Dr. Wang, Shu-Ping 's publons link picture

Dr. Wang, Shu-Ping

Assistant Research Fellow
  • 02-2789-9122 (Lab) (Room No: 540)
  • 02-2652-3073 (Office)

Specialty:
  • Molecular oncology, Cancer epigenetics
  • Biochemically defined cell free system
  • CRISPR-based genetic/epigenetic editing

Education and Positions:
    • B.S. National Sun Yat-Sen University
    • M.S. National Tsing-Hua University
    • Ph.D. National Defense Medical Center
    • Postdoc. The Rockefeller University, New York

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A novel therapeutic strategy for lung cancer treatment: Priming a favorable tumor microenvironment by chemotherapeutic agents for cancer immunotherapy with immune checkpoint inhibitor

Dr. Wang, Shu-Ping
Journal for ImmunoTherapy of Cancer (JITC), Nov 26, 2020

Introduction

Immunotherapy is the use of methods to improve human immune system by inducing the body’s own immune system to re-identify cancer cells, thereby adjusting the tumor microenvironment to effectively inhibit the survival of cancer cells. There are different types of immunotherapy, among which immune checkpoint blockade (ICB) treatment has recently been considered as a way to improve the overall response rate in clinical cancer treatment.

When cancer cells appear in the body, it should be recognized by the immune system and attacked by cytotoxic T cells. However, cancer cells developed defensive mechanisms that can suppress immune cells, causing cytotoxic T cells to lose their ability to attack cancer cells. This mechanism is due to a specific protein receptor (PD-L1) produced on the surface of cancer cells, which can bind to the “immune checkpoint protein PD-1” on the surface of cytotoxic T cells to activate immune checkpoints and make T cells inactive. Therefore, as long as inhibitors that block their binding is applied, we can keep the immune checkpoints inactivated and make cytotoxic T cells aggressive towards cancer cells. This method is called "immune checkpoint blockade (ICB)" therapy. Among them, monoclonal antibodies against PD-1 or PD-L1 have become a commonly used immune checkpoint inhibitor around the world. However, not all ICB therapies are beneficial to lung cancer patients, partly due to the low or no expression of PD-L1 on lung tumors.

Rationale

In the past, patients with advanced lung cancer could not use targeted therapies and can only rely on chemotherapy if the genetic test results of cancer cells showed no gene mutations such as EGFR or ALK mutations. However, when tumor immunotherapy emerged, whether it is combined with chemotherapy or used alone in patients with high PD-L1 expression, the survival time is significantly better than that of chemotherapy alone, which has become a new treatment option for lung cancer patients. However, there are many kinds of chemotherapeutic drugs but not all combinations are beneficial to patients with lung cancer. Some ineffective combinations of treatments bring more side effects and pain on cancer patients. Therefore, finding a chemo drug that can be perfectly combined with ICB treatment has become a new opportunity for the treatment of lung cancer.

Recently, the combination of Pemetrexed and platinum-based chemotherapeutics with ICB therapy has demonstrated convincing clinical activity in non-small cell lung cancer (NSCLC), and clinical results have led to the accelerated approval of this immunochemotherapy combination for advanced NSCLC patients. Although this novel combination therapy has become the first-line treatment for advanced NSCLC, little is known about the potential anti-tumor mechanism of the combination between Pemetrexed and immunotherapy.

Experimental Designs & Research Findings

In this study, we assessed that the most effective first-line NSCLC chemotherapeutic agents, including antimetabolic agents and antimitotic agents, to check whether they can regulate PD-L1 expression and prime a favorable tumor microenvironment for immune check point blockade (ICB) therapy. The long-term objective of this study is to investigate the effect and mechanism of chemotherapeutics on the priming of immune tumor microenvironment in NSCLCs, to provide a better molecular basis for chemoimmunotherapy.

 First, we administered different first-line NSCLC chemotherapeutic agents, including anti-metabolic agents and anti-mitotic agents, and tested their PD-L1 prime activities to NSCLC cells. The results showed that compared with other chemotherapeutic agents, such as cisplatin and paclitaxel, the low-dose antimetabolitic chemotherapy drugs Pemetrexed and Fluorouracil (5-FU) significantly increased the expression of PD-L1 in lung cancer cells, priming a tumor microenvironment conducive to ICB treatment.

