類別:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/144
2024-03-29T09:28:06Z阻斷WNT訊息傳導以遏制KRAS引起肺癌轉移
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58450
標題: 阻斷WNT訊息傳導以遏制KRAS引起肺癌轉移; The Inhibition of Wnt Restrain KRASG12V-Driven Metastasis in Non-Small-Cell Lung Cancer
作者: Pei-Shan Hung; 洪珮珊
摘要: KRAS基因變異而導致癌症的發生約佔30% ,特別是發生在胰臟癌、大腸癌、肺癌等這些高度惡性癌症。針對KRAS基因突變的治療標的是有相當的困難度。KRAS的基因變異主要為錯義突變(missense mutation),其中,在胺基酸12、13和61的密碼子是突變的熱點。KRAS的錯義突變,會提高抵抗GTPase activating protein (GAP) 所引起的 GTP 水解能力,與降低內生性GTP 水解能力,進而導致RAS-GTP鎖定在活化狀態之下,且持續活化下游訊息傳遞。近來許多文獻指出,KRAS基因的突變亞型,並不能被視為同一種基因突變。然而,卻缺乏對於各別KRAS基因突變亞型的特性,與相關致癌機制的研究與探討。在本論文裡,我們驗證在非小細胞肺癌中,KRAS基因突變亞型KRASG12V 與 KRASG12D所引起的癌症轉移機轉差異,並且使用Wnt/β-catenin pathway 抑制劑可以降低KRASG12V所引起的非小細胞肺癌細胞轉移。
我們使用H838等基因系細胞株 (isogenic cell line),以排除癌細胞內基因背景的干擾。首先,我們發現KRASG12V 與KRASG12D,在細胞實驗 (in vitro) and與動物實驗 (in vivo) 中,觀察到不同的轉移 (metastasis) 能力。透過基因集(GSEA)的分析後,在H838KRASG12V細胞所表現的基因集與RhoA-related signaling基因集相比,比對結果呈現負相關。我們也進一步發現,在H838KRASG12D細胞中,KRASG12D反而引起較高的RhoA活性,且具有較低的Wnt/β-catenin 活化。當抑制H838KRASG12D細胞內的RhoA活性後,則可以偵測到提高的Wnt/β-catenin 的活化。此外,當給予Wnt/β-catenin抑制劑後,可降低H838KRASG12V細胞的爬行 (migration) 能力。在本論文,我們闡明了在KRAS 所引起的非小細胞肺癌裡,KRAS/RhoA/Wnt/β-catenin所調控的轉移機轉。KRASG12V所引起的轉移對於Wnt/β-catenin機制有較高的依賴。我們的研究結果證實,KRASG12V 與KRASG12D基因變異亞型所引起的非小細胞肺癌轉移,在設計治療方法上,應被視為獨立的疾病; The KRAS mutations has been an obstacle to identify therapeutic targets in cancer treatment. In this work, we clarified the distinct metastasis pattern of Non-Small-Cell Lung Carcinoma (NSCLC) induced by KRASG12V/KRASG12D mutations and inhibited the KRASG12V mediated metastasis by Wnt inhibitor. First, we found that KRASG12V induced more aggressive phenotype in Vitro and in Vivo experiments. The Gene Set Enrichment Analysis (GSEA) results of H838 KRASG12V cells showed a significant negative correlation with RhoA-related signaling. Following this clue, we observed KRASG12D induced higher activation of RhoA and suppressed activation of Wnt/β-catenin in H838 KRASG12D cells. The restored activation of Wnt/β-catenin in H838KRASG12D cells could be detected when expression with a dominant-negative mutant of RhoA or treatment with RhoA inhibitor. Furthermore, the Wnt inhibitor abolished the KRASG12V -induced migration. We elucidated the importance of the axis of RhoA/Wnt in regulatory NSCLC metastasis driven by KRAS mutations. Our data indicate that KRASG12V driven NSCLC metastasis is Wnt-dependent and the mechanisms of NSCLC metastasis induced by KRASG12V/KRASG12D is distinct.2020-01-01T00:00:00Z阻斷Wnt/β-catenin訊息傳遞路徑以治療肝細胞癌
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59667
