探討骨髓細胞在放射線與介白素-12治療肝細胞癌扮演的角色

Abstract

肝癌目前在全球造成癌症的流行率位居第六位，也是造成癌症死亡最常見的疾病之一。Hepatocellular carcinoma (HCC)是最主要肝癌的種類，造成HCC的主要原因為B型肝炎病毒與C型肝炎病毒感染。在不同階段的HCC中，有不同的治療方法。目前主要治療HCC的方法為手術切除肝癌腫瘤、肝臟移植、放射治療、化學栓篩治療、或是給予Sorafenib進行治療，但是病人治療的預後並不佳，癌症晚期病人存活延長時間短，因此本研究希望能夠研發出更有效的肝癌治療法。 癌症的免疫治療為可能治療HCC的方法，其原理是改變病患免疫系統，使免疫系統產生對抗癌症的效果。其中一種有效的免疫治療方法為介白素-12 (IL-12)，在許多小鼠腫瘤模式，IL-12可以有效地在腫瘤免疫抑制環境下刺激免疫反應，其機制為活化抗原呈獻細胞、毒殺型T細胞以及NK細胞，並且調控免疫抑制細胞的功能以及抑制腫瘤部位的血管新生。雖然IL-12免疫治療可以引發抗腫瘤免疫反應，但是其療效在治療大型腫瘤時成效有限。放射線治療(Radiotherapy, RT)也是常見的治療癌症的方法，放射線可以殺死腫瘤細胞，讓腫瘤細胞的DNA斷裂、活化caspase pathway以及釋放danger signals使dendritic cells (DCs)成熟，吞噬因為放射治療從腫瘤細胞釋放的腫瘤抗原。然而，臨床研究中發現，部分的HCC對放射線治療具有抗性，而使療效受到限制。此外，肝臟以及腫瘤是免疫功能抑制的環境，由IL-12免疫治療或是放射治療引起的免疫反應會受到抑制。 在此篇研究中，我們將放射線合併IL-12進行癌症的免疫治療，主要目的是利用放射線破壞腫瘤細胞以及滲入腫瘤的免疫抑制細胞，使腫瘤細胞死亡並且釋放出腫瘤抗原提供DCs吞噬，加強對抗腫瘤的專一性免疫反應。同時在腫瘤中注射腺病毒(Ad)/IL-12，將表現IL-12引起的免疫反應限制在腫瘤的位置。在此研究中，我們在小鼠皮下以及原位肝臟上種植BNL HCC腫瘤細胞，在腫瘤分別生長至第14日以及第10日時，以10 Gy放射線、1 x 108 pfu Ad/IL-12或是合併放射線以及Ad/IL-12治療(合併治療)大型BNL腫瘤。在皮下與原位肝癌的治療中，比較未治療組，分別以放射治療或是Ad/IL-12治療的腫瘤生長較慢，治療腫瘤的療效有限。但是合併治療可以使腫瘤縮小，甚至使部分腫瘤完全消失。在長期觀察下，以合併治療比較未治療組與單一治療的組別，顯著的提升存活率。 為了研究治療在腫瘤部位的細胞組成是否受放射線或是IL-12影響，我以流式細胞儀分析治療後腫瘤部位的白血球族群改變。在合併治療的肝腫瘤中，NK細胞比例雖然呈現下降的趨勢，但是CD8+ T細胞的比例明顯地上升。進一步分析腫瘤內免疫抑制myeloid細胞的白血球族群，合併治療可以減少MDSCs與TAMs進入腫瘤的比例。 為了了解治療後腫瘤中免疫抑制myeloid cell是否受治療的影響而改變其功能，我進一步分析腫瘤中骨髓細胞表現的activation marker，發現合併治療的肝腫瘤中，MDSCs、TAMs以及DCs表現的MHC class II、CD40與CD86分子等activation marker的細胞比例以及每一個細胞的表現量明顯的增加。接著我利用磁珠分離的方式純化出脾臟以及腫瘤中的Gr-1+細胞，研究腫瘤治療後的Gr-1+細胞對抑制T細胞增生的能力是否會改變，我發現合併治療後，腫瘤中的MDSCs比起未治療組的MDSCs，其免疫抑制的能力下降，但是在脾臟中的Gr-1+細胞抑制T細胞增生的能力則不會受治療影響。合併治療亦會抑制腫瘤中的MDSCs以及TAMs表現reactive oxygen species (ROS)，但並不會影響脾臟中的Gr-1+細胞以及巨噬細胞表現ROS，因此，這些實驗證明合併治療的效果主要作用在腫瘤部位。 總結以上的資料，結合放射線以及IL-12治療HCC，除了可以使腫瘤消退，也可以延長小鼠存活時間。在治療的機制分析中，合併治療也會改變腫瘤微環境，使腫瘤中的T細胞增加，並且使myeloid細胞的比例減少且降低其免疫抑制的能力。

