癌細胞是一種不正常增生的突變細胞，不受控制迅速增殖的腫瘤團塊限制了周邊血液以及氧氣供應，使其局部氧氣含量明顯低於健康組織，出現了｢缺氧｣的狀態，幾乎所有實體腫瘤都具有相似的微環境特徵。近年來的研究顯示，腫瘤缺氧的狀態下會刺激缺氧誘導因子HIF(hypoxia-inducible factor)表現，並造成許多影響，包括:血管生成、腫瘤轉移擴散、多重抗藥性產生、調節細胞增殖及抑制細胞凋亡。進而使腫瘤細胞延長存活並產生抗藥性及影響治療成功率。
本研究針對不同病人、腫瘤缺氧狀況彼此的差異，建立一項仿缺氧微環境之微型晶片，利用聚二甲基矽氧烷PDMS(Polydimethylsiloxane) 對於氣體的通透性，使用還原劑亞硫酸鈉還原吸收細胞培養區的的氧分子，在化學還原劑不與細胞直接接觸的前提下同時形成四種不同的氧氣含量。使用生物相容水膠材料GelMA作為癌細胞A549的3D生長支架，比較不同缺氧環境下對於免疫細胞Jurkat的遷移能力影響以及使用化療藥物Pemetrexed合併免疫藥物Pembrolizumab在這些環境中的治療效果。
實驗結果顯示，氧分子含量最低之腔室[O2]Chamber1=2%的免疫細胞遷入數量最少，並依序遞增到正常培養箱【O2】=20%，接著免疫細胞遷入數量與免疫治療藥物效果成正比，推測是透過低氧環境抑制免疫細胞之活性，再進而降低藥物療效。

Cancer cells are abnormal, mutated cells that undergo uncontrolled and rapid proliferation, forming tumor masses that restrict the supply of blood and oxygen to the surrounding tissues. This leads to significantly lower oxygen levels compared to healthy tissues, resulting in a state of "Hypoxia," which is a common micro-environmental characteristic in nearly all solid tumors. Recent research has shown that the hypoxic conditions in tumors stimulate the expression of Hypoxia-Inducible Factor (HIF) and have various effects, including promoting angiogenesis, facilitating tumor metastasis, inducing multidrug resistance, regulating cell proliferation, and inhibiting apoptosis. This ultimately allows tumor cells to survive longer, develop drug resistance, and impact the success rates of chemotherapy and radiotherapy.
In this study, we aimed to establish a microchip mimicking the hypoxic tumor microenvironment to investigate the differences in hypoxia among different patients and tumor conditions. We designed a microchip using PDMS (polydimethylsiloxane), which allows gas permeability, and employed sodium bisulfite as a reducing agent to reduce oxygen molecules within the cell culture chambers. Four different oxygen levels were generated simultaneously without direct contact with the cells. We utilized a biocompatible hydrogel material called GelMA as a 3D growth scaffold for cancer cells(A549.
We compared the impact of different hypoxic environments on the migration capability of immune cells (Jurkat) and assessed the therapeutic efficacy of chemotherapy drug Pemetrexed in combination with the immune drug Pembrolizumab within these environments.
The experimental results revealed that the chamber with the lowest oxygen concentration [O2]Chamber1=2% had the fewest immune cells migrating into it. The number of immune cells gradually increased up to the normal culture condition [O2] = 20%. Furthermore, the number of immune cells migrating and the effectiveness of immunotherapy were positively correlated, suggesting that the low oxygen environment inhibited the activity of immune cells, subsequently reducing the efficacy of the drugs.

[1] 衛生福利部. "111年國人死因統計結果." https://dep.mohw.gov.tw/dos/lp-5069-113-xCat-y111.html
[2] Wan L, Neumann CA, and L. PR, "Tumor-on-a-chip for integrating a 3D tumor microenvironment: chemical and mechanical factors," Lab Chip, vol. 20, no. 5, pp. 873-888, Mar 3, 2020.
[3] Wang, Y., Roche, O., Xu, C., Moriyama, E. H., Heir, P., Chung, J., Roos, Frederik C, Chen, Y. , Greg F., Milosevic, & Ohh, M. (2012). Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor–mediated upregulation of caveolin-1. Proceedings of the National Academy of Sciences, 109(13), 4892-4897.