標題: 阻斷Wnt/β-catenin訊息傳遞路徑以治療肝細胞癌; Targeting the Wnt/β-catenin Signaling Pathway in the Treatment of Hepatocellular Carcinoma
作者: Hsiao-Hui Lin; 林曉慧
摘要: 肝細胞癌(Hepatocellular Carcinoma,簡稱HCC)為十分致命且預後非常差的癌症之一。過去幾十年來,在肝細胞癌的篩檢、早期診斷、以及局部治療上,雖然已有長足的進步;但大多數的肝細胞癌病人仍終將發展至晚期(advanced stage),而必須考慮接受全身性藥物治療。目前晚期肝細胞癌的標準治療,也是唯一經衛生主管機關核可的藥物是sorafenib (商品名為Nexavar【蕾莎瓦】)。Sorafenib是一種多重激酶抑制劑,可抑制Raf激酶及對抗血管內皮生長因子受體。整體而言,sorafenib在肝癌的治療上,腫瘤縮小的機率不高,腫瘤控制時間短暫,其治療效果仍有相當進步的空間。
本論文研究鎖定「Wnt/β-catenin訊息傳遞路徑」做為肝細胞癌嶄新的治療標的。Wnt/β-catenin訊息傳遞路徑在胚胎發育、組織結構的恆定、以及肝細胞癌癌化的過程上,都扮演十分重要的角色。本論文研究嘗試驗證以下兩個假說:一、抑制Wnt/β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療療效;二、肝細胞癌β-catenin 基因上特殊的活化性突變,可能導致該癌細胞對特定的Wnt/β-catenin訊息傳遞路徑抑制劑特別敏感。
在本論文的第一部分中,我們證實了無論於細胞培養上或活體內,抑制Wnt/ β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療效果。ICG-001是Wnt/β-catenin訊息傳遞路徑的小分子抑制劑,它經由阻斷的β-catenin與轉譯共同激活因子CBP之間的交互作用而達到抑制效果。在多株肝細胞癌的體外培養實驗中,我們發現伴隨著ICG-001使用的濃度提高,肝細胞癌細胞株生長受到sorafenib治療後生長抑制的情況及走向細胞凋亡的細胞比例隨之增加。進一步以RNA干擾的技術抑制β-catenin後,我們亦發現肝細胞癌細胞株Huh7對sorafenib作用的敏感度明顯提升;反之將β-catenin過度表現,則可降低Huh7對sorafenib作用的敏感度。此外,我們發現經短髮夾RNA(shRNA)處理導致β-catenin表現低下之Huh7對sorafenib作用的敏感度提升,再將該細胞的β-catenin過度表現之後,其敏感度則再度降低。就機制上而言,合併使用sorafenib與ICG-001可促使更多肝細胞癌細胞株走向細胞凋亡,並導致Mcl-1表現的更顯著下降。在Huh7肝細胞癌細胞小鼠皮下腫瘤的動物模式,合併使用sorafenib與ICG-001的組別相較於單獨使用sorafenib亦或ICG-001的組別,更能顯著地抑制腫瘤的生長。
在本論文的第二部分中,我們則闡明了CTNNB1基因之第三外顯子具有錯義突變(missense mutation)之肝細胞癌細胞株,對特定的Wnt/β-catenin訊息傳遞路徑抑制劑的抑制作用有較高的敏感度。我們使用SNU398與Huh6(分別於CTNNB1基因的第三外顯子的S37C及G34V有錯義突變)、Huh7及HepG2(在CTNNB1基因第三外顯子處有缺失的片段[interstitial deletion])、及PLC5、Hep3B、HLE及SK-Hep1 (在第三外顯子的位置無基因異常)等肝細胞癌細胞株;並分別處理包括ICG-001、XAV939 (為tankyrase的抑制劑,可藉此達到AXIN-1的穩定,以抑制β-catenin的訊息傳遞活性)、以及LGK974 (為porcupine的抑制劑;porcupine可促使Wnt ligands的成熟與分泌)等Wnt/β-catenin訊息傳遞路徑抑制劑。在XAV939及LGK974的處理下,不同肝細胞癌細胞株生長受到抑制的程度差異並不顯著;然而在ICG-001的處理下,我們發現SNU398的生長抑制作用最為顯著,緊追其後為Huh6。除此之外, ICG-001對於SNU398的細胞聚落形成的抑制力、經ICG-001處理後誘發sub-G1波峰提升的程度,皆高於其他肝細胞癌細胞株。最後,不同的化學治療藥物,包含doxorubicin、cisplatin、及5-fluorouracil對不同肝細胞癌細胞株的抑癌效果,並無發現顯著的差異。
我們的實驗研究結果顯示抑制Wnt/β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療療效,而具有β-catenin第三外顯子有錯義突變的肝細胞癌細胞株對ICG-001的抑癌效果最為敏感。本論文研究的發現支持持續鎖定Wnt/β-catenin訊息傳遞路徑研發抑制劑(無論是與sorafenib合併使用,或針對特定分子標記族群做單一治療)以治療肝細胞癌。; Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. Although a lot of advances in screening, early diagnosis, and loco-regional therapies for HCC have been made over the past decades, most HCC patients would eventually develop advanced diseases that need systemic therapy. Currently, sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is the only approved drug for HCC. However, its efficacy is limited, with low tumor response rate and short tumor control duration.
