol, vol. 54, no. 6, pp. 1–42, 2010, doi: 10.1088/0031-9155/54/6/R01.Ultrasound.
[60] C. Y. Ting et al., “Concurrent blood-brain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment,” Biomaterials, vol. 33, no. 2, pp. 704–712, 2012, doi: 10.1016/j.biomaterials.2011.09.096.
[61] T. D. Reusser, D. Ramirez, R. K. P. Benninger, V. Papadopoulou, P. A. Dayton, and M. A. Borden, “Designing Oxygen Microbubbles for Treating Tumor Hypoxia,” IEEE Int. Ultrason. Symp. IUS, vol. 2019-Octob, pp. 1338–1341, 2019, doi: 10.1109/ULTSYM.2019.8926242.
[62] S. M. Fix et al., “Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model – A preliminary study,” pp. 1–18, 2018.
[63] J. R. Eisenbrey et al., “Sensitization of Hypoxic Tumors to Radiation Therapy Using Ultrasound-Sensitive Oxygen Microbubbles,” Radiat. Oncol. Biol., vol. 101, no. 1, pp. 88–96, 2018, doi: 10.1016/j.ijrobp.2018.01.042.
[64] A. Bisazza, P. Giustetto, A. Rolfo, I. Caniggia, S. Balbis, and R. Cavalli, “Microbubble-Mediated Oxygen Delivery to Hypoxic Tissues As a New Therapeutic Device,” pp. 2067–2070, 2008.
[65] J. J. Kwan, M. Kaya, M. A. Borden, and P. A. Dayton, “Theranostic Oxygen Delivery Using Ultrasound and Microbubbles,” vol. 2, no. 12, 2012, doi: 10.7150/thno.4410.
[66] J. R. Eisenbrey et al., “Development of an ultrasound sensitive oxygen carrier for oxygen delivery to hypoxic tissue,” Int J Pharm, vol. 478, no. 1, pp. 361–367, 2019, doi: 10.1016/j.ijpharm.2014.11.023.Development.
[67] D. Wang et al., “A Mesoporous Nanoenzyme Derived from Metal–Organic Frameworks with Endogenous Oxygen Generation to Alleviate Tumor Hypoxia for Significantly Enhanced Photodynamic Therapy,” Adv. Mater., vol. 31, no. 27, pp. 1–9, 2019, doi: 10.1002/adma.201901893.
[68] Z. Yang, J. Wang, S. Ai, J. Sun, X. Mai, and W. Guan, “Self-generating oxygen enhanced mitochondriontargeted photodynamic therapy for tumor treatment with hypoxia scavenging,” Theranostics, vol. 9, no. 23, pp. 6809–6823, 2019, doi: 10.7150/thno.36988.
[69] J. Chen et al., “Oxygen-Self-Produced Nanoplatform for Relieving Hypoxia and Breaking Resistance to Sonodynamic Treatment of Pancreatic Cancer,” ACS Nano, vol. 11, no. 12, pp. 12849–12862, 2017, doi: 10.1021/acsnano.7b08225.
[70] H. Tian et al., “Cancer Cell Membrane-Biomimetic Oxygen Nanocarrier for Breaking Hypoxia-Induced Chemoresistance,” Adv. Funct. Mater., vol. 27, no. 38, pp. 1–7, 2017, doi: 10.1002/adfm.201703197.
[71] C. F. Thorn et al., “Doxorubicin pathways: pharmacodynamics and adverse effects,” Pharmacogenet. Genomics, vol. 21, no. 7, pp. 440–446, 2011, doi: 10.1097/fpc.0b013e32833ffb56.
[72] J. Das, Y. J. Choi, J. W. Han, A. M. M. T. Reza, and J. H. Kim, “Nanoceria-mediated delivery of doxorubicin enhances the anti-tumour efficiency in ovarian cancer cells via apoptosis,” Sci. Rep., vol. 7, no. 1, pp. 1–12, 2017, doi: 10.1038/s41598-017-09876-w.