胰管腺癌的侵襲性非常強，五年存活率僅有 8%。胰管腺癌的特徵是具有高度的組織間質增生，即細胞外基質堆積和纖維化組織的增加，這已被證明會干擾藥物遞送效果和促進腫瘤進展。然而，到目前為止造成組織間質增生的機制仍然不清楚。細胞外囊泡是由細胞釋放的奈米大小的粒子，它能攜帶多種生物分子進行遠端調控，並且影響腫瘤微環境。我們先前已經發現雙特異性磷酸酶-2的表達下降會促進細胞外囊泡的分泌。此外，小鼠胰腺中的組織特異性之雙特異性磷酸酶-2剔除表現出組織間質增生。然而，雙特異性磷酸酶-2如何調節細胞外囊泡的生成以及對胰管腺癌腫瘤形成的影響仍然未知。因此，本篇研究想要去探討生成細胞外囊泡的潛在機制以及其對組織間質增生的影響。首先，我們使用蛋白質轉漬法確定了四跨膜蛋白（細胞外囊泡的標誌蛋白）的表達，發現與對照組相比，在雙特異性磷酸酶-2表達下降的胰臟癌細胞中的CD63表達量增加。除此之外還觀察到高分子量的CD63，代表抑制雙特異性磷酸酶-2可能會使得CD63發生了轉譯後修飾。因此，利用醣基化抑製劑（衣黴素）和重組糖苷酰胺酶來證明 CD63在雙特異性磷酸酶-2 表達下降的胰臟癌細胞中是高度糖基化的。透過衣黴素去阻斷 CD63 醣基化，減少了由雙特異性磷酸酶-2表達下降所誘導的胰臟癌細胞外囊泡的分泌，證明了四跨膜蛋白的糖基化是細胞外囊泡生物發生的關鍵步驟。接下來，從雙特異性磷酸酶-2表達下降的胰臟癌細胞中分離出細胞外囊泡，並將其用於處理胰腺星狀細胞。我們發現用細胞外囊泡處理後導致胰腺星狀細胞活化，其中的特徵為脂滴的流失。為了在老鼠模型中驗證結果，我萃取了來自雙特異性磷酸酶-2表達下降的胰臟癌細胞的細胞外囊泡，並將它們透過皮下注射到小鼠的胰臟癌腫瘤中。結果與對照組的腫瘤相比，細胞外囊泡治療的腫瘤中堆積了更多的膠原蛋白，證明了細胞外囊泡會促進了組織間質增生。最後，我們利用一種新型組織蛋白去乙醯酶抑制劑 (B390)，透過給予組織蛋白去乙醯酶抑制劑可以誘導雙特異性磷酸-2重新的在胰臟癌細胞中表達，也抑制了雙特異性磷酸酶-2表達下降的胰臟癌細胞中的 CD63 醣基化。此外，與對照組小鼠相比，用組織蛋白去乙醯酶抑制劑處理患有原位腫瘤的小鼠後，發現有較少的膠原纖維沉積於腫瘤中。綜合以上的數據，我們發現雙特異性磷酸酶-2的缺失調節了細胞外囊泡的生物發生的機制，並為抑制胰腺癌中的組織間質增生提供了新的治療方向。

Pancreatic ductal adenocarcinoma (PDAC) is very aggressive cancer with a five-year survival rate of 8 %. The unique feature of PDAC is desmoplastic reaction, an increase of extracellular matrix deposition and fibrotic cells, which has been shown to affect drug delivery and tumor progression. However, the mechanism of desmoplasia is still largely unknown. Extracellular vesicles (EVs) are nano-sized particles released by cells that carry many kinds of biomolecules to distal regions. We have previously demonstrated that loss of dual-specificity phosphatase-2 (DUSP2) promotes EV secretion. In addition, tissue-specific DUSP2 knockout in the mouse pancreas showed desmoplastic reaction with abundant collagen synthesized. However, how DUSP2 regulates EV biogenesis and the impact on PDAC tumorigenesis remain unknown. This study is designed to investigate underlying mechanism of EV biogenesis and subsequent effect on desmoplasia. First, I quantified the expression of tetraspanin proteins, markers of EV, by Western blotting and found that CD63 expression was increased in DUSP2 knockdown (KD) PANC-1 cells when compared to control cells. Western blotting result also showed extra higher molecular weight bands than expected, indicating post-translational modification occurs. I thus used glycosylation inhibitor (tunicamycin) and PNGaseF amidase to prove that CD63 is highly N-link glycosylated in DUSP2 KD PANC-1 cells. Treatment with tunicamycin to block CD63 glycosylation decreased loss-of-DUSP2-induced EV secretion, suggesting that glycosylation of tetraspanin is a critical step for EV biogenesis. Next, I isolated EVs from DUSP2 KD cells and used that to treat pancreatic stellate cells (HPaSteC). Treatment with EV fraction resulted in HPaSteC activation with loss of lipid droplet. To verify this effect in vivo, I isolated EVs derived from DUSP2-KD cells and injected them subcutaneously into PANC-1 tumors. The result showed more collagen accumulation in EV-treated tumors compared to PBS-treated tumors, suggesting EVs promote the desmoplasia reaction. Finally, administration of a novel HDAC inhibitor (B390), which was found by our lab to induce DUSP2 re-expression, inhibited CD63 glycosylation in DUSP2 KD cells. Furthermore, treatment of mice bearing orthotopic tumor with B390 resulted in less collagen fibers deposition as compared to vehicle treated ones. In conclusion, these data reveal a mechanism that loss-of-DUSP2 regulates EV biogenesis and provide a therapeutic direction to inhibit desmoplasia in pancreatic cancer.

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