MRI of combination immunotherapy in an epithelial ovarian cancer preclinical model

MRI of combination immunotherapy in an epithelial ovarian cancer preclinical model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MRI of combination immunotherapy in an epithelial ovarian cancer preclinical model Jessica T. Gosse, Caitrin Sobey Skelton, Marie-Laurence Tremblay, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7714918/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Background Novel treatments are needed for epithelial ovarian cancer, the most lethal gynecologic malignancy due to late diagnosis, resistance to treatment, and high relapse rate. Immunotherapies such as checkpoint inhibitors (i.e, anti-PD-1) and peptide-based therapies (DPX-Survivac) have strong potential to improve responses. Magnetic resonance imaging (MRI) can be used to track tumor growth and iron-labelled immune cells longitudinally at the individual level. We studied MRI immune cell tracking in response to the combination of DPX-Survivac, anti-PD-1, and an intermittent low dose of Cyclophosphamide (CPA), which has been shown to suppress cancer growth in a preclinical model of ovarian cancer. Methods HHD-DR1 mice were orthotopically implanted with mouse ovarian surface epithelial (MOSE) cancer cells. Myeloid and activated CD8 + cells were isolated from disease- and treatment-matched donor mice, labelled with superparamagnetic iron oxide (SPIO) and intravenously injected on 41, 48, and 55 days post-implant with either type of cells. Mice were scanned using MRI approximately 24h after SPIO-labeled cell injections. Results Tumor volumes in the treatment group were significantly lower than in the control group as measured by MRI ( p < 0.01). The density of SPIO-labelled myeloid and CD8 + T cells in tumors was higher in the treatment group than in the control group. Furthermore, ascitic fluid in treated mice has a significantly higher frequency of CD45 + leukocytes. Conclusion Using MRI, our study has shown that this combination treatment can slow down ovarian tumor growth and increase the recruitment of myeloid and CD8 + cells to tumors. This study provides insights into how MRI can be used in concert with biological assays to study how immunotherapy and chemotherapy combinations exert their antitumor effects. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology Magnetic resonance imaging (MRI) immunotherapy cell tracking cytotoxic T lymphocytes ovarian cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Epithelial ovarian cancer is the most lethal gynecological cancer: the disease is highly aggressive and commonly diagnosed at advanced stages. Most cases are diagnosed at stages III and IV when symptoms have spread to the abdominal area 1 , 2 , such as ascites and pelvic discomfort. Ascites are often associated with intraperitoneal metastasis caused by cell shedding from the primary tumor 3 . Late-stage diagnoses are primarily responsible for the global five-year survival rate between 30 and 40% 1,4 . One of the most significant challenges associated with ovarian cancer is that many patients do not respond to current treatment methods. Even if advanced ovarian cancer cases respond to traditional procedures, such as chemotherapy, radiation, and surgery, an estimated 75% of patients will relapse 1 . In light of these challenges, immunotherapies are a promising class of cancer therapies. Immunotherapies aim to harness and enhance the immune system's natural ability to detect and fight off cancerous cells. Some therapies currently in clinical use include immune checkpoint inhibitors/blockers and peptide-based T cell-activating molecules. Various blockers of checkpoint inhibitors have been FDA-approved and have shown success in clinical trials for skin, lung, and pancreatic cancers 5 . Immune checkpoint receptors act as "gatekeepers" of the immune response, preventing overstimulation of the immune system 6 . However, in tumor microenvironments, this interaction can have a suppressive effect on the activity of T cells, which can result in the exhaustion of their immune function 6 – 8 . Checkpoint blockers work by blocking surface-expressed immune cell checkpoints that regulate the duration and intensity of immune responses. One