Characterization of the interaction between inflammation and cancer progression

本项目的目标是研究细胞因子在体内调节炎症和宿主抗肿瘤免疫反应的细胞和分子机制，特别是它们与肿瘤和器官微环境之间复杂的细胞相互作用有关。我们的第一种方法是描述肿瘤进展过程中肿瘤浸润白细胞的特征。使用可移植的转移性肾细胞癌，我们正在分析肾脏以及肺和肝脏中发展的原发肿瘤中包含的白细胞，它们分别作为肿瘤转移的原发和继发部位。趋化因子是一种趋化细胞因子，用于招募特定的白细胞亚群进入正在进行的炎症反应区域。我们正在利用缺乏各种趋化因子受体的小鼠来确定单核细胞、T细胞和其他炎症细胞被募集到肿瘤部位的机制。一旦出现在发展中的肿瘤部位，这些细胞能够产生许多不同的可溶性介质，如干扰素γ (IFNg)、一氧化氮和VEGF，它们可能影响肿瘤的进展。通过调节这些细胞类型在肿瘤内积累的能力，我们正在确定这些细胞在原发性肿瘤进展和转移到远处器官中所起的作用。我们的免疫治疗方案包括IL-2或IL-15与CD40激动抗体联合使用。在用这种组合治疗荷瘤小鼠后，我们已经确定了巨噬细胞和T细胞的募集，它们似乎是抗肿瘤反应的关键介质。我们的数据表明，单独抗cd40与与IL-2联合使用介导的生物学效应存在巨大的机制差异，包括IFNg和控制肿瘤负荷的一氧化氮表达的协同上调。迄今为止，我们已经证明IL-2/aCD40诱导增强的抗肿瘤反应依赖于效应CD8+ T细胞浸润已建立的肿瘤，并伴随ifng依赖性降低CD4+/FoxP3+调节性T细胞（Tregs）、髓源性抑制细胞（MDSC）和肿瘤微环境中Th2趋化因子的表达。这些结果可能有助于解释aCD40作为单一药物的有限临床疗效，因为它不能特异性地从肿瘤微环境中去除Tregs和MDSC，它们表明抗cd40与其他选定的免疫药物（如IL-2）联合使用可能更有益。我们还将我们早期使用IL2和IL12联合的临床前研究结果转化为人类临床试验。我们当前和未来的研究旨在阐明对aCD40观察到的生物学效应至关重要的细胞和分子事件，以及aCD40与合理选择的分子靶向药物互补使用的潜力。在另一种方法中，我们将抗cd40与阿斯利康公司开发的ATP竞争性mTOR药物AZD8055结合使用。这一策略的基本原理有两个方面。首先，mTOR抑制是目前RCC的主要临床靶点。我们还假设AZD8055与aCD40抗体联合使用可以通过直接杀死肿瘤并随后将肿瘤相关抗原释放到抗原提呈细胞和同步调节体内免疫细胞功能来诱导更有效的抗肿瘤作用。我们最近发表的研究结果表明，在同基因小鼠转移性肾细胞癌（RCC）模型中，AZD8055和aCD40通过激活巨噬细胞和dc，并在肿瘤微环境中诱导强烈的Th1免疫反应，协同肿瘤消退。在另一种方法中，我们已经开始分析树突状细胞在免疫疗法实现的抗肿瘤反应中所起的作用。IL-12是一种有效的免疫调节细胞因子，其本身具有显著的抗肿瘤作用，与脾脏等淋巴器官相比，IL-12治疗可增加肝脏中的树突状细胞。我们目前正在分析IL-12与其他免疫调节细胞因子（如IL-2或IL-15）联合治疗是否通过增强这些重要效应细胞的抗原呈递能力，提供更有效的免疫应答，以对抗肾细胞癌。此外，树突状细胞与NK细胞和NK-T细胞之间存在动态串扰，调节这些细胞的数量或功能的疗法可能对DC启动T细胞特异性抗肿瘤反应的能力产生重大影响。此外，我们已经确定了肿瘤微环境中T调节细胞的特异性积累，这可能有助于抑制抗肿瘤反应。我们已经确定了肿瘤坏死因子α (TNFa)，一种关键的促炎细胞因子，可能参与T调节细胞的募集和/或扩增。我们正在通过分子和生化方法靶向这种细胞因子，这可能最终在临床中用于对抗抑制性肿瘤微环境的建立。我们还确定了NK-T细胞的推定免疫抑制作用，我们现在正在开发免疫治疗方案，以减少不同器官中NK-T细胞的数量和活性。通过表征炎症在肿瘤进展中的作用，这些重叠的方法有望导致免疫治疗方法的发展，从而扭曲炎症反应以对抗肿瘤生长。

The goal of this project is to investigate the cellular and molecular mechanisms by which cytokines regulate inflammation and host anti-tumor immune responses in vivo, particularly as they relate to the complex cellular interactions between the tumor and organ microenvironments. Our first approach is to characterize the profile of tumor-infiltrating leukocytes during tumor progression. Using a transplantable, metastatic renal cell carcinoma, we are analyzing leukocytes contained within the primary tumors that develop in the kidney as well as those in the lung and livers, which serve as primary and secondary sites of tumor metastases, respectively. Chemokines are chemotactic cytokines that serve to recruit specific leukocyte subsets into regions of ongoing inflammatory responses. We are utilizing mice deficient in various chemokine receptors to identify the mechanisms whereby monocytes, T cells and other inflammatory cells are recruited to the tumor site. Once present at the developing tumor site, these cells are capable of producing many different soluble mediators, such as interferon gamma (IFNg), nitric oxide, and VEGF that may influence tumor progression. By regulating the ability of these cell types to accumulate within tumors, we are identifying the role that these cells play during both primary tumor progression and metastasis to distant organs. Our immunotherapeutic regimens include IL-2 or IL-15 in combination with agonistic antibody to CD40. After treating tumor-bearing mice with this combination, we have identified the recruitment of macrophages and T cells that appear to be critical mediators of anti-tumor responses. Our data have illustrated the potential for dramatic mechanistic differences in biological effects mediated by anti-CD40 alone versus its use in combination with IL-2 that includes the synergistic upregulation of IFNg and nitric oxide expression that controls tumor burden. To