[4] Bollinger, T., Gies, S., Naujoks, J., Feldhoff, L., Bollinger, A., Solbach, W., & Rupp, J. (2014). HIF-1α-and hypoxia-dependent immune responses in human CD4+ CD25high T cells and T helper 17 cells. Journal of leukocyte biology, 96(2), 305-312.
[5] B. Keith, R. S. Johnson, and M. C. Simon, "HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression," Nature Reviews Cancer, vol. 12, no. 1, pp. 9-22, 2012.
[6] I. Singh, I. S. Chohan, M. Lal, P. K. Khanna, M. C. Srivastava, R. B. Nanda, J. S. Lamba & M. S. Malhotra(1977). Effects of high altitude stay on the incidence of common diseases in man. International journal of biometeorology, 21, 93-122.
[7] Yu, A. Y., Frid, M. G., Shimoda, L. A., Wiener, C. M., Stenmark, K., & Semenza, G. L. (1998). Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. American Journal of Physiology-Lung Cellular and Molecular Physiology, 275(4), L818-L826.
[8] Kent, B. D., Mitchell, P. D., & McNicholas, W. T. (2011). Hypoxemia in patients with COPD: cause, effects, and disease progression. International journal of chronic obstructive pulmonary disease, 199-208.
[9] Vogel, J., Thiel, C. S., Tauber, S., Stockmann, C., Gassmann, M., & Ullrich, O. (2019). Expression of hypoxia-inducible factor 1α (HIF-1α) and genes of related pathways in altered gravity. International journal of molecular sciences, 20(2), 436.
[10] V. S. Shirure, S. F. Lam, B. Shergill, Y. E. Chu, N. R. Ng, and S. C. George, "Quantitative design strategies for fine control of oxygen in microfluidic systems," Lab Chip, vol. 20, no. 16, pp. 3036-3050, Aug 11 2020.
[11] Caldwell, C. C., Kojima, H., Lukashev, D., Armstrong, J., Farber, M., Apasov, S. G., & Sitkovsky, M. V. (2001). Differential effects of physiologically relevant hypoxic conditions on T lymphocyte development and effector functions. The Journal of Immunology, 167(11), 6140-6149.
[12] 醫學百科. "缺氧." http://cht.a-hospital.com/w/%E7%BC%BA%E6%B0%A7.
[13] Liu, C. H., Chang, H. D., Li, K. H., Lin, C. H., Hsu, C. J., Lin, T. Y., Chou, H. H., Huang, H. C. & Liao, H. Y. (2013, May). Adaptable and integrated packaging platform for MEMS-based combo sensors utilizing innovative wafer-level packaging technologies. In 2013 IEEE 63rd Electronic Components and Technology Conference (pp. 1675-1681). IEEE.
[14] M. Sánchez-Ortega, A. C. Carrera, and A. Garrido, "Role of NRF2 in lung cancer," Cells, vol. 10, no. 8, p. 1879, 2021.
[15] L. Jianhan, "肺癌分類與分期：小細胞癌與非小細胞癌," 2019. [Online]. Available: https://helloyishi.com.tw/cancer/lung-cancer/types-symptoms-and-risk-of-lung-cancer/.
[16] F. Al Dayel, "EGFR mutation testing in non-small cell lung cancer (NSCLC)," Journal of infection and public health, vol. 5, pp. S31-S34, 2012.
[17] L. Ye, X. Chen, and F. Zhou, "EGFR-mutant NSCLC: emerging novel drugs," Current Opinion in Oncology, vol. 33, no. 1, pp. 87-94, 2021.
[18] Cao, H., Yu, S., Chen, D., Jing, C., Wang, Z., Ma, R., Liu, S. , Ni, J., Feng, J. & Wu, J. (2017). Liver X receptor agonist T0901317 reverses resistance of A549 human lung cancer cells to EGFR‐TKI treatment. FEBS Open Bio, 7(1), 35-43.
[19] V. Petrova, M. Annicchiarico-Petruzzelli, G. Melino, and I. Amelio, "The hypoxic tumour microenvironment," Oncogenesis, vol. 7, no. 1, pp. 1-13, 2018.
[20] N. Li Jeon, H. Baskaran, S. K. Dertinger, G. M. Whitesides, L. Van de Water, and M. Toner, "Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device," Nat Biotechnol, vol. 20, no. 8, pp. 826-30, Aug 2002.
[21] Paul J. Hung, Philip J. Lee, Poorya Sabounchi, Robert Lin, and L. P. Lee, "Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays," Biotechnol Bioeng, vol. 89, pp. 1-8, Jan 5, 2005.