significant checkpoint inhibitor protein is the programmed cell death-1 (PD-1) receptor. PD-1 receptors are upregulated on the surface of T cells following activation and modulate cell function, survival, and proliferation upon ligand binding 9 . Ligand binding to PD-1 will induce a signaling cascade that ultimately inactivates T cell receptor (TCR) associated proteins essential for T cell activation. Many malignant cell types have evolved the ability to subvert host immune responses by expressing a PD-1 ligand (PD-L1) 9 . Upon binding to PD-1, PD-L1 inhibits the ability of cytotoxic T cells to target and attack tumor cells. Immunotherapeutic checkpoint inhibitor blockers, such as anti-PD-1, are monoclonal antibodies (mAb) that bind and block the interaction between PD-1 and PD-L1, enhancing T cell antitumor activity by preventing PD-L1 tumor-mediated shutoff of T cells 10 . Peptide-based therapies enhance the immune system's ability to recognize and respond to specific tumor antigens. The novel immunotherapy DPX™ (IMV Inc., Halifax, NS, Canada) is a specialized formulation that stimulates strong and specific immune responses 11 . DPX's oil-based, water-free formulation enhances immune responses by providing the immune system with prolonged exposure to antigens 11 . Specifically, DPX-Survivac combines the DPX formulation and a mixture of survivin peptides. Survivin, a protein necessary for inhibiting apoptosis, is highly expressed in many cancer types with solid tumors, including ovarian cancer 11 . DPX-Survivac therefore enhances antitumor immune responses by stimulating host immune systems 12 , 13 . One of the most significant limitations of single-agent therapies is the increased likelihood of acquired therapeutic resistance in malignant cells 1 . Combination therapies avoid acquiring resistance by enhancing antitumor activity through multiple mechanisms 14 . For example, positive outcomes have been demonstrated in ovarian cancer patients treated with a combination of DPX-Survivac and low-dose cyclophosphamide (CPA) 11 . Low-dose CPA is a chemotherapeutic agent regimen that has been shown to selectively reduce the number of regulatory T cells and subsequently enhance targeted immune responses 15 , 16 . Studies demonstrated that low-dose CPA enhanced antigen-specific immune responses induced by DPX-Survivac in ovarian cancer patients 11 . Furthermore, Weir et al. 17 found that triple combination therapy with DPX, low-dose CPA, and anti-PD-1 provided better long-term control of established tumors via slower growth in a derived preclinical model of human papillomavirus (HPV) cancer compared to treatment with anti-PD-1 alone. Given these results, this triple combination therapy may also be beneficial for epithelial ovarian cancer. Evaluating the efficacy of novel combination therapies like this triple combination therapy in preclinical models is vital for their translation into a clinical setting. Tumor progression and cell recruitment can be tracked using magnetic resonance imaging (MRI). Once acquired, anatomical MR images can be used to identify the volume and properties of specific regions of interest (ROI), such as tumors and lymph nodes. Quantification and recruitment of cells to particular ROIs can be performed using the MRI pulse sequence, TurboSPI 18 . TurboSPI is used to create R 2 * maps that can be overlaid onto anatomical images and is particularly sensitive to the field inhomogeneities caused by the encapsulation of superparamagnetic iron oxide (SPIO) within cells. SPIO nanoparticles can be used to label a wide variety of immune cells, including dendritic cells, T cells, and natural killer (NK) cells, amongst others 12 , 18 – 20 . Our lab has previously demonstrated the use of TurboSPI to semi-quantitatively evaluate SPIO-labeled T cells and myeloid-derived suppressor cells 12 , 18 . MRI and molecular imaging in general have proven to be particularly useful in better understanding the roles of treatment and interaction with the immune system on tumor growth and individual outcomes 9 . The objectives of this study were to use MRI in combination with biological assays to (1) study immune cell recruitment in an orthotopic preclinical model of ovarian cancer and (2) evaluate the effects of