The current thesis work focuses on developing new therapeutic strategies for HCC by targeting the Wnt/β-catenin signaling pathway, which plays an important role in embryonic development and tissue homeostasis, as well as in hepatocarcinogenesis. The thesis work has tested two hypotheses: (1) inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC; (2) activating mutations of β-catenin gene may confer sensitivity to specific Wnt/β-catenin pathway inhibitors in HCC.
In the first part of the current thesis work, we demonstrated that inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib against HCC in vitro and in vivo. ICG-001, a small molecule that blocks the interaction of β-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis- induction effects of sorafenib in multiple HCC cell lines. Downregulation of β-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of β-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was attenuated by β-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001.
In the second part of the current thesis work, we demonstrated that HCC cells with point mutation at exon 3 of the CTNNB1 gene exhibited an increased sensitivity to antitumor effects of certain Wnt/β-catenin inhibitors in vitro. SNU398 and Huh6 are HCC cells harboring missense somatic mutations in exon 3 of the CTNNB1 gene, resulting in S37C and G34V mutation, respectively; Huh7 and HepG2 are HCC cells with interstitial deletion of exon 3; while other HCC cells including PLC5, Hep3B, HLE, and SK-Hep1 are HCC cells containing no mutations in exon 3. Several classes of Wnt/β-catenin pathway inhibitors were tested including ICG-001, XAV939 (an inhibitor of tankyrases which would stabilize AXIN-1 and thus β-catenin transactivation activity), and LGK974 (an inhibitor of porcupine which could help maturation and secretion of Wnt ligands). The sensitivity to growth suppressive effects of XAV939 and LGK974 did not vary significantly among all tested HCC cells. However, the growth-suppressive effect to ICG-001 was most sensitive in SNU398 cells, followed by Huh6 cells, and less sensitive in other HCC cells. ICG-001 also induced a more significant suppression of colony-formation, and a more significant increase of sub-G1 fraction by flow cytometry in SNU398 than other HCC cells. Finally, the cytotoxic effects of multiple chemotherapy drugs including doxorubicin, cisplatin, and 5-fluorouracil did not significantly differ among tested HCC cells.
