在小鼠模型中Semaphorin 6A 在抗癌免疫上的機制及作用

To study semaphorin 6A regulated the anti-cancer immune response and its mechanism in mouse model

陳語謙(Yu-Chien Chen)

指導教授 ： 李心予

共同指導教授 ： 莊曜宇

國立臺灣大學/生命科學院/生命科學系/碩士(2020年)

https://doi.org/10.6342/NTU202000215

摘要

全世界癌症死亡率不斷攀升，世界衛生組織WHO（World Health Organization）2018 年統計指出肺癌死亡率為所有癌症死亡率中最高。目前較常見肺癌治療方式有手術移除、放療、化療及標靶治療，雖然已有明顯提升肺癌存活率，但晚期肺癌存活率仍偏低為19%。近幾年癌症治療的方法中有一種新興的治療方式稱為免疫療法，免疫治療可以促進自身的免疫系統來攻擊腫瘤細胞藉此阻止癌症發展。為了使免疫治療可以發揮最佳效果，找出具有正向免疫調節性質且能夠對抗癌症的指標基因是很重要的。根據我們實驗室之前的研究，在肺癌組織中Semaphorin 6A (SEMA6A) 表現量較周邊正常組織低。而我們先前ex vivo 的研究中，SEMA6A 能夠降低調節型 T 細胞的增生能力，以及減輕SEMA3A 抑制毒殺型 T 細胞及自然殺手細胞毒殺能力的效果。因此推測SEMA6A 在癌症免疫中可能扮重要角色。因為已知SEMA3A 抑制T 細胞所使用的受體為Neuropilin1(NRP1)/PlexinA4(PLXNA4) 也是SEMA6A 的受體， 所以提出SEMA6A 可能是經由與SEMA3A 競爭NRP1/PLXNA4 ，而避免SEMA3A 的免疫抑制的假設。本篇研究目的是進一步以小鼠模型探討SEMA6A 的抗癌免疫功能以及與SEMA3A 在免疫的相互作用。首先，我們研究SEMA6A 及SEMA3A是否會競爭NPR1/PLNXA4 受體，並影響NRP1/PLNXA4 下游基因表現，結果顯示SEMA6A 會與SEMA3A 競爭NRP1 的結合且會抑制掉SEMA3A/NRP1 下游路徑，所以證實SEMA6A 應是經由與SEMA3A 競爭NRP1/PLNXA4 受體來避免肺癌細胞藉由SEMA3A 引發的免疫抑制。而小鼠模型實驗中，將不同處理之小鼠肺癌細胞KLN205，以皮下注射打進小鼠體內，4-5 周後犧牲測量腫瘤大小，並以流式細胞儀檢測免疫細胞對KLN205 細胞活性，結果表示SEMA6A 可有效的降低腫瘤的成長，在免疫細胞的調控方面SEMA6A 可以抑制SEMA3A 對毒殺性T 細胞的抑制作用，並且藉由抑制SEMA3A 來降低調節型T 細胞的增生，除此之外SEMA6A 也可減輕SEMA3A 減少腫瘤浸潤淋巴細胞比例的現象。統整以上結果，SEMA6A 可以透過競爭SEMA3A 的受體NRP1 來抑制SEMA3A/NRP1 的下游基因表現，進而抑制SEMA3A 的抑制免疫效果回復免疫細胞的增生、移動能力、抑制腫瘤的形成。

關鍵字

肺癌 ； Semaphorin 6A ； Semaphorin 3A ； Neuropilin1 ； 毒殺型T細胞 ； 調節型T 細胞 ； 腫瘤浸潤淋巴細胞 ； 細胞性免疫 ； 抗癌免疫

並列摘要

In 2018, 9.6 million people died because of cancer worldwide. Lung cancer is the leading cause of cancers death. Although some therapies have been used to treat lung cancer, the prognosis of patients remains poor. Thus, the therapy strategies of lung cancer have to be developed. Immunotherapy is a revolutionary cancer therapy that uses the natural capability of immune system to detect and destroy cancer cells. Therefore, discovering stimulators of immunity is an important task. Our previous study showed that the level of semaphorin (SEMA)-6A was lower in cancer tissues than in adjacent normal tissues. The results of our ex vivo study also showed that SEMA6A reduced the SEMA3A-led inhibition of anti-cancer activity of cytotoxic T (Tc) cells and natural killer (NK) cells and decreased the proliferation of regulatory T (Treg) cells. However, the mechanism of how SEMA6A reduces the effects of SEMA3A in immune cells is still unclear. In the study, I firstly investigated whether SEMA6A decreased SEMA3A-derived immunosuppression by disrupting the binding of SEMA3A between NRP1/PLNXA4, which is the co-receptor of both SEMA3A and SEMA6A and is used by SEMA3A to suppress T cells. Then, we further validated the ex vivo experiments of immunoregulatory of SEMA6A in mouse models. The results of competition assay revealed that the level of SEMA3A and NRP1 bindings was negatively correlated with the dose of SEMA6A. The results of GSEA also showed that SEMA6A lowered the SEMA3A/NRP1-derived pathways. In the in vivo trial, mice were injected with SEMA3A and KLN205KD cells with/without SEMA6A overexpression. After 4-5 weeks, the tumors were smaller in the mice injected with SEMA6A overexpressing KLN205KD cells. The splenocytes of mice were subsequently co-cultured with KLN205KD cells, and the immune effects of SEMA6A were evaluated by the proliferation of Tc, Th, and Treg cells. Moreover, the proportion of tumor-infiltrating lymphocytes (TILs), Tc and NK cells, were measured in tumor. The results indicated that SEMA3A reduced the proliferation of Tc and induced which of Treg. The TILs were also decreased by SEMA3A. Moreover, SEMA6A modulated the effects of SEMA3A in immune cells. Therefore, the in vivo study double confirmed the effects of SEMA6A on anti-cacner immunity. In conclusion, our results suggest that SEMA6A attenuates tumor formation, supresses the downstream gene expression of SEMA3A/NRP1, and further inhibits the immune effect of SEMA3A to restore the proliferation and migration ability of immune cells.

並列關鍵字

lung cancer ； Semaphorin 6A ； Semaphorin 3A ； Neuropilin1 ； cytotoxic T cells ； regulatory T cells ； Tumor-infiltrating lymphocytes ； cellular immunity ； anti-cancer immunity

參考文獻

1. Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018. 68(6): p. 394-424.

2. Siegel, R.L., K.D. Miller, and A.J.C.a.c.j.f.c. Jemal, Cancer statistics, 2019. 2019. 69(1): p. 7-34.

3. Shames, D.S. and Wistuba, II, The evolving genomic classification of lung cancer. J Pathol, 2014. 232(2): p. 121-33.

4. Furrukh, M., Tobacco Smoking and Lung Cancer: Perception-changing facts. Sultan Qaboos Univ Med J, 2013. 13(3): p. 345-58.

5. Zhong, L., et al., A case-control study of lung cancer and environmental tobacco smoke among nonsmoking women living in Shanghai, China. Cancer Causes Control, 1999. 10(6): p. 607-16.