combination therapy on survival and tumour progression. We hypothesized that mice treated with DPX-Survivac, anti-PD-1, and low-dose CPA would have better tumor control (i.e., slower tumour growth) and increase immune cell recruitment to tumors. Methods Mice Female humanized transgenic mice (HLA-A2.I-HLA-DRI-transgenic (HHD) H-2 class I/II knockout), 6–23 weeks old, were obtained from Charles River Laboratory (France) and bred in-house. All mice were housed in filter-top cages and were provided food and water ad-libitum . Recipient mice were either untreated (n = 10) or treated (n = 14) with DPX-Survivac, anti-PD-1, and low-dose CPA. Experiments were conducted under ethics protocols approved by the University Committee on Laboratory Animals at Dalhousie University, Halifax, N.S., Canada. Cancer cell line and implant Mouse ovarian surface epithelial (MOSE) cancer cells were provided by Dr. Vanderhyden and were genetically modified in-house to express the survivin epitope and cryopreserved in Calf Bovine Serum with 10% dimethyl sulfoxide (DMSO). Cells were thawed and cultured in DMEM (Dulbecco's Modification of Eagle's Medium; Corning, Corning NY) supplemented with 100 Units/mL Penicillin, 100µg/mL streptomycin (Gibco, Burlington, ON), and 10% Calf Bovine Serum (Hyclone) and grown at 37°C in an atmosphere of 5% CO 2 . After the first passage, cells were selected for survivin by adding 10 µg/mL of puromycin. On intrabursal surgery day, cells were resuspended at 5x10 6 cells/mL in 1X PBS (Phosphate Buffered Solution; Corning), and 1x10 4 MOSE cells were injected into the left ovary. Animals received frequent detailed clinical examinations (DCEs) until the pre-surgery weight was reached. Once mice regained weight (approximately 3 weeks post-surgery), DCEs were conducted twice/week. Tumors were monitored throughout the study by palpating the abdomen. Mice were terminated if they lost 15% of their pre-surgery weight, presented severe ascites or ulcerations, or showed signs of pain and lethargy. Implant and treatment timelines can be found in Fig. 1 . DPX-Survivac Treatment DPX-Survivac was prepared at IMV Inc. using their proprietary DPX formulation described elsewhere 11 . Detailed methodology can be found in the Supplementary Materials. Doses of 50 µL were given subcutaneously to each mouse on days 28 and 49 post-MOSE implants (Fig. S1 ). The dosing schedule for all treatments was chosen based on previous work done by Weir et al. 17 demonstrating that this dosing combination and schedule worked well in an HPV model. Cyclophosphamide (CPA; MilliporeSigma, Oakville, ON, Canada) was reconstituted in PBS and delivered to mice in drinking water at 0.133 mg/mL for seven consecutive days, beginning on days 14, 28, and 42 post-MOSE implants (Fig. 1 ). Mice received 20 mg/kg/day, assuming a 20 g mouse consumes 3 mL water/day. Anti-PD-1 (clone RPM1-14; BioXCell; West Lebanon, NH, USA) was diluted to 2 mg/mL in 1x PBS. Treated donor mice received six intraperitoneal injections of anti-PD-1 at 200 µg per dose on days 21, 24, 27, 42, 45, and 48 post-MOSE implants. Treated recipient mice received injections on days 28, 31, 34, 49, 52, and 55 post-MOSE implants. (Fig. 1 ). CD8 Cytotoxic T Lymphocyte (CTL) Isolation & Labeling Inguinal, axial, brachial, and mesenteric lymph nodes were collected from disease-matched and treatment-matched donor mice for isolation of CD8 + T cells. CD8 + T cells were isolated from lymph nodes using the EasySep™ Mouse CD8 + T cell Isolation Kit (Stemcell Technologies, Cambridge, MA, United States). Cells were suspended at 5 x 10 5 cells/mL in complete RPMI (cRPMI) media (RPMI 1640 (Corning), 10% FBS (Hyclone), 1% penicillin/streptomycin (Gibco), and 55µM β-mercaptoethanol (Gibco)) and incubated in a CD3-coated cell culture plate with human IL-2 (20 U/mL), mouse IL-12 (100 ng/mL), hamster anti-mouse CD28 (1 µg/mL), and Gentamicin (5 µg/mL). Cells were monitored and kept at a density of 0.5 to 1 x 10 6 . Fresh cRPMI with IL-2 (20 U/mL) was added as required. Four days following CTL isolation, antigen-presenting cells (APC) were isolated from the spleens of disease- and treatment-matched donor mice. Splenocytes were incubated with LPS (10 µg/mL) in media (DMEM + 10% FBS + 55µM β-mercaptoethanol + 1% Penicillin-Streptomycin + 1% L-glutamine) for 48 hours. Non-adherent cells were then treated with mitomycin-c (50 µg/mL) for 20 mins, washed, and added to the CTL culture at a ratio of 1:6 (APC: CTL) with survivin peptides (10 µg/mL). Following a 48-hour incubation with the APCs, a sample of cells was collected to assess cell purity via flow cytometry, and the other cells were used for in vivo studies. Activated CTLs were collected, washed, and incubated with SPIO-Rhodamine B Molday ION (0.075 mg/mL; Biopal Inc., Worchester, MA, USA) and IL-2 (100 U/mL) at 4 x 10 6 cells/mL in cRPMI for 22–24 hours. Cell viability of CTLs post SPIO labeling was found to be > 90%. Previous work done in 21 demonstrated that labeling CTLs using this methodology does not affect the cytotoxicity ability of CTLs. A subset of labeled CTLs were also removed for flow cytometry analysis to assess the effects of labeling on functionality. Myeloid Cell (MC) Isolation & Labeling Bone marrow was collected from the femurs and tibias of disease- and treatment-matched donor mice for isolation of myeloid cells. Red blood cells were lysed with 1x RBC lysis buffer (Tonbo Biosciences, San Diego, CA, USA). Three million cells were incubated in Petri dishes with 10 mL of media containing RPMI, 10% FBS, 1% penicillin-streptomycin, 20 mM HEPES (MilliporeSigma), and 20 ng/mL of granulocyte monocyte-colony stimulating factor for 72 hours (GM-CSF; Peprotech, Rocky Hill, NJ, USA). After incubation, 10 mL of media with 20 ng/mL of GM-CSF was added to each plate. Following another 72 hours, cells were collected, centrifuged, resuspended in fresh media with 20 ng/mL of GM-CSF, and returned to the plates. The next day, cells were stimulated overnight with survivin peptide SurA2.M (20 µg/mL). Forty-eight hours later, cells were collected, resuspended at 4 x 10 6 cells/mL, and a sample of cells was assessed for cell purity using flow cytometry, with remaining cells being used for in vivo studies. The purified myeloid cells were incubated with 0.030 mg/mL of SPIO-Rhodamine B Molday ION for 18–20 hours. Cell viability of myeloid cells (MCs) post SPIO labeling was found to be > 90%. A subset of labeled MCs were also removed for flow cytometry analysis to assess the effects of labeling on functionality. Cell Injection & Preparation SPIO-labeled cells (CTLs or MCs) were collected, washed twice with 1x PBS, twice with HBSS++ (Hank's Balanced Salt Solution; Corning), and then resuspended in HBSS + + with 20 mM HEPES at 5 x 10 6 cells/mL (MCs) and 25 x 10 6 cells/mL (CTLs). All mice received 200 µL of CTLs or MCs through intravenous tail vein injections. Cells were injected on days 41, 48, and 55 post-implant (24 hours before MRI). Iron loading was assessed in the remaining cells using a Prussian blue assay 21 ; cells were lysed overnight in 100 µL of 1M HCl, and then 100 µL of K 4 Fe(CN) 6 was added to each sample. The absorbance ( \(\:\lambda\:\) = 620 nm) was recorded on SpectraMax i3 (Molecular Devices, San Jose, CA, USA) and compared to a standardized in-vitro calibration curve. Tumor Dissociation for Tumor-Infiltrating Lymphocyte Assay Tumors were resected from mice following their final MRI scans. In Petri dishes, the tumor was chopped into smaller pieces with a scalpel and then incubated in digestion buffer [1 mg/mL collagenase type 1 (Gibco) + 0.1 mg/mL DNase I (MilliporeSigma) + 5% FB Essence in HBSS++] at 37°C for 30 min. Samples were then filtered through a 70 µm strainer with separation buffer [2% FB essence + 1mM EDTA in 1x PBS (Gibco)]. Red blood cell (RBC) lysis was performed on the suspension as required. Cells were washed with 1x PBS and used for flow cytometry. Ascites Sample Preparation At the endpoint, mice were euthanized, and ascites fluid was collected using a 25G needle and a 5 mL syringe. Red blood cells were lysed with an equal volume of 1x RBC lysis buffer, and samples were centrifuged to collect cells. These remaining cells were washed thoroughly with 1x PBS and used for flow cytometry. Flow Cytometry Cell samples were blocked in 5% normal rat serum (NRS) for 10 min and then incubated with antibody cocktails at 4°C for 20 min (Supplementary Tables 1). Samples requiring intracellular staining were permeabilized with permeabilization