Department of Pharmacology, Boston University School of Medicine, Boston, MA, USA.
Whitaker Cardiovascular Institute and Department of Medicine, Boston University School of Medicine, Boston, MA, USA.
BMC Cancer. 2021 Apr 14;21(1):407. doi: 10.1186/s12885-021-08107-w.
Pancreatic peritoneal carcinomatosis (PPC), with the worst median overall-survival (mOS), epitomizes the incurability of metastatic cancer. Cancer stem cells (CSCs) underpin this incurability. However, inhibitors of CSC-stemness fail to increase mOS in cancer patients despite preclinical tumor-reduction. This shortfall reinforces that preclinical efficacy should be defined by increased mOS in the presence of cancer comorbidities, CSC-heterogeneity and plasticity. The primary objectives of this study are: to test the dual endothelin-1/signal peptide receptor, DEspR, as a nodal therapeutic target in PPC, given DEspR induction in anoikis-resistant pancreatic CSCs, and to validate humanized anti-DEspR antibody, hu-6g8, as a potential therapeutic for PPC.
We used heterogeneous pools of CSCs selected for anoikis resistance from reprogrammed Panc1 and MiaPaCa2 tumor cells (TCs), and adherent TCs reprogrammed from CSCs (cscTCs). We used multiple anti-DEspR blocking antibodies (mAbs) with different epitopes, and a humanized anti-DEspR recombinant mAb cross-reactive in rodents and humans, to test DEspR inhibition effects. We measured DEspR-inhibition efficacy on multiple prometastatic CSC-functions in vitro, and on tumorigenesis and overall survival in a CSC-derived xenograft (CDX) nude rat model of PPC with comorbidities.
Here we show that DEspR, a stress-survival receptor, is present on subsets of PDAC Panc1-TCs, TC-derived CSCs, and CSC-differentiated TCs (cscTCs), and that DESpR-inhibition decreases apoptosis-resistance and pro-metastatic mesenchymal functions of CSCs and cscTCs in vitro. We resolve the DNA-sequence/protein-function discordance by confirming ADAR1-RNA editing-dependent DEspR-protein expression in Panc1 and MiaPaCa2 TCs. To advance DEspR-inhibition as a nodal therapeutic approach for PPC, we developed and show improved functionality of a recombinant, humanized anti-DEspR IgG4 antibody, hu-6g8, over murine precursor anti-DEspR mabs. Hu-6g8 internalizes and translocates to the nucleus colocalized with cyto-nuclear shuttling galectins-1/3, and induces apoptotic cell changes. DEspR-inhibition blocks transperitoneal dissemination and progression to peritoneal carcinomatosis of heterogeneous DEspR±/CD133 ± Panc1-derived CSCs in xenografted nude rats, improving mOS without chemotherapy-like adverse effects. Lastly, we show DEspR expression in Stage II-IV primary and invasive TCs in the stroma in PDAC-patient tumor arrays.
Collectively, the data support humanized anti-DEspR hu-6g8 as a potential targeted antibody-therapeutic with promising efficacy, safety and prevalence profiles for PPC patients.
胰腺腹膜癌转移（PPC）的中位总生存期（mOS）最差，是转移性癌症无法治愈的典型代表。癌症干细胞（CSC）是导致这种无法治愈的原因。然而，尽管在临床前肿瘤减少方面取得了成功，但CSC 干性抑制剂并未增加癌症患者的 mOS。这一不足表明，临床前疗效应通过存在癌症合并症、CSC 异质性和可塑性时增加 mOS 来定义。本研究的主要目的是：鉴于在抗凋亡的胰腺 CSC 中诱导了 DEspR，测试双内皮素-1/信号肽受体 DEspR 作为 PPC 的治疗靶点的能力，验证人源化抗 DEspR 抗体 hu-6g8 是否可能成为 PPC 的潜在治疗方法。
我们使用从重编程的 Panc1 和 MiaPaCa2 肿瘤细胞（TC）中选择的抗凋亡抗性的异质 CSC 池，以及从 CSC 重编程的贴壁 TC（cscTC），来研究 CSC 异质性。我们使用多种具有不同表位的抗 DEspR 阻断抗体（mAb），以及一种在啮齿动物和人类中具有交叉反应性的人源化抗 DEspR 重组 mAb，来测试 DEspR 抑制作用。我们在体外测量了 DEspR 抑制对多种促转移 CSC 功能的作用，以及在具有合并症的 CSC 衍生异种移植（CDX）裸鼠 PPC 模型中的肿瘤发生和总生存期。
在这里，我们证明了应激生存受体 DEspR 存在于 PDAC Panc1-TC、TC 衍生的 CSC 和 CSC 分化的 TC（cscTC）的亚群中，并且 DEspR 抑制降低了 CSC 和 cscTC 的体外抗凋亡和促转移间充质功能。我们通过确认 ADAR1-RNA 编辑依赖性 DEspR 蛋白表达来解决 DNA 序列/蛋白功能不一致的问题，在 Panc1 和 MiaPaCa2 TC 中。为了将 DEspR 抑制作为 PPC 的治疗靶点，我们开发并展示了重组人源化抗 DEspR IgG4 抗体 hu-6g8 的改进功能，优于鼠源前体抗 DEspR mAb。Hu-6g8 内化并转位到与细胞质核穿梭半乳糖凝集素-1/3 共定位的核内，并诱导细胞凋亡变化。DEspR 抑制阻止了异质的 DEspR+/CD133+Panc1 衍生 CSC 在异种移植裸鼠中的腹膜扩散和进展为腹膜癌转移，提高了 mOS，而没有化疗样的不良反应。最后，我们显示在 PDAC 患者肿瘤阵列中的原发性和侵袭性 TC 的基质中存在 DEspR 表达。
总的来说，数据支持人源化抗 DEspR hu-6g8 作为一种潜在的靶向抗体治疗方法，具有有希望的疗效、安全性和普遍性，适用于 PPC 患者。