Our studies have demonstrated that targeting of Wnt/β-catenin signaling pathway improves the anti-tumor effects of sorafenib against HCC and the presence of a missense mutation in exon 3 of β-catenin may confer increased sensitivity to ICG-001 in certain HCC cells. These data support further investigations on developing Wnt/β-catenin inhibitors as potential cancer therapeutics for HCC by either combination with sorafenib or single-agent approach focusing on biomarker-enriched population.2017-01-01T00:00:00Z鈣網蛋白突變在骨髓增生性腫瘤的角色
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/2538
標題: 鈣網蛋白突變在骨髓增生性腫瘤的角色; The Roles of Calreticulin Mutations in Myeloproliferative Neoplasms
作者: Ken-Hong Lim; 林建鴻
摘要: 骨髓增生性腫瘤是克隆性造血幹細胞疾病,並且可以分類為“典型”和“非典型”骨髓增生性腫瘤。典型骨髓增生性腫瘤通常會在周邊的血液中表現出終端骨髓細胞的擴增,包括真性紅血血球增多症、原發性血小板增多症、原發性骨髓纖維化和慢性骨髓性白血病。近年來,在大約30%的JAK2/MPL-未突變的骨髓增生性腫瘤中發現鈣網蛋白(Calreticulin, CALR)突變。目前,鈣網蛋白突變已經成為診斷原發性血小板增多症和原發性骨髓纖維化的重要克隆標誌物。
這項研究的第一個目的是開發一種快速和敏感的篩選工具,用於檢測鈣網蛋白突變。我們使用CFX Connect即時系統成功開發了高分辨率熔解分析,以檢測原發性血小板增多症病人的鈣網蛋白第9外顯子突變。我們的高分辨率熔解分析系統在識別病人基因組DNA中的鈣網蛋白第1型和第2型突變體的最大敏感性為2.5%。我們的高分辨率熔解分析系統假陽性率為3%,並且無假陰性出現。
本研究的第二個目的是評估台灣原發性血小板增多症病人鈣網蛋白突變和JAK2 /CALR共突變的臨床和預後意義。我們在92例成年原發性血小板增多症病人中利用高分辨率熔解分析篩選了鈣網蛋白第9外顯子的改變,隨後並且進行了TA克隆。我們在21例(22.8%)病人中鑑定出典型的鈣網蛋白插入/刪除型突變。鈣網蛋白突變與年齡較輕(p = 0.025),血小板數較高(p <0.001)和較低的血紅素(p = 0.016)有相關。有趣的是,我們檢測到在59例JAK2突變的原發性血小板增多症病人中有13例(22%)的鈣網蛋白第9外顯子的改變。與這些具有JAK2 /CALR共突變的原發性血小板增多症病人有相關的因子包括年齡較大(p = 0.025),診斷後發生血栓事件較多(p = 0.048),診斷後主要動脈血栓事件較多(p = 0.022),且較多屬於血栓出血併發症高風險組病人(p = 0.023)。我們的研究顯示JAK2突變的原發性血小板增多症病人可以出現頻繁的鈣網蛋白第9外顯子的改變,並且界定出一群具有血栓事件風險增加的病人亞群。
本研究的第三個目的是要探討鈣網蛋白突變之原發性血小板增多症病人的B細胞免疫特徵。我們篩選了54例台灣成年原發性血小板增多症病人的鈣網蛋白突變並評估了其B細胞免疫特徵。在這54例原發性血小板增多症病人中有19例(35.2%)具有8種不同類型的鈣網蛋白笫9外顯子突變,其中包括4例(7.4%)同時伴有JAK2V617F共突變的病人。經過年齡、性別、追踨期和血液學參數校正後,我們進行的多變量分析證實,與健康成年人相比,活化的B細胞在JAK2突變,鈣網蛋白突變和三陰性原發性血小板增多症病人中都有增加的現象。因此,活化的B細胞增加在不同突變亞組的原發性血小板增多症病人中是一種普遍存在的現象。
本研究的第四個目的是使用斑馬魚動物模型研究鈣網蛋白突變的分子發病機制。我們確定了3種與人類鈣網蛋白直系同源的斑馬魚基因,稱為calr,calr3a和calr3b。CALR-del52和CALR-ins5突變體的表達使斑馬魚早期的造血幹/前驅細胞增加,並進而造成血小板球增多但不影響正常的血管生成。我們發現使用morpholino降低mpl但不是epor或csf3r可以顯著的減弱CALR突變體對血小板球增多的影響。此外,CALR突變體的表達也活化jak/stat信息傳遞路徑,而使用JAK抑製劑(ruxolitinib和fedratinib)可以抑制此活化現象。這些研究結果說明CALR突變體通過mpl依賴機制活化jak信息傳遞路徑導致斑馬魚致病性之血小板球生成。我們研究的結果也說明與突變型CALR腫瘤發生相關的訊息傳遞機制在人與斑馬魚之間是保守的。; The myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders and can be classified into 'classic' and 'atypical' MPNs. Classic MPNs usually exhibit terminal myeloid cell expansion in the peripheral blood and include polycythemia vera, essential thrombocythemia (ET), primary myelofibrosis and chronic myeloid leukemia. Calreticulin (CALR) mutations have recently been discovered in about 30% JAK2/MPL-unmutated myeloproliferative neoplasms (MPN), and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis.