buffer and stained with the intracellular antibody for 40–50 min at 4°C. The same staining procedure was followed as in 21 . After staining, samples were fixed with 4% paraformaldehyde (PFA). OneComp ebeads (eBioscience) were used for controls. Data were acquired with a FACS Celesta or FACS Canto II equipped with FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA) at the Dalhousie University Flow Cytometry Core Facility. Samples were analyzed using FlowJo v10.6.2 (Vicro, Torrance, CA). Immunohistochemistry Samples (spleen, tumor, and lymph nodes) were frozen immediately after termination in an Optical cutting temperature (OCT; Fisher)/Sucrose (1:1) solution, and stored in a -80 o C freezer until sectioning. Samples were taken to the Dalhousie immunohistochemistry core for processing; tissues were sectioned on the cryostat and placed on slides. Slides were then fixed in -20 o C cold acetone for 2 min, dried, and stored in a -20 o C freezer until staining. For IHC staining, slides were brought to RT, dried, and fixed in cold acetone for 10 min and air-dried again for 30 min. They were then washed in a Tris buffered saline (TBS)/Bovine serum albumin (BSA) wash and blocked with 20% horse serum for 1 hour. They were rinsed again in the TBS/BSA solution before staining with the Avidin-Biotin Vector kit for 15 min. The samples were stained with the biotinylated primary antibodies overnight (CD8-biotin for the CD8 T cells and CD11-biotin for the MCs, 1:50 dilution). Samples were washed with the TBS/BSA and stained with the Avidin-Alexa Fluor 633 fluorophore (1:200) for one hour, washed with TBS/BSA and TBS alone. Slides were mounted with antifade mounting media with 4',6-diamidino-2-phenylindole (DAPI) and visualized on the Zeiss LSM 710 (upright) laser-scanning confocal microscope at the Dalhousie CDMI core facility. MRI Acquisition Mice were imaged with MRI using a 3T preclinical Agilent MRI (Varian Inc., Santa Clara, CA, USA). The MRI contained a 21-cm inner diameter gradient coil (200 mT/m; Magnex Scientific, Oxford, UK) and was interfaced with a Varian DD Console (Varian Inc.). Mice were anesthetized and secured in an animal holder immediately before imaging. Temperature and respiration rates were monitored throughout imaging using a rectal probe and breathing monitor. Anatomical images were acquired using a balanced steady-state free precession (bSSFP) pulse sequence. The bSSFP parameters were set at a repetition time of 8ms, echo time of 4 ms, and a flip angle of 30°. The field of view (FOV), 256 x 170 x 170 matrix, was set at an isotropic resolution of 200 µm and was centred over the torso. The TurboSPI parameters were set with a FOV of 32 x 32 x 32 mm and a slab size of 30 mm 12,22 . The repetition time (TR) was 250 ms, the echo train length (ETL) was 8, and the echo spacing (ESP) was 10 ms. Mice were scanned on days 42, 49, and 56 post-implant, approximately 24h after SPIO-labeled cell injections. Imaging Analysis MRI Images were loaded in VivoQuant (InVicro, Ma, US), and regions of interest (ROI) were drawn on the tumor and lymph nodes using the bSSFP image for each imaging time point as in 12 . Cell density in tumors and lymph nodes was obtained by extracting frequency histograms of the R 2 * signal from the ROIs, and converted from R 2 * values per voxel to cell density per mm 3 using the calibration curve for either CTLs or MCs. Each voxel was then summed over the ROI, resulting in total cell density for each tumor and lymph node ROI (same methods as 12,18 ). Data were then imported into GraphPad Prism 8 (San Diego, CA, USA) for statistical analysis. Statistical Analysis To compare the results between the two treatment groups, we used a student's t -test with Bonferroni correction for multiple comparisons, and to evaluate group-level results across both time and treatment groups, we used a two-way ANOVA. Significance is designated as * p \(\:\le\:\) 0.05, ** p \(\:\le\:\) 0.01, *** p \(\:\le\:\) 0.001. Results Imaging demonstrates the success of triple combination therapy The average tumor volume of mice treated with DPX-Survivac, anti-PD-1, and CPA was smaller than the average tumor volume of untreated mice at all three imaging timepoints (Fig. 2 ). There was a statistically significant