Purpose In vivo immune cell tracking using MRI is a valuable tool for studying the mechanisms underlying successful cancer therapies. Current cell labeling methods using superparamagnetic iron oxide (SPIO) lack the specificity and persistence needed to track the fate and location of transplanted cells long-term. Magnetospirillium magneticum is a commercially available, iron-producing bacterium that can be taken up by, and live harmoniously within, mammalian cells as magneto-endosymbionts (MEs). MEs have shown promise as labeling agents for in vivo stem and cancer cell tracking but have yet to be evaluated in immune cells. This pilot study examined ME labeling in myeloid-derived suppressor cells (MDSCs), cytotoxic T lymphocytes (CTLs) and dendritic cells (DCs) and its effects on cell purity, function and MRI contrast. Procedures: MDSCs, CTLs and DCs were incubated with MEs at various ME labelling ratios (MLR) and various biological metrics and iron uptake were assessed. For in vivo imaging, MDSCs were labeled overnight with either MEs or SPIO (Molday ION Rhodamine B) and injected into C3 tumor-bearing mice via tail vein injection 24 days post-implant and scanned daily with MRI for one week to assess cellular quantification. Results Following incubations MDSCs contained 0.62 and 2.22 pg Fe/ cell. CTLs achieved Fe loading of < 0.5 pg/ cell and DCs achieved Fe loading of ~ 1.4pg/cell. The suppressive functionality of MDSCs at 1000MLR was not affected by ME labeling but was affected at 2000MLR. Markers of CTL dysfunction were not markedly affected by ME labeling, nor were DC markers. In vivo data demonstrated that the MDSCs labeled with MEs generated sufficient contrast to be detectable using TurboSPI, similar to SPIO-labeled cells. Conclusions Cells can be labeled with pre-clinically relevant amounts of MEs without compromising cell viability. Care must be taken at higher concentrations of MEs, which may affect the functional activity and/or morphology of some cell types. Immune cells with minimal phagocytic behaviour have much lower iron content per cells after incubation with MEs vs SPIO; however, MEs can successfully be used as a contrast agent for phagocytic immune cells.

Introduction: Epithelial ovarian cancers are highly aggressive and often diagnosed at late stages. To date, a limited number of these patients benefit from immunotherapies, and recurrent ovarian cancer remains an area of unmet medical need. Novel immunotherapies that generate and drive activated T cells into tumors, such as DPX-Survivac, represent a promising approach. To better understand the underlying mechanisms of action of this novel immunotherapy and how it may correlate with clinical outcomes, we used an orthotopic ovarian cancer model and a dual approach using molecular imaging to track immune cells in vivo followed by biological assays. Methods: Humanized transgenic mice (HLA-A2.1-/HLA-DR1; H-2 class-I/II knockout) were implanted with 104 syngeneic mouse ovarian surface epithelial cells in the left ovarian bursa and were either untreated or treated with DPX-Survivac, intermittent low dose cyclophosphamide and anti-PD-1. The CD8+ cytotoxic T cells (CTLs) and myeloid cells (MCs) were isolated from disease matched mice, cultured in vitro, loaded with superparamagnetic iron oxide (SPIO) nanoparticles, and injected i.v. in recipient mice. The next day, recipient mice were injected with 500µCi of 18F-fluorodeoxyglucose and imaged by magnetic resonance (MRI) and positron emission tomography (PET). Anatomical images and tumor volumes were collected with a balance steady-state free precession sequence, cells were semi-quantified using R2* maps from a multi-echo single point imaging sequence (TurboSPI) and tumor metabolism was assessed by simultaneous acquisition of PET. Mice were imaged days 41, 49, and 56 post-implant. CTLs and MCs were characterized by flow cytometry (FC) before injection into recipient mice. Following imaging completion, tumors and lymph nodes (LN) were assayed for tumor infiltrating lymphocytes. Ascites were analyzed for tumor cell to lymphocyte content by FC. Results: PET/MRI images showed that the combination immunotherapy significantly decreased primary tumor burden and improved survival rates. The recruitment of isolated CTLs and MCs was detected in both tumors and LNs. CTL recruitment to tumors was increased in mice receiving combination immunotherapy while MCs recruitment was decreased. CTLs were recruited at a higher rate to the DPX-Survivac draining LN than tumor draining LN and MCs were equally recruited to both. The expression of checkpoint inhibitor molecules on CTL did not appear to vary with treatment. CTLs from treated mice expressed higher rates of Ki67 (a proliferative marker) than CTLs from untreated mice. The treatment increased the lymphocyte:tumor cell ratio within the tumors of treated animals and included a population of CD8/CD4 double positive (DP) T cells. Differences in tumor infiltrating lymphocyte phenotype correlated with tumor burden. Conclusions: DPX-Survivac combination immunotherapy induces the recruitment of CD8+ T cells into tumors, resulting in tumor control in an orthotopic ovarian cancel model. Citation Format: Marie-Laurence Tremblay, Caitrin Sobey-Skelton, Hailey Wyatt, Victoria Gonzalez, Andrea Nuschke, Christa Davis, Alecia Mackay, Kim Bobbit, Andrea West, Barbara Vanderhyden, Genevieve Weir, Alexandra Merkx-Jacques, Kimberly Brewer, Marianne Stanford. T cell infiltration into tumors induced by DPX-Survivac combination immunotherapy demonstrated by PET/MRI imaging in an orthotopic ovarian cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 384.