利用Semaphorin6A基因剔除小鼠模型探討Semaphorin6A為基礎之重組蛋白於肺癌免疫治療的效果

Using Semaphorin6A Knockout Mouse Model to Examine Antitumor Immune Response Triggered by Semaphorin6A-Based Recombinant Proteins in Lung Cancer Immunotherapy

黃信和(Shin-Her Huang)

指導教授 ： 蔡孟勳

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102173

摘要

全球肺癌於癌症排名中致死率高居第一、發生率位居第二，五年存活率僅21％。肺癌患者通常接受手術、化學療法、放射療法或標靶療法，而近期較新的免疫療法正蓬勃發展，免疫療法可以藉由刺激抗癌免疫反應或抵消由癌細胞誘導的免疫抑制機制達成治療功效。先前研究證實Semaphorin6A（Sema6A）可以透過競爭Semaphorin3A (Sema3A)與Neuropinlin1（Nrp1）/ Plexin-A4共受體的結合而增強細胞毒殺型T細胞的增生能力同時減少調節型T細胞的增生能力，Sema6A成為免疫治療的潛在目標。由於Sema6A的分子量高、結構複雜且為穿膜蛋白，製程困難，因此，本研究設計兩種Sema6A為基礎的重組蛋白—SEMA-PSI與SEMA，並使用Sema6A基因剔除C57BL/6J小鼠探討Sema6A蛋白、SEMA-PSI重組蛋白及SEMA重組蛋白對於抗癌免疫反應的影響。競爭試驗顯示Sema6A蛋白、SEMA-PSI和SEMA重組蛋白於濃度為5至20 μg/ml時可競爭Sema3A與Nrp1的結合。透過共培養LL/2小鼠肺癌細胞株與來自皮下注射LL/2的Sema6A基因剔除以及野生型小鼠的脾臟細胞，顯示SEMA重組蛋白均可顯著減少調節型T細胞的增生能力，也代表抗癌免疫的提升，SEMA-PSI重組蛋白可降低調節型T細胞的增生能力但未達顯著，而Sema6A蛋白未顯著影響免疫細胞的增生能力。活體實驗顯示，LL/2腫瘤於Sema6A基因剔除小鼠的生長較野生型小鼠快速，且腫瘤浸潤毒殺型T細胞的比例也顯著低於野生型小鼠。為了評估在活體中Sema6A、SEMA-PSI和SEMA重組蛋白治療後的抗癌免疫情形，皮下注射LL/2細胞至Sema6A基因剔除小鼠，每兩天腫瘤周邊皮下注射20μg的蛋白，與對照組相比，SEMA重組蛋白可顯著降低腫瘤的生長，而Sema6A和SEMA-PSI重組蛋白未顯著降低腫瘤的生長，SEMA重組蛋白治療的組別中，腫瘤浸潤毒殺型T細胞無顯著差異，而腫瘤浸潤調節型T細胞有下降趨勢但未達顯著，取其脾臟細胞發現毒殺型T細胞及自然殺手細胞的增生能力有上升趨勢但未達顯著，調節型T細胞的增生能力也有下降但未達顯著。另外於野生型小鼠模型中，SEMA-PSI及SEMA重組蛋白治療後未顯著降低LL/2腫瘤生長，但SEMA重組蛋白治療後的腫瘤浸潤毒殺型T細胞有上升趨勢，且調節型T細胞增生能力也有下降趨勢。為建立化學物質誘導的肺癌致癌模型，本研究將致癌物4-（甲基亞硝胺）-1-（3-吡啶基）-1-丁酮 （NNK）腹腔注射至14隻Sema6A基因剔除及8隻野生型小鼠，觀察第16周及32周時鼠肺中結節的形成，僅一隻Sema6A基因剔除小鼠肺部外觀於第16周出現明顯腫瘤結節，依據病理組織型態判讀為骨肉瘤，而野生型小鼠肺部外觀皆未觀察到明顯腫瘤結節，經蘇木精-伊紅染色後，無論第16周或32周，小鼠肺部損傷程度於Sema6A基因剔除小鼠皆高於野生型小鼠，且未觀察到肺腺癌細胞或鱗狀上皮癌細胞的形成。
總結而論，LL/2腫瘤於Sema6A基因剔除小鼠中生長較野生型小鼠快速，且腫瘤浸潤毒殺型T細胞顯著降低。Sema6A蛋白、SEMA-PSI重組蛋白和SEMA重組蛋白可以競爭Sema3A與Nrp1的結合。活體外共培養結果顯示SEMA重組蛋白可顯著減少調節型T細胞的增生能力，意謂抗癌免疫反應的提升。活體試驗指出SEMA重組蛋白有提升抗癌免疫的趨勢，顯著降低LL/2腫瘤於Sema6A基因剔除小鼠的生長，綜上所述，SEMA重組蛋白的抗癌效果較Sema6A蛋白及SEMA-PSI重組蛋白優異，成為潛在的肺癌免疫治療蛋白。