The first aim of this study is to develop a rapid and sensitive screening tool for the detection of CALR mutations. We successfully developed a high-resolution melting analysis (HRMA) with the CFX Connect real-time system to detect CALR exon 9 mutations in ET patients. The maximal sensitivity of our HRMA system in identifying both CALR type 1 and type 2 mutants from patients’ genomic DNA was 2.5%. Our HRMA has a false positive rate of 3% and no false negative.
The second aim of this study is to evaluate the clinical and prognostic significance of CALR mutations and JAK2/CALR co-mutations in Taiwanese ET patients. We screened for CALR exon 9 alterations with HRMA followed by TA-cloning in 92 adult ET patients. We identified classic CALR indel mutations in 21 (22.8%) patients. CALR mutations were associated with younger age (p=0.025), higher platelet count (p<0.001) and lower hemoglobin level (p=0.016). Interestingly, we detected various CALR exon 9 alterations in 13 (22%) of 59 JAK2-mutated ET patients. JAK2-mutated ET patients with concomitant CALR alterations were associated with oldest age (p=0.025), higher thrombotic events after diagnosis (p=0.048), higher major arterial thrombotic events after diagnosis (p=0.022) and more patients being high risk group for thrombo-hemorrhagic complications (p=0.023). Frequent CALR exon 9 alterations in JAK2-mutated ET patients define a specific subgroup of patients with increased risk of thrombotic events.
The third aim of this study is to determine the B cell immune profiles in CALR mutated ET patients. We screened for CALR mutations and evaluated B cell immune profiles in a cohort of 54 adult Taiwanese ET patients. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. In conclusion, increased B cell activation is present in ET patients across different mutational subgroups.
The fourth aim of this study is to investigate the molecular pathogenesis of CALR mutations using zebrafish animal models. We identified 3 zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. Expression of the CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.2017-01-01T00:00:00Z肝細胞癌的免疫組成:從標靶藥物到免疫治療
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66770
標題: 肝細胞癌的免疫組成:從標靶藥物到免疫治療; Immune Contexture of Hepatocellular Carcinoma: From Sorafenib to Immune Checkpoint Inhibitors
作者: Li-Chun Lu; 呂理駿
摘要: 肝細胞癌(肝癌)一直以來都是全球死亡率前幾名的癌症之一。晚期肝癌病患無法接受局部治療,而全身性治療,包括「抗血管新生標靶藥物」和「抗計畫性細胞死亡蛋白-1 (PD-1)為主的免疫檢查點抑制劑(ICIs)」,是標準治療。近來,腫瘤微環境(TME)和全身性治療療效間的關係已成為主要的研究課題。蕾莎瓦 (sorafenib)為一抑制多重激酶的標靶藥物 (包含抗血管新生的療效),也是第一個被核准在晚期肝癌患者使用的藥物,過去研究指出sorafenib具有多重免疫調節的作用,但是其對晚期肝癌TME內的計畫性細胞死亡配位基-1 (PD-L1)表現的影響並不清楚。另外,TME的免疫組成會影響ICIs的療效,但是在肝癌不同器官的免疫組成,是否會造成ICIs在不同器官轉移腫瘤的差別反應也不清楚。
