Modulating the immuno-metabolic interplay in liver cancer with cryoablation

摘要 晚期癌症的全身治疗使用免疫检查点抑制剂来利用患者自身的免疫功能。 免疫系统产生强大的抗肿瘤免疫应答。该方法已成功应用于 黑色素瘤和肺癌，但对肝细胞癌（HCC）患者效果较差。由于大多数HCC 当治愈性治疗选择有限时，患者被诊断为中期或晚期， 免疫疗法驱动的治疗是非常需要的。 由高糖酵解肿瘤代谢驱动的肿瘤微环境的酸性代表了一个关键 抵抗机制，并有助于免疫细胞耗竭，从而帮助癌细胞逃避局部免疫。 免疫反应我们的假设是，对免疫疗法有反应的肿瘤具有可检测的特征，如 肿瘤酸度正常化和免疫细胞密度更高。这些参数如果一起测量， 帮助预测哪些患者会对免疫疗法有反应和/或了解为什么有些患者没有反应。成像 为了更好地监测肿瘤的细胞外pH（pHe），有必要沿着评估局部免疫细胞募集 并指导肝癌免疫治疗。然而，为了准确地将pHe信息与免疫共定位， 细胞，需要高空间分辨率的pHe成像。该提案的目标是建立高分辨率 在小鼠肝脏中进行体内pHe成像，以研究冷冻消融对肿瘤侵袭性的影响， 与局部免疫细胞的募集有关。 在目标1中，我们将建立和验证一种高分辨率的pHe成像技术，在小鼠肝脏中使用 多药耐药基因2敲除（Mdr2 KO）肝癌模型，其中慢性- 炎症-纤维化模拟了人类中所见的肝损伤的重要阶段。由于反复冻结 肿瘤触发抗原释放以产生抗肿瘤免疫应答，在目标2中，我们将确定冷冻消融是否 可调节肿瘤的免疫原性状态。因此，冷冻消融可以提供原位免疫系统， 通过将侵袭性免疫抑制性肿瘤转化为更具免疫容许性的肿瘤， 肿瘤工作假设是MRI引导的冷冻消融将通过杀死肿瘤细胞来提高pHe，并且将 通过释放抗原调节肿瘤的免疫原性程度。为了研究冷冻消融术的效果， 在冷冻消融前1天和冷冻消融后7天在Mdr2 KO小鼠肝脏中采集高分辨率pHe图像。 我们将使用具有多个肝肿瘤的小鼠，仅冷冻消融一个，以研究靶点和远端效应。 组织学将用于揭示肿瘤内免疫细胞募集活化的T细胞，辅助T细胞， 效应T细胞、吞噬细胞和M2巨噬细胞。如果成功，这种方法将允许未来的调查， 通过将冷冻消融与免疫检查点抑制剂组合来实现协同抗肿瘤作用， 免疫效应。

ABSTRACT Systemic therapies for advanced stage cancer use immune checkpoint inhibitors to exploit patient's own immune system to generate a robust anti-tumor immune response. This approach is successfully applied for melanoma and lung cancer, but is less effective for hepatocellular carcinoma (HCC) patients. Since most HCC patients are diagnosed at intermediate or advanced stages when curative treatment options are limited, a novel immunotherapy driven treatment is highly desirable. The acidity of the tumor microenvironment driven by hyperglycolytic tumor metabolism represents a key mechanism of resistance and contribute to immune cell exhaustion, thereby helping cancer cells evade the local immune response. Our hypothesis is that tumors responding to immunotherapies have detectable features like normalization of tumor acidity and higher density of immune cells. These parameters, if measured together, can help predict which patients will respond to immunotherapy and/or understand why some patients do not. Imaging extracellular pH (pHe) of tumors along with assessing local immune cell recruitment is necessary to better monitor and guide liver cancer immunotherapies. However, to accurately co-localize the pHe information with the immune cells, high spatial resolution pHe imaging is needed. The goal of this proposal is to establish high resolution in vivo pHe imaging in mouse liver to investigate the effect of cryoablation on tumor aggressiveness in relation to recruitment of local immune cells. In Aim 1 we will establish and validate a high resolution pHe imaging technique in mouse liver using a multidrug resistance gene 2 knockout (Mdr2 KO) model of HCC on a cirrhotic background, where the chronic- inflammation-fibrosis mimics the important stages of liver injury seen in humans. Since repeated freezing of a tumor triggers release of antigens for an anti-tumor immune response, in Aim 2 we will determine if cryoablation can modulate the state of tumor's immunogenicity. Thus, cryoablation may provide an in situ immune system amplification instrument by converting an aggressive immuno-suppressive tumor into a more immuno-permissive tumor. The working hypothesis is that MRI-guided cryoablation will raise pHe by killing tumor cells, and will modulate tumor's degree of immunogenicity by releasing antigens. To investigate the effects of cryoablation, high resolution pHe images will be acquired in Mdr2 KO mice livers 1 day before and 7 days after cryoablation. We will use mice with multiple liver tumors and cryoablate only one to investigate both target and distal effects. Histopathology will be used to reveal intratumoral immune cell recruitment of activated T-cells, helper T-cells, effector T-cells, phagocytes and M2 macrophages. If successful, this approach will allow future investigations of synergistic anti-tumor effects achieved by combining cryoablation with immune checkpoint inhibitors for improved immunotherapeutic effects.