Sitta Juliana, De Carlo Flavia, Kirven Imani, Tackett John H, Penfornis Patrice, Dobbins George Clement, Barbier Mallory, Del Valle Luis, Larsen Clayton T, Schutt Ernest G, Li Rhodemann, Howard Candace M, Claudio Pier Paolo
Department of Radiology, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Department of Biomedical Sciences, Imaging Track, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Int J Mol Sci. 2024 Dec 21;25(24):13697. doi: 10.3390/ijms252413697.
Oncolytic virotherapy has shown great promise in mediating targeted tumor destruction through tumor-selective replication and induction of anti-tumor immunity; however, obstacles remain for virus candidates to reach the clinic. These include avoiding neutralizing antibodies, preventing stimulation of the adaptive immune response during intravenous administration, and inducing sufficient apoptosis and immune activation so that the body's defense can work to eradicate systemic disease. We have developed a co-formulation of oncolytic viruses (OVs) with Imagent lipid-encapsulated, perfluorocarbon microbubbles (MBs) to protect the OVs from the innate and adaptive immune system. Once inside the MB, the viral particles become acoustically active such that external ultrasound can target the delivery of the virus locally within the tumor. Humanized NSG female mice (Hu-CD34 NSG-SGM3) engrafted in their flanks with MDA-MB-231-Luc triple-negative breast cancer (TNBC) cells were transduced with MB/OVs, with or without adjuvant Pembrolizumab treatment, and tumor sizes and tumor necrosis were assessed. The presence of CD8 (cytotoxic T-cells), CD4 (helper T-cells), and CD25 (Tregs) tumor-infiltrating lymphocytes (TILs) was quantified in the tumor samples by immunohistochemistry. In an in vivo model of humanized mice engrafted with a human immune system, we observed significantly greater tumor necrosis and smaller tumor mass in human TNBC xenografts systemically treated with MB/OV complexes in the presence or absence of pembrolizumab adjuvant treatment, compared to controls. Additionally, we observed a low ratio of CD4/CD8 TILs and a high ratio of CD8/CD25 TILs in the MDA-MB-231 xenografts treated with MB/OVs complexes with or without pembrolizumab adjuvant treatment, compared to controls. Our study demonstrated the feasibility of using MBs to target OVs to TNBC through diagnostic ultrasound, which decreased tumor mass by increasing tumor necrosis and stimulated a local and systemic antitumoral immune response by increasing intratumoral CD8 T-cytotoxic lymphocyte infiltration and decreasing CD25 Treg cells.
溶瘤病毒疗法在通过肿瘤选择性复制和诱导抗肿瘤免疫来介导靶向肿瘤破坏方面显示出巨大潜力；然而，候选病毒进入临床仍存在障碍。这些障碍包括避免中和抗体、在静脉给药期间防止刺激适应性免疫反应，以及诱导足够的细胞凋亡和免疫激活，以便机体的防御机制能够发挥作用根除全身性疾病。我们开发了一种溶瘤病毒（OVs）与Imagent脂质包裹的全氟碳微泡（MBs）的联合制剂，以保护OVs免受先天性和适应性免疫系统的影响。一旦进入微泡内，病毒颗粒就会具有声学活性，这样外部超声就可以将病毒局部递送至肿瘤内。将携带MDA-MB-231-Luc三阴性乳腺癌（TNBC）细胞的人源化NSG雌性小鼠（Hu-CD34 NSG-SGM3）双侧接种肿瘤，用MB/OVs进行转导，同时给予或不给予辅助性帕博利珠单抗治疗，并评估肿瘤大小和肿瘤坏死情况。通过免疫组织化学对肿瘤样本中CD8（细胞毒性T细胞）、CD4（辅助性T细胞）和CD25（调节性T细胞）肿瘤浸润淋巴细胞（TILs）的存在情况进行定量分析。在植入人类免疫系统的人源化小鼠体内模型中，我们观察到，与对照组相比，在有或没有帕博利珠单抗辅助治疗的情况下，用MB/OV复合物进行全身治疗的人TNBC异种移植瘤中，肿瘤坏死明显增加，肿瘤体积明显减小。此外，与对照组相比，在有或没有帕博利珠单抗辅助治疗的情况下，用MB/OV复合物治疗的MDA-MB-231异种移植瘤中，我们观察到CD4/CD8 TILs比例较低，CD8/CD25 TILs比例较高。我们的研究证明了通过诊断超声将OVs靶向TNBC的可行性，这通过增加肿瘤坏死减少了肿瘤体积，并通过增加肿瘤内CD8 T细胞毒性淋巴细胞浸润和减少CD25调节性T细胞刺激了局部和全身抗肿瘤免疫反应。
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