(G-280) Utilizing Lipid Nanoparticles to Disrupt CD47-SIRPα Signaling Axis in Ovarian Cancer

Introduction:: Introduction: Ovarian cancer (OvCa) is a leading cause of cancer-related death among women in the world that commonly presents in advanced stages. Due to limited effective therapeutic options, the 5-year survival of patients with OvCa liver metastases (OCLM) is less than 30%. Macrophages represent a large cell population within the OvCa microenvironment. Macrophages promote tumor growth/invasion and enhance OvCa platinum chemo-resistance. OvCa cells express surface immunoglobulin CD47 that binds to signaling receptor protein-α (SIRPα) on macrophages to suppress the macrophages innate phagocytic functions, thus enhancing pro-tumoral capabilities. Thus, the disruption of the CD47- SIRPα signaling axis presents a highly relevant immunotherapeutic target to curtail unregulated tumor growth and invasion in conjunction with conventional chemotherapies. We hypothesize that disruption of the CD47- SIRPα signaling axis between OvCa cells and tumor microenvironment macrophages can decrease the metastatic potential of OvCa and increase the efficiency of carboplatinum therapy. The objective of this study is to design, characterize, and assess cellular interactions of lipid nanoparticles (LNP) targeted to disrupt the CD47-SIRPα signaling axis with the aim to develop a novel and efficient immunotherapy for advanced OvCa. LNP are utilized as a delivery vehicle due to their biocompatibility and biodegradability, along with their ability to encapsulate and efficiently deliver the proposed therapeutics to macrophages in the metastatic OvCa microenvironment.


Materials and Methods:: Methods: Tumor microarrays from OvCa patient samples were utilized to immunofluorescently stain and quantify the presence of CD47 and SIRPα at different metastatic stages. Two types of immuno-nanoparticles were developed to disrupt the CD47-SIRPα pathway: LNP1 CD47- SIRPα siRNA encapsulating LNP for silencing SIRPα expression and LNP2, with anti-CD47 antibody conjugated onto the LNP surface aimed to obstruct binding of the receptor. LNP1 and LNP2 were designed and characterized for size, zeta potential and particle concentration by Dynamic Light Scattering and NanoSight, respectively. LNP1 were prepared using a microfluidic device, NanoAssemblr (Precision Nanosystems) and LNP2 by a thin-film hydration method. The encapsulation efficiency of siRNA in LNP1 was assessed by RiboGreen assay. LNP stability of both formulations were assessed up to 2 weeks. To evaluate in vitro biocompatibility and cellular uptake of LNP, two- and three-dimensional cultures, containing single cell type or hetero-cultures, containing both cancer cells and macrophages, were utilized. For efficacy evaluation, OVCAR3 (human high grade serous OvCa) and THP-1 monocyte-derived macrophages were combined on hanging drop spheroid arrays to generate OvCa mono-spheroids or OvCa/macrophage hetero-spheroids. Hetero-spheroids were treated with either chemotherapy drug carboplatin (0-1000 µM), siRNA-containing NPs (0-50 nM) or both for 48 hours. LNP efficacy on hetero-spheroids invading bio-engineered decellularized porcine liver matrices were also evaluated. Cell viability after LNP treatment was evaluated by MTS assay. RT-qPCR was performed to quantify expression of CD47 and SIRPα as well as metastasis-indicating genes in OvCa cells on the different models.


Results, Conclusions, and Discussions:: Results: The CD47-SIRPα targeting approach for OvCa was validated with increased expression of CD47 and SIRPα in metastatic patient samples versus normal ovarian tissue. Co-localization of CD47-SIRPα was seen in ascites of metastatic patients. LNP1 and LNP2 were successfully formulated with narrow size distribution (polydispersity index< 0.14) and diameter within 64-165nm range. Both formulations showed consistent size distribution, zeta potential, and concentration across the evaluated time period. RNA content in LNP1 showed >95% encapsulation efficiency and minimal degradation in non-lyophilized systems up to day 12 of cold storage. Both LNP1 and LNP2 showed high in vitro biocompatibility and very efficient cellular uptake in 2D cultures. Macrophages contributed to carboplatin chemoresistance in hetero-spheroids (1.7-fold more resistant than OvCa mono-spheroids). Our results indicate that LNP1 siRNA nanoparticles treated OvCa/macrophage hetero-spheroids can reduce SIRPα expression by 50%. Disruption in macrophage SIRPα signaling via LNP1 and LNP2 demonstrates a significant drop in OvCa cell proliferation, 48h post-treatment. Both LNP1 and LNP2 inhibited OvCa invasion of decellularized porcine biomatrix.


Conclusions: The study confirms the ability to design and generate LNP systems that target and disrupt the CD47-SIRPα signaling axis through siRNA and antibody-based pathway inhibition to potentially treat advanced ovarian cancer. Efficacy in disruption of the CD47- SIRPα axis in both LNP types was seen in decreased gene expression of both CD47 and SIRPα expression in mono-spehroids and hetero-spheroids. The LNPs generated validate a potentially new avenue of immune-nanoparticle adjuvant therapy for late-stage ovarian cancer patients to reduce metastatic tumor burden and improve prognosis. In vivo studies with metastatic OvCa in immunocompetent mice is underway.


Acknowledgements (Optional): : Acknowledgment: The authors acknowledge kind support of Dr. and Mrs. Alan Kaplan Gynecologic Fund through the Houston Methodist Cancer Center.


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