在本研究的第一部份,要驗證的假設為:sorafenib會對肝癌TME內的PD-L1表現有影響。我們回顧有接受過sorafenib的晚期肝癌病患,收集其sorafenib治療前後的配對腫瘤檢體,利用免疫組織染色,半定量分析檢體內腫瘤細胞(TCs)及免疫細胞(ICs)其細胞膜上PD-L1的表現。在23對可分析的檢體中,我們發現ICs的PD-L1表現在sorafenib治療惡化後的檢體顯著高於治療前的;然而,TCs的PD-L1表現在sorafenib治療前後並無顯著差異。我們進一步證明,這些表現PD-L1的ICs,也同步表現巨噬細胞的標記CD68,暗示這些表現PD-L1的巨噬細胞在sorafenib治療後而惡化的肝細胞癌可能有特殊意義。
在本研究的第二部份,要驗證的假設為:各器官TME不同的免疫組成,會造成在各器官內的肝癌腫瘤對ICIs的差別反應。我們回顧曾接受ICIs治療並有可量測腫瘤大小的晚期肝癌病患,分別評估ICIs對其肝、肺、淋巴結、及腹腔內轉移腫瘤的反應率。在75位接受分析的晚期肝癌病患中,有58、34、19及18位患者有可量測之肝、肺、淋巴結、及腹腔內轉移腫瘤,其相對應的特定器官腫瘤反應率分別為22.4%、41.2%、26.3%、及38.9%。在39位同時有肝臟腫瘤及肝外轉移的患者中,12位其肝外轉移腫瘤有獲得控制,但是肝臟腫瘤卻惡化;反之只有四位患者肝臟腫瘤獲得控制,但是肝外轉移腫瘤有惡化(P = .046, McNemar test)。而在16位治療前只有肝臟腫瘤和肺部轉移的患者中,8位其肺部轉移獲得控制,但是肝臟腫瘤卻惡化;反之沒有一位患者有肝臟腫瘤獲得控制,但是肺部轉移腫瘤卻惡化的情形(P = .005)。在本部分的研究,我們發現肝內的肝癌反應率似乎比肝外轉移的腫瘤來得差,而肺部轉移的肝癌對ICIs的反應率最好,暗示TME的免疫組成,會影響對ICIs的療效。
為了進一步探究上述「ICIs對肝癌之不同器官轉移的療效差別反應」之可能機轉,我們利用BNL小鼠肝癌細胞株,分別建立包括原位肝臟肝癌、皮下、及肺臟植入肝癌的同源小鼠模式。我們分析未接受治療的不同器官肝癌小鼠模式,分離腫瘤浸潤CD45陽性白血球和分析其次族群細胞組成,發現肝臟腫瘤比肺部腫瘤有趨勢上較高比例的巨噬細胞及骨髓抑制細胞,不過CD4及CD8陽性T細胞在肝臟及肺部腫瘤的比例則相近。然而,肝臟腫瘤浸潤的CD8 T細胞,比起其他器官腫瘤的有較高比例會表現衰竭標記。此外,我們發現VEGF和其他一些免疫抑制細胞激素,在未接受治療的原位肝腫瘤內比皮下腫瘤來的高;而在PD-L1抗體治療後,其他免疫抑制激素在原位肝腫瘤內有下降,但VEGF和IDO等的表現仍持續比皮下腫瘤顯著的高。在接受PD-L1抗體治療後,我們發現皮下植入的肝癌腫瘤療效好,而原位肝臟植入的肝癌腫瘤反應較差。整體而言,我們的小鼠模式研究結果發現原位肝癌的TME,比植入其他器官的更傾向免疫抑制,支持「不同器官相異的免疫組成,可能影響ICIs的治療效果」。
總結上述研究,首先我們發現在sorafenib治療後惡化的肝癌檢體,其ICs (特別是巨噬細胞) 的PD-L1會顯著上升。其次,我們利用臨床肝癌病人的資料證實,各器官內的肝癌腫瘤對ICIs有差別反應,肝內的肝癌反應率比肝外轉移的腫瘤來得差。在機轉的研究上,我們分析小鼠原位肝癌,有較高比例的巨噬細胞、有較多的衰竭CD8 T細胞、及較高表現的VEGF,使得肝內TME傾向免疫抑制,因此可造成ICIs的療效比其他器官的肝轉移來得差。我們的研究成果可提供未來晚期肝癌全身性治療發展的參考。; Hepatocellular carcinoma (HCC) has been the leading cause of cancer mortality globally for decades. In cases of advanced HCC, for which locoregional therapy is unsuitable, systemic anticancer therapy through either antiangiogenic agents or anti-programmed-cell-death protein 1 (PD-1)-based immune checkpoint inhibitors (ICIs) is the standard of care. Recently, the interaction between the tumor microenvironment (TME) and systemic therapy response has been a major focus of research. Sorafenib, a multikinase inhibitor with antiangiogenic properties and the first systemic therapy approved for HCC, has been demonstrated to exhibit various immunomodulatory effects. However, the impact of sorafenib on programmed death ligand-1 (PD-L1) expression in the TME of advanced HCC was unclear. In addition, the antitumor effects of ICIs are influenced by the immune contexture of the TME. However, whether distinct immune contextures in different organ systems contribute to the variable tumor response to ICIs in HCC was unknown.