date, we have shown IL-2/aCD40 induces enhanced antitumor responses that depend on the infiltration of established tumors by effector CD8+ T cells and a concomitant IFNg-dependent reduction in CD4+/FoxP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC) and Th2 chemokine expression within the tumor-microenvironment. These results may help to explain the limited clinical efficacy of aCD40 as a single agent based on its inability to remove Tregs and MDSC specifically from within the tumor microenvironment and they suggest that anti-CD40 may be more beneficial in combination with other selected immune agents, such as IL-2. We have also translated our earlier preclinical findings using IL2 in combination with IL12 to human clinical trials. Our current and future studies seek to clarify the cellular and molecular events critical for the observed biological effects of aCD40, and the potential for complementary use of aCD40 with rationally selected molecularly targeted agents. In another approach, we have combined anti-CD40 with an ATP competitive mTOR drug, AZD8055, developed by AstraZeneca. The rationale for this strategy is two-fold. First, mTOR inhibition represents is currently a leading clinical target for RCC. We also hypothesized that combining AZD8055 with aCD40 antibody would induce more efficient antitumor effects by a combination of direct tumor killing and subsequent release of tumor-associated antigens to antigen presenting cells and coincident modulation of immune cell functions in vivo. The results of our recently published study show that in a syngeneic mouse metastatic renal cell carcinoma (RCC) model, AZD8055 and aCD40 synergize for tumor regression by activating macrophages and DCs and inducing strong Th1 immune responses in the tumor microenvironment. In another approach, we have begun to analyze the role that dendritic cells play during the anti-tumor responses achieved by immunotherapies. Treatment with IL-12, a potent immunoregulatory cytokine with dramatic anti-tumor effects on its own, increases dendritic cells differently in the liver as compared to lymphoid organs such as the spleen. We are currently analyzing whether therapies that combine IL-12 with other immunomodulatory cytokines such as IL-2 or IL-15 offer a more effective immune response against renal cell carcinoma by enhancing the antigen presenting capability of these important effector cells. Furthermore, there is dynamic cross-talk between dendritic cells and NK and NK-T cells and therapies that modulate the numbers or function of these cells may have significant impact upon the ability of DC to prime T cells for specific anti-tumor responses. In addition, we have identified a specific accumulation of T regulatory cells within the tumor microenvironment, which may serve to suppress anti-tumor responses. We have identified tumor necrosis factor alpha (TNFa), a critical proinflammatory cytokine, as possibly being involved in the recruitment and/or expansion of T regulatory cells. We are targeting this cytokine by molecular and biochemical approaches that may ultimately be useful in the clinic for counteracting the establishment of a suppressive tumor microenvironment. We have also identified a putative immunosuppressive role for NK-T cells and we are now developing immunotherapeutic regimens that reduce the numbers and activity of these cells in different organs. By characterizing the role of inflammation upon tumor progression, these overlapping approaches will hopefully lead to the development of immunotherapeutic approaches that skew the inflammatory response to counter tumor growth.