difference in tumor volume between groups on day 56 post-implant ( p < 0.01, Fig. 2 B). Additionally, the tumor growth percentage was calculated for all mice imaged across the three-time points (Fig. 2 C). Percent tumor growth between days 42–56 post-implant in untreated mice was significantly increased compared to the treated group (335% increase vs 93% increase, ** p < 0.01, Student's t- test, Fig. 2 C). Taken together, these results suggested that a combination therapy of DPX-Survivac, anti-PD-1, and CPA slows the growth of primary tumors, resulting in reduced tumor volumes. However, there was no significant change in survival due to treatment (Supp Fig. 1 ). In addition to quantifying primary tumor volumes, the volumes of both inguinal lymph nodes were quantified at each imaging time point (Fig. 3 A, quantitative values in Supp Fig. 2 ). The average volume of the DPX-draining LN (i.e., the left inguinal lymph node) trended higher than the tumor-draining LN (i.e., the right inguinal lymph node) within the treated group. We then calculated the volumetric ratio between DPX-draining and tumor-draining lymph nodes by dividing the volume of the DPX-draining LN by the tumor-draining LN. Therefore, a ratio > 1 indicated increased swelling in the DPX-draining LN relative to the tumor-draining LN. At each imaging time point, the average volumetric ratio was > 1 in the treated group, whereas it was < 1 in the untreated group (Fig. 3 B). Using a two-way ANOVA, we found there were significant group-level differences due to treatment (p < 0.0001). As DPX-draining lymph nodes were generally more swollen than the tumor-draining LNs, we then assessed whether increased swelling correlated with tumor volume. The lymph node volumetric ratio of the treated animals negatively correlated with tumor volume, whereas the opposite was observed in the untreated group (Fig. 3 C). We found there was a significant difference (p < 0.05) between the slope of the untreated and treated mice. These results suggested that DPX-Survivac induces lymph node swelling in the DPX-draining lymph node, which correlates with a smaller tumor volume. Cell culture phenotyping and SPIO labelling Flow cytometry was used on samples not tagged with SPIO to assess the purity of cell cultures. Cultures of marrow-derived myeloid cells from treated mice appeared to have a higher proportion of macrophages and fewer monocytes and dendritic cells. Still, they were not significantly different than those in untreated mice (data not shown). Furthermore, the percentage of myeloid cells cultured from treated mice appeared to be higher than that of untreated mice expressing MHCII. There were no evident differences between treated and untreated cultures in the CD11b/CD11c subsets of macrophages, monocytes, and dendritic cells. Cultured CTLs were very pure: >90% of CD3 + cells were CD8 + (Supplementary Fig. 3A). There were no differences in the expression of PD-1, CTLA-4, or TIM3 between CTLs cultured from treated and untreated mice (Supp Fig. 3 B). However, CTLs cultured from untreated mice appeared to express higher levels of Ki67 (Supp Fig. 3 B). Uptake of SPIO by labeled CTLs and MCs was evaluated using a Prussian Blue assay measured on a spectrophotometer, validated against a known concentration curve 21 . CTLs were found to have approximately 4pg of iron/cell, and MCs were found to have approximately 7pg of iron/cell. Viability for all labeled cells was > 90%. Semi-quantitative analysis of SPIO-labeled cells in the tumor and inguinal lymph nodes To quantify the recruitment of SPIO-labeled MCs and CTLs, R 2 * maps were generated with the TurboSPI MRI pulse sequence and overlaid onto anatomical MR images. R 2 * values within the specific ROI were converted into the number of SPIO-labeled cells per mm 3 of ROI using R 2 * relaxivity curves generated from cell phantoms made with SPIO-labeled MCs and CTLs (same isolation and labeling procedures as in vivo studies). Treatment with DPX-Survivac, low dose CPA, and anti-PD-1 significantly increased MC recruitment to the tumor, as measured by MCs per mm 3 of tumor (p = 0.0211, two-way ANOVA; Fig. 4 A). Tumor MCs were recruited to the tumors of treated mice at a density of approximately 750 cells/mm 3 (day 42), 590 