[22] K. Ziółkowska, R. Kwapiszewski, and Z. Brzózka, "Microfluidic devices as tools for mimicking the in vivo environment," New J. Chem, pp. 979-990, 2011.
[23] Coluccio, M. L., Perozziello, G., Malara, N., Parrotta, E., Zhang, P., Gentile, F., Limongi , T., Michael Raj, P. , Cuda, G. , Candeloro, P. & Di Fabrizio, E. (2019). Microfluidic platforms for cell cultures and investigations. Microelectronic Engineering, 208, 14-28.
[24] Swartz, M. A., Iida, N., Roberts, E. W., Sangaletti, S., Wong, M. H., Yull, F. E., Coussens, L. M. & DeClerck, Y. A. (2012). Tumor microenvironment complexity: emerging roles in cancer therapy.
[25] Y. G. Zhuyun Xu, Yuanyuan Hao, E.nchengLi, Yan Wang, Jianing Zhang, Wenxin Wang, Zhancheng Gao, and Qi Wang, "Application of a microfluidic chip-based 3D co-culture to test drug sensitivity for individualized treatment of lung cancer," Biomaterials, vol. 34 no.16, pp. 4109-4117, 2013 May.
[26] M. Adler, M. Polinkovsky, E. Gutierrez, and A. Groisman, "Generation of oxygen gradients with arbitrary shapes in a microfluidic device," Lab Chip, vol. 10, no. 3, pp. 388-91, Feb 7, 2010.
[27] Zirath, H., Rothbauer, M., Spitz, S., Bachmann, B., Jordan, C., Müller, B., Ehgartner, J., Priglinger, E., Mühleder, S., Redl, H., Holnthoner, W., Harasek, M., Mayr, T. & Ertl, P. (2018). Every breath you take: non-invasive real-time oxygen biosensing in two-and three-dimensional microfluidic cell models. Frontiers in physiology, 9, 815.
[28] Z. H. Wang, Z. X. Liu, L. L. Li, and Q. L. Liang, "Investigation into the hypoxia-dependent cytotoxicity of anticancer drugs under oxygen gradient in a microfluidic device," (in English), Microfluidics and Nanofluidics, vol. 19, no. 6, pp. 1271-1279, Dec 2015.
[29] Zhong, H., De Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D., Buechler P., Isaacs, W. B., Semenza, G. L. & Simons, J. W. (1999). Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases. Cancer research, 59(22), 5830-5835.
[30] M. Sitkovsky and D. Lukashev, "Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors," Nat Rev Immunol, vol. 5, no. 9, pp. 712-21, Sep 2005.
[31] Y. Li, L. Zhao, and Xiao-Feng, "Targeting Hypoxia: Hypoxia-Activated Prodrugs in Cancer Therapy," Front Oncol, vol. 11, p. 700407, 2021.
[32] "Wikipedia_HIF-1A." https://en.wikipedia.org/wiki/HIF1A#cite_note-40.
[33] G. L. Semenza, "Targeting HIF-1 for cancer therapy," Nature reviews cancer, vol. 3, no—10, pp. 721-732, 2003.
[34] J.-W. Lee, S.-H. Bae, J.-W. Jeong, S.-H. Kim, and K.-W. Kim, "Hypoxia-inducible factor (HIF-1) α: its protein stability and biological functions," Experimental & molecular medicine, vol. 36, no. 1, pp. 1-12, 2004.
[35] Munkácsy, G., Sztupinszki, Z., Herman, P., Bán, B., Pénzváltó, Z., Szarvas, N., & Győrffy, B. (2016). Validation of RNAi silencing efficiency using gene array data shows 18.5% failure rate across 429 independent experiments. Molecular Therapy-Nucleic Acids, 5.
[36] Batra, U., Sharma, M., Nathany, S., Bansal, A., Pasricha, S., Jain, P., Mehta, A.& Singh, H. (2021). EGFR and PDL1: A Match (Not) Made in Heaven—A Real-World Retrospective Analysis of PDL1 Expression in EGFR-Mutated NSCLC. Advances in Therapy, 38, 1791-1800.
[37] Chaft, J. E., Rimner, A., Weder, W., Azzoli, C. G., Kris, M. G., & Cascone, T. (2021). Evolution of systemic therapy for stages I–III non-metastatic non-small-cell lung cancer. Nature reviews Clinical oncology, 18(9), 547-557.