Liver cancer is the sixth-most-common cancer worldwide and the second-most-frequent cause of cancer death. Among primary liver cancers, hepatocellular carcinoma (HCC) represents the major histological subtype. Most cases of HCC are due to hepatitis B virus (HBV) and HCV infection. HCC could be classified in different stages that require different therapeutic straegies, such as resection, transplantation, radiation, chemoembolization and sorafenib. However, patients with late-stage HCC usually have poor prognosis and low survival rate. Therefore, an alternative therapy for HCC is urgently needed. Cancer immunotherapy is capable of eliciting potent immune responses against cancer and represents an alternative therapy for HCC. One of the most powerful immunotherapy is Interleukin-12 (IL-12). In many mouse tumor modals, IL-12 can stimulate effective immune response even in the immunosuppressive tumor microenvironment. The antitumor effect of IL-12 involves in activating antigen-presenting cells, cytotoxic T lymphocytes (CTL), natural killer (NK) cells as well as reprograming immune suppressive myeloid cells, and induce a potent antiangiogenic effect in tumor site. Although IL-12 immunotherapy can induce antitumor immune response, its effect is largely limited to small tumors. Radiotherapy is one of the most-common modalities for cancer therapies. Radiation can kills tumor cells by triggering cell DNA damage through activation of the caspase pathway, resulting in release of danger signals. These lead to maturation of dendritic cells (DCs) for uptaking and presenting tumor antigens released from apoptotic tumor cells. However, resistance of HCC to radiotherapy is commonly observed, which limits its therapeutic efficacy to HCC. Moreover, immunosuppressive environment in livers and tumors may hinder the therapeutic efficacy by radiation and IL-12. We propose to apply radiation and IL-12 for cancer therapy. The rationale of combination therapy is to use radiation to kill tumor cells, releasing tumor antigens, which then are uptaken by DCs for inducing tumor-specific antitumor response as well as by alleviating the immune suppressive functions of the tumor infiltrating myeloid cells. At the same time, we applied immunotherapy by intratumoral injection of adenoviral vectors (Ad)-expressing IL-12 to further boost in the local tumor region the antitumor immune response. We first implanted BNL murine HCC tumor cells subcutaneously and orthotopically in live lobe on syngeneic BALB/c mice for 14 and 10 days, respectively, then these large BNL tumors were treated by 10 Gy local irradiation, 1 x 108 pfu Ad/IL-12 or a combination of radiotherapy and Ad/IL-12 (combination therapy). Compared with tumors in the non-treatment group, both radiation and Ad/IL-12 therapy resulted in suppression of tumor growth of in both the subcutaneous and othotopic BNL tumors models. The most significant antitumor effect was achieved by combination therapy, which not only delayed tumor growth but also led to tumor regression in most animals, some were completely eliminated. Combination therapy also extent the survival rate of the treated tumor-bearing animals. To investigate whether combination therapy influence the infiltration of immune cells in tumor site, I analyzed the populations of tumor-infiltrating cells (TILs) by flow cytometry. In the combination therapy group, CD8+ T cells were most significantly increased while the percentage of tumor-infiltrating NK cells was decreased. The percentage of MDSCs and TAMs were markedly reduced after combination therapy. To study the influence of combination therapy on the functions of tumor-infiltrating myeloid cells, I analyzed the activation markers expressed on these cells. I found that after combination therapy, expression of MHC class II, CD40 and CD86 molecules was significantly increased on tumor-infiltrating MDSCs, TAMs and DCs. Then I purified Gr-1+ cells from the tumor and spleen by magnetic microbeads and co-cultured these Gr-1+ cells with T cells to investigate their ability to suppress T cells proliferation. Compared with MDSCs from non-treated tumors, MDSCs from tumors treated by combination therapy showed significantly lower suppressive activity. However, similar immune suppressive activity was not observed in splenic Gr-1+ cells isolated from non-treated or combination treated mice. Combination therapy also decreased expression of reactive oxygen species (ROS) in tumor-infiltrating MDSCs and TAMs, but not in Gr-1+ cells and macrophages from spleen of the same mice. These data provided strong evidence that the major therapeutic impact of combination therapy is on immune cells at the tumor site. Taken together, the data generated in this study showed that combination of radiation and IL-12 not only led to tumor regression in most animals but also significantly extended the survival rate of HCC-bearing mice. Mechanism analysis reveals that combination therapy has altered the tumor microenvironment by increasing infiltration of CD8+ T cell and decreasing the percentage and immune suppressive functions of tumor-infiltrating myeloid cells.