關鍵字

肺癌 ； 免疫療法 ； Semaphorin6A ； Semaphorin3A ； 基因剔除 ； NNK

並列摘要

Lung cancer is the leading cause of cancer-related death and 5-year survival rate for all people with all types of lung cancer is 21% worldwide. Patients with lung cancer are usually treated with surgery, chemotherapy, radiation therapy, or targeted therapy. The immunotherapy becomes popular nowadays. The immunotherapy is performed either to stimulate the anti-cancer immune response or to counteract the immune suppression induced by cancer cells. Our previous study showed that Semaphorin6A (Sema6A) could enhance proliferating cytotoxic T cells (Tc) and reduce proliferating regulatory T cells (Treg) through competing with the binding of Semaphorin3A (Sema3A) to Neuropinlin1 (Nrp1)/Plexin-A4 co-receptor. Therefore, we hypothesized that Sema6A may be a potential target for immunotherapy. However, due to the high-molecular-weight and complex structure of Sema6A, it is very difficult to be manufactured. Thus, two recombinant proteins—SEMA-PSI domain and SEMA domain alone, which were only the functional domains located at the extracellular region of Sema6A were investigated for their anti-cancer ability in this study. Sema6A knockout (KO) C57BL/6J mice are utilized to elucidate the antitumor immune response triggered by mouse full-length Sema6A protein, SEMA-PSI recombinant protein, or SEMA recombinant protein. The result of the competitive assay indicated full-length Sema6A protein, SEMA-PSI, and SEMA recombinant proteins at the concentration of 5 to 20 ug/ml could reduce the binding of Sema3A to Nrp1. Ex vivo assay was conducted by co-culturing LL/2 mouse lung cancer cells with splenocytes collected from LL/2-bearing Sema6A KO or wild-type (WT) mice. Indicated that SEMA recombinant protein could significantly reduce proliferating Treg cells and SEMA-PSI recombinant protein showed the downtrend of proliferating Treg cells. However, full-length Sema6A protein did not take effect. In vivo assay indicated that LL/2 tumor growth in Sema6A KO mice was significantly faster than in wild-type (WT) mice and it was related to the significantly lower proportion of tumor-infiltrating Tc. To evaluate the treatment effect of Sema6A, SEMA-PSI, and SEMA recombinant proteins in vivo, Sema6A KO mice were subcutaneously injected with LL/2 cells and then peritumorally injected 20 μg of each protein every two days. The result showed that SEMA recombinant protein could significantly reduce tumor growth compared to the control group, whereas treatment of full-length Sema6A and SEMA-PSI recombinant proteins did not reduce the tumor growth significantly. In WT mouse model, treatment of SEMA-PSI and SEMA recombinant proteins did not reduce LL2 tumor growth, but tumor-infiltrating Tc increased and proliferating Treg decreased after SEMA recombinant protein was treated. To examine the treatment efficacy of chemically-induced lung carcinogenesis, the feasibility of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was assessed by peritoneally injecting into Sema6A KO and WT mice, then observing the formation of nodules in lungs at 16th and 32nd weeks. However, only one Sema6A KO mouse showed solid tumor nodules at the 16th week which were diagnosed with osteosarcoma and none of WT mice showed solid tumor nodules. After hematoxylin-eosin staining of each lung was performed, the degree of lesions was higher in Sema6A KO mice than in WT mice without the growth of adenocarcinoma cells or squamous carcinoma cells.
In conclusion, LL/2 tumor growth in Sema6A KO mice was significantly faster than in WT mice and it was related to the significantly lower proportion of tumor-infiltrating Tc. Full-length Sema6A protein, SEMA-PSI recombinant protein, and SEMA recombinant protein could compete for the binding of Sema3A to Nrp1. Ex vivo assay showed SEMA recombinant proteins could enhance antitumor immune response through reducing proliferating Treg. In vivo assay indicated SEMA recombinant protein could reduce LL/2 tumor growth in Sema6A KO mice and trigger antitumor immune response in WT mice. Taken together, SEMA recombinant protein performed better anti-cancer effect in both ex vivo and in vivo assay than full-length Sema6A and SEMA-PSI recombinant proteins, consequently becoming the potential target for immunotherapy in lung cancer.

並列關鍵字

Lung cancer ； immunotherapy ； Semaphorin6A ； Semaphorin3A ； knockout ； NNK

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