Second, we used the cancer/T-cell co-culture system to test whether using first-line NSCLC chemotherapeutic agents really have the function of regulating immune cell activation in tumor microenvironment. We found that Pemetrexed and anti-PD-L1 antibody synergistically induced T cell activation in vitro, mainly through restoring the exhausted T-cell activities. In addition, to dissect the crosstalk mechanism between chemotherapeutic agent and ICB, we selected CL1-5-derived subclones that displayed different Pemetrexed (PEM) responses and classified them into two groups, PEM responder (PEM-R) and PEM non-responder (PEM-NR). RNA-sequencing (RNA-seq) for each CL1-5 subclone were also performed. Gene Ontology (GO) enrichment analysis was applied for identification of the candidate regulatory pathways. Through cell-based experiments, we have revealed a novel molecular mechanism of how Pemetrexed induces the up-regulation of PD-L1 in lung cancer cells. We found that Pemetrexed suppresses the activity of thymidylate synthase (TS), and in turn increases the intracellular level of ROS, which further triggers the NF-κB signaling pathway followed by the activation of PD-L1 gene transcription in NSCLC cells.

Finally, we confirmed the combined therapeutic effect of the chemotherapeutic agent Pemetrexed and anti-PDL1 antibodies by using syngeneic mouse models. Compared with monotherapy, we observed that the combined treatment of Pemetrexed and anti-PD-L1 antibody can induce the accumulation of tumor-infiltrating T lymphocytes, synergistically inhibiting tumor growth; followed by increased PD-L1 expression, the recruitment of tumor infiltrating T lymphocytes, and reverse exhaustion of T cell activities by inducing antitumor cytokine IL2, TNF-α, and IFN-γ’s expression, rendering the tumor microenvironment more favorable for immunotherapy. In contrast, data from the syngeneic mouse lung tumor model indicates that platinum-based chemotherapy, e.g., cisplatin, cannot induce the expression of PD-L1 in NSCLC cells and provides no advantage in the combinatory therapy of Pemetrexed and anti-PD-L1 antibody for lung cancer treatment. This strongly suggests that Pemetrexed can be combined with ICB therapy without platinum-based chemotherapy.

Summary & Therapeutic Implications

Our findings showed that Pemetrexed increases the expression of PD-L1 in lung cancer cells, thereby creating a tumor microenvironment that is more suitable for immunotherapy. We revealed a novel mechanism that Pemetrexed can activate and increase PD-L1 gene transcription in cancer cells by inducing the TS−ROS−NF-κB signaling pathway, which provides a mechanistic basis for the FDA-approval combinatory chemoimmunotherapy in NSCLC treatment. Our results also provide more detailed instructions to avoid unnecessary treatment (for example, the use of Platinum drugs) and to improve the therapeutic value of cancer patients. We believe that our findings will put forward more specific concepts and clinical therapeutic significance for the direction of cancer immunotherapy.

背景介紹

免疫療法一種誘發人體自身的免疫系統,使之能重新辨識癌細胞,藉此調整腫瘤微環境來有效抑制癌細胞存活的方法。免疫療法有不同的類型,其中免疫檢查點封鎖(immune checkpoint blockade,ICB)最近被認為是改善臨床癌症治療中總體反應率 (overall response rate) 的最有效方法。

當身體出現癌細胞時,癌細胞理論上應該要被免疫系統辨識並且發動殺手 T 細胞攻擊。然而癌細胞自身發展出一套防禦機制可以抑制免疫細胞,讓殺手 T 細胞喪失攻擊癌細胞的能力。此機制就是癌細胞表面產生出特異的蛋白受體 (PD-L1),可與殺手 T 細胞表面的「免疫檢查點蛋白」 (PD-1) 結合,啟動免疫檢查點使T細胞失去活性。因此只要投入可破壞「免疫檢查點蛋白結合」的抑制劑,就可以有效封鎖免疫檢查點,使殺手 T 細胞持續保有攻擊性,這種方法稱為「免疫檢查點封鎖」(ICB) 治療。其中針對PD-1或PD-L1的單株抗體抑制劑已經成為全球普遍使用的一種免疫檢查點抑制劑, 然而目前並非所有的ICB療法都對肺癌患者有益,部分是由於肺癌腫瘤中PD-L1的表現量較低甚或是沒有表現導致反應率低的結果。

研究假說與原理

以往肺癌晚期患者,若癌細胞的基因檢測結果無EGFR或是ALK等基因突變,就無法使用標靶藥物,而只能仰賴化學治療。但是當免疫療法問世之後,無論是合併化療或是單獨使用於PD-L1高表現的肺癌患者,其存活時間都明顯比單獨使用化學治療來得好,因而成為肺癌病患的治療新選擇。然而,化學治療的藥物有許多樣,並非所有的組合方式都對肺癌病人有益,一些無效的治療組合反而會為癌症病患帶來更多副作用和痛苦。因此,找出可與ICB治療完美結合的化癌藥物成為治療肺癌的新契機。