cells/mm 3 (day 49), and 540 cells/mm 3 (day 56). MCs were recruited to the tumors of untreated mice at a density of 185 cells/mm 3 (day 42), 40 cells/mm 3 (day 49), and 55 cells/mm 3 (day 56). Given that lymph nodes are immune cell infiltration and priming sites, it was hypothesized that the combination therapy might increase the recruitment of MCs to lymph nodes. However, treatment did not significantly impact MC recruitment to the DPX-draining lymph node (i.e., left LLN, p = 0.1598; two-way ANOVA) (Fig. 5 A). In addition, treatment did not substantially affect MC recruitment to the tumor-draining lymph node (p = 0.8903; two-way ANOVA; Fig. 5 B). CTL recruitment was also quantified using the same methods. Treatment with DPX-Survivac, anti-PD-1 and low dose CPA significantly increased CTL recruitment to the tumor, as measured by CTLs per mm 3 (p = 0.0155, two-way ANOVA; Fig. 4 B). Average CTL recruitment to the tumors of treated mice was 2500 cells/mm 3 (day 42), 2300 cells/mm 3 (day 49), and 2300 cells/mm 3 (day 56; Fig. 4 ). Average CTL recruitment to tumors in the untreated group was 2200 cells/mm 3 (day 42), 450 cells/mm 3 (day 49), and 83 cells/mm 3 (day 56; Fig. 4 B). In untreated mice, only 1 of 5 mice had CTLs present in the tumors on days 49 and 56. These results suggest that treatment with DPX-Survivac, low-dose CPA, and anti-PD-1 increases the recruitment of CTLs to the tumors of treated mice. We found that treatment did not significantly impact CTL recruitment to the DPX-draining lymph node (p = 0.2405, two-way ANOVA; Fig. 5 C) or the tumor-draining lymph (p = 0.9820, two-way ANOVA; Fig. 5 D). However, it did appear that the recruitment of CTLs to the DPX-draining lymph node decreased in untreated mice as the study progressed, whereas it remained relatively consistent in treated mice. This resulted in far more CTLs in the DPX-draining lymph node in treated mice at day 56 compared to untreated mice. There was a single outlier mouse in the untreated group, but the other 4 of 5 untreated mice had no detectable CTLs in the DPX-draining lymph node at day 56. TurboSPI validation IHC was used to validate that the injected cells labeled with SPIO were the cells of interest in the terminal tissues. Lymph nodes were utilized as the stains were more homogeneous. Supp Fig. 4 shows lymph nodes from mice that received either CD8 + T cells (top) or CD11 + myeloid cells (bottom). Immune cells were stained with Alexa Fluor 633 Avidin and biotinylated anti-CD8 (top) or biotinylated anti-CD11 (bottom). The SPIO used had a rhodamine B tag also visible with IHC. All of the rhodamine B positive cells are also CD8 or CD11 positive in the lymph nodes (as indicated by the green arrows), indicating that the cells visualized with MRI are the cells of interest. Characterization of Ascites The total proportion of immune cells in the ascitic fluid was determined using the CD45 marker (common leukocyte antigen). On average, treated mice had a higher proportion of leukocytes than untreated mice, albeit not statistically significant (p = 0.1429; unpaired t -test, Fig. 6 A). The cellular composition of ascitic fluid collected from treated mice had an average of 97.16% CD45 + cells, while the untreated mice had an average of only 83.3% CD45 + cells (Fig. 6 A). Within the CD45 + population in ascites from treated mice, an average of 22.02% expressed F4/80, 27.90% expressed CD11c, and 16.76% expressed CD3ε (Fig. 6 B). In the untreated ascites samples, 34.89% of the CD45 + population expressed F4/80, 32.88% expressed CD11c, and 17.39% expressed CD3ε (Fig. 6 B). There were no statistical differences in the percentage of F4/80 (p = 0.1712), CD11c (p = 0.6212), and CD3ε (p = 0.9310) populations between treated and untreated samples (unpaired t -test, Fig. 6 B). The CD3ε marker encompasses all T cell populations; therefore, we also examined the proportions of CD4 and CD8 cells within the CD3ε population (Fig. 6 D). There were more CD4 + T cells than CD8 + T cells in the ascitic fluid collected from treated and untreated mice (Fig. 6 D). Interestingly, a population of double-positive T cells also expressed both CD4 and CD8. In the treated ascites samples, an average of 44.37% of the CD3ε population was CD4 + CD8+ (Fig. 6 D). Only