PO.IM01.12 · 免疫学
剖析溶瘤病毒在胶质瘤中的抗病毒免疫与抗肿瘤免疫：来自患者来源类器官模型的见解
Dissecting oncolytic virus anti-virus vs anti-tumor immunity in glioma: Insights from a patient derived organoid model
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言。瘤内注射溶瘤单纯疱疹病毒（oHSV）CAN-3110可重塑复发性胶质母细胞瘤的免疫抑制性微环境。CAN-3110治疗引发的免疫浸润与生存期延长相关，尤其在HSV1血清阳性个体中（Ling等，2023）。然而，oHSV治疗诱导的免疫应答主要是抗病毒还是抗肿瘤仍不清楚。我们旨在使用离体患者来源胶质瘤类器官（pGBO）-免疫共培养模型来剖析这一区别，以研究oHSV治疗对抗肿瘤免疫的影响。
方法。pGBO由手术切除的胶质瘤标本生成，并通过高度多重循环免疫荧光（CycIF）用（截至目前）29种标志物进行表征。使用表达GFP的oHSV rQNestin34.5v.1评估这些pGBO中的病毒感染动力学。在完成pGBM表征后，我们建立了若干pGBO克隆与rQNestin34.5v.1以及来自健康供者或pGBO患者的外周血单个核细胞（PBMC）的共培养模型。该系统用于评估病毒和非病毒条件下肿瘤-免疫相互作用的特征和动力学。
结果。组织学和免疫荧光分析显示，pGBO在培养数周内保留了组织微结构，如血管结构——以含红细胞的内皮壁为特征——即使在模型冻融后也是如此。pGBO中的细胞多样性反映了体内所见的异质性，包括神经干样细胞、胶质细胞和残留的固有免疫细胞群。这些不同的细胞类型表现出代谢活性、上皮-间质转化（EMT）和增殖。在三个月的培养期内，其结构和表型变得更具侵袭性的胶质瘤特征，胶质瘤细胞扩增增加，血管丧失。值得注意的是，潜在的细胞异质性在整个过程中得以保留。pGBO与同种异体PBMC共培养在病毒和非病毒条件下均触发了CD3+、CD8+和CD4+淋巴细胞的浸润。切割型caspase-3和granzyme B染色显示granzyme+ CD8 T细胞对肿瘤细胞的主动杀伤，且该杀伤似乎不局限于受感染的胶质瘤细胞。此外，在受感染和未受感染的pGBO中均检测到CD20+淋巴细胞。
结论。我们将pGBO表征为一种生理学上有效的离体模型，观察到与原位胶质瘤中相同的血管结构和细胞组成。在PBMC×pGBO共培养中，无论有无病毒治疗，均通过成像和分子读出检测到免疫介导的肿瘤细胞杀伤。该模型能够研究溶瘤HSV感染如何调节免疫应答，并提供了一个可行的系统来剖析抗肿瘤与抗病毒免疫应答。
查看英文原文 English abstract
Introduction. Intratumoral injection of the oncolytic herpes simplex virus (oHSV) CAN-3110 remodels the immunosuppressive microenvironment of recurrent glioblastoma. The immune infiltration triggered by CAN-3110 treatment is correlated with prolonged survival, especially in HSV1-seropositive individuals (Ling et al., 2023). However, it is still unclear if the immune response induced by oHSV therapy is mainly anti-virus or anti-tumor. We aim to dissect this distinction using ex vivo patient-derived glioma organoid (pGBO)-immune co-culture models to study the impact of oHSV treatment on anti-tumor immunity.
Methods. pGBOs were generated from surgically resected glioma specimens and characterized by highly multiplexed cyclic immunofluorescence (CycIF), via (up to date) 29 markers. Viral infection kinetics were assessed in these pGBOs using the GFP-expressing oHSV, rQNestin34.5v.1. Following pGBM characterization, we established a co-culture model of several pGBOs clones with rQNestin34.5v.1 and peripheral blood mononuclear cells (PBMCs) from either healthy donors or the pGBO patients. This system was used to assess tumor-immune interaction characteristics and kinetics under viral and non-viral conditions.
Results. Histological and immunofluorescent analyses showed that pGBOs retain tissue microstructures, such as vascular architecture - characterized by endothelial walls containing erythrocytes - for several weeks in culture, even after freezing and thawing the models. The cellular diversity in pGBOs mirrors the heterogeneity found in in vivo , comprising neural stem-like, glial, and residual innate immune cell populations. These distinct cell types exhibited metabolic activity, epithelial-to-mesenchymal transition (EMT), and proliferation. Over a three-month culture period, the architecture and phenotype changed to more aggressively glioma, with increased glioma cell expansion and loss of vasculature. Notably, the underlying cellular heterogeneity remained throughout the process. Co-culture of pGBOs with allogeneic PBMCs triggered infiltration of CD3+, CD8+, and CD4+ lymphocytes under viral and non-viral conditions. Staining for cleaved caspase-3 and granzyme B showed active tumor cell killing by granzyme+ CD8 T cells, which appears unrestricted to infected glioma cells. Further, CD20+ lymphocytes were detected in both infected and uninfected pGBOs.
Conclusion. We characterized pGBOs as a physiologically valid ex vivo model, observing both vasculature and cellular composition as found in in situ gliomas. Immune-mediated tumor cell killing was detected, both with and without virus treatment in PBMC x pGBO co-culture, using imaging and molecular readouts. This model enables investigation of how oncolytic HSV infection modulates immune response and provides a tractable system to dissect anti-tumor vs. anti-virus immune responses.
利益披露 Disclosure
C. Linke, None..
E. Chen, None..
J. Gantchev, None..
A. Chauhan, None..
C. Jannotta, None.
K. L. Ligon,
Travera Other Business Ownership.
Bristol Myers Squibb Independent Contractor.
Blaze Bioscience Independent Contractor.
Integran Independent Contractor.
MIT/DFCI Patent, (PCT/US2022/051514).
A. Santos, None.
N. Agar,
Bruke Daltonics Other, Key opinion leader.
Thermo Finnigan Other, Collaboration.
EMD Serono Independent Contractor.
iTeos Therapeutics Independent Contractor.
BondZ g., Board of Directors, non-salaried role).
E. Chiocca,
Alliance for Cancer Gene Therapy ).
Bionaut Labs Stock, Other, Advisor.
reignite Therapeutics Stock.
Seneca Therapeutics Stock, Other, Advisor.
Theriva Other, Advisor.
Ternalys Therapeutics g., Board of Directors, non-salaried role), Stock, Other, co-founder.
NIH ).
Department of Defense ).
Candel Therapeutics Other, Advisor and patent inventor (patent no. US10,806,761 B2, date: 20 October 2020; patent no. 6,897,057, date 24 May 2005; patent no. 7,214,515 4 January 2002).
A. L. Ling, None.