近年來,以鉑金藥物 (Platinum-based chemotherapeutics) 與培美曲塞 (Pemetrexed) 為基礎的化學療法與免疫療法合併使用已證明在非小細胞肺癌(NSCLC)極具治療潛力,良好的臨床治療成果更促使這種免疫化學聯合療法獲得加速批准使用於晚期的非小細胞肺癌病患。儘管這種新穎的免疫化學聯合療法已成為晚期非小細胞肺癌的一線治療方法,但對於化學療法與免疫療法相結合的潛在抗腫瘤機制所知仍很有限。

實驗方法與研究發現

在本研究中,我們評估了第一線肺癌化學治療藥物,包括抗代謝藥劑和抗有絲分裂藥劑,檢查它們是否可以調節非小細胞肺癌細胞中PD-L1的表現並為免疫檢查點抑製劑 (ICB) 治療提供良好的腫瘤微環境。 本研究的長期目標是研究化學療法對肺癌引發免疫腫瘤微環境的作用和機制,以便為化學免疫聯合療法提供更好的依據。

在這項研究中,我們首先給予非小細胞肺癌細胞不同的第一線化療藥物,結果顯示了相較於其他化療藥物(例如順鉑和紫杉醇),低劑量的抗代謝化療藥物--培美曲塞(Pemetrexed)和氟尿嘧啶 (Fluorouracil, 5-FU)--明顯地增加肺癌細胞中PD-L1 的表現量,進而創造有利於ICB治療的腫瘤微環境。

我們接著利用肺癌細胞與免疫細胞的共同培養實驗來測試化療藥物是否真的具備調節免疫細胞活化的功能,我們發現培美曲塞(Pemetrexed)和PD-L1抗體可協同誘導T細胞活化,恢復失能的T細胞活性。為了分析化學治療劑與免疫抑制劑之間的交互影響機制,我們也分選出對化學治療劑培美曲塞(Pemetrexed)具有不同反應的CL1-5肺癌細胞株,並進行RNA序列分析。經由細胞實驗,我們揭示了培美曲塞(Pemetrexed)如何誘導肺癌細胞中PD-L1表現增加的新穎分子機制。我們發現培美曲塞(Pemetrexed)經由抑制胸苷酸合酶(TS)的活性,而增加了細胞體內的活性氧物質 (Reactive oxygen species,ROS) ,進而觸發NF-κB訊息傳遞路徑,最後導致非小細胞肺癌細胞中的PD-L1基因轉錄活化。

最後, 我們利用同源小鼠肺腫瘤模型,證實了化療藥物培美曲塞(Pemetrexed)和PD-L1抗體的聯合治療效果。相較於單一療法, 我們觀察到培美曲塞(Pemetrexed)和PD-L1 抗體的聯合治療可以引發腫瘤浸潤性 T 淋巴細胞的聚集,進而對於肺癌腫瘤生長達到加成的抑制作用。透過腫瘤微環境中抑癌細胞激素IL-2、TNF-α和 IFN-γ的表現增加,我們推論失能T 細胞的活性已產生逆轉,進而使腫瘤為環境更適合於免疫治療。相比之下,同源小鼠肺腫瘤模型的數據表明鉑金類化學藥劑(例如順鉑),因為不能誘導非小細胞肺癌細胞中PD-L1的表現,因此並沒有額外增加培美曲塞和PD-L1抗體的聯合治療效果。我們的研究提出強而有力的證據,說明了在無需鉑金類化療藥劑的輔佐下, 培美曲塞可以單獨與ICB治療結合用來治療非小細胞腺癌。

治療意義 (研究重要性)

本研究結果表明培美曲塞增加肺癌細胞內PD-L1的表現,進而創造出更適於免疫治療的腫瘤微環境。我們揭示了培美曲塞可以透過誘發TS-ROS-NF-κB訊息傳遞路徑而活化並增加癌細胞內PD-L1的基因轉錄,此新穎作用機制的揭示為已經批准的非小細胞肺癌化學免疫聯合療法提供了扎實的理論基礎。我們的研究結果也為避免不必要的治療 (例如: 柏金類藥劑的使用與否) 和提高癌症患者的治療價值提供了更詳細的指示。相信本研究的發現對於癌症免疫治療方向提出更具體的概念以及臨床上的治療意義。