The first part of the thesis addressed the hypothesis that sorafenib treatment affects the expression of PD-L1 in the TME of HCC. We identified patients who received sorafenib for advanced HCC and who had paired tumor tissues obtained before and after sorafenib treatment. We analyzed the tissue slides using immunohistochemistry to semiquantitatively score the membrane PD-L1 staining in tumor cells (TCs) or tumor-infiltrating immune cells (ICs). In 23 paired HCC tissues, PD-L1 expression in ICs was increased in HCC tissues after sorafenib treatment. However, PD-L1 expression in TCs did not increase significantly after sorafenib treatment. We also demonstrated that PD-L1-expressing ICs were highly co-localized with CD68-positive tumor-infiltrating macrophages, suggesting that PD-L1-expressing macrophages play roles in HCC progression after treatment with sorafenib.
The second part of the thesis explored the hypothesis that the distinct immune contexture of the TME is associated with variable tumor response to ICIs in HCC. We reviewed data from patients with advanced HCC who had received ICIs and had measurable diseases. The objective response to ICIs in tumors located in different organ systems—such as the liver, lung, lymph nodes, and other intra-abdominal sites—was evaluated independently. Among the 75 enrolled patients, 58, 34, 19, and 18 patients had measurable hepatic tumors and lung, lymph node, and other intra-abdominal metastases, respectively, with the corresponding organ-specific objective response rates being 22.4%, 41.2%, 26.3%, and 38.9%. Among the 39 patients who had both hepatic and extrahepatic tumors, 12 had disease control in the extrahepatic tumors but progressive disease (PD) in the hepatic tumors, whereas only 4 exhibited disease control in the hepatic tumors but PD in the extrahepatic tumors (P = .046). Among the 16 patients with evaluable tumors in only the liver and lung at baseline, 8 had disease control in the lung and PD in the liver, and none had disease control in the liver but PD in the lung (P = .005). In the present study, we demonstrated that hepatic tumors of HCC are less responsive to ICIs than extrahepatic lesions, and lung metastases respond most favorably to ICIs, suggesting that the TME influences the efficacy of ICIs.
To explore further the mechanisms underlying the aforementioned organ-specific differential responses to ICI treatment in HCC, we employed a syngeneic mouse liver cancer model in immunocompetent mice. We used BNL mouse liver cancer cells to establish orthotopic, subcutaneous, and lung HCC mouse models independently. We isolated tumor-infiltrating CD45+ leukocytes from untreated tumors at various organ sites. The percentages of overall macrophages and myeloid-derived suppressor cells in liver tumors were numerically higher than those in lung tumors. Conversely, the overall percentages of CD4+ and CD8+ T cells were similar between liver and lung tumors. However, the tumor-infiltrating CD8+ T cells were more exhausted in the liver tumors than in tumors of other organs. In addition, we evaluated the soluble factors in untreated liver and subcutaneous tumors. We observed that the levels of vascular endothelial growth factor (VEGF) and some other immunosuppressive cytokines were higher in the liver than in subcutaneous tumors. After treatment with PD-L1, although the levels of other immunosuppressive cytokines were decreased in the liver, the levels of VEGF and indoleamine 2,3- dioxygenase remained high in liver tumors. Anti-PD-L1 antibody or isotype control was administered to the orthotopic, subcutaneous, or combination models. The results showed that orthotopic HCC tumors responded more poorly to anti-PD-L1 therapy than subcutaneous HCC tumors. Overall, the data demonstrated that BNL mouse liver cancer cells, when implanted in the liver, were associated with a more immunosuppressive TME than those implanted in other organs. The results supported that the distinct immune contextures of different organ sites influenced the efficacy of ICI treatment in mouse HCC models.
Overall, we found that increased PD-L1 expression in ICs, mainly macrophages, was associated with HCC progression after treatment with sorafenib. In addition, we demonstrated organ-specific responses to ICIs in patients with advanced HCC. HCC in the liver was less responsive to ICIs than were extrahepatic metastases. We demonstrated mechanistically that orthotopic liver tumors had higher percentages of macrophages, more exhausted CD8+ T cells, and higher VEGF levels, which could render the TME more immunosuppressive and lead to a poorer ICI response than in other organ systems. Our findings provide insights that could facilitate the development of systemic therapy for advanced HCC in the future.2020-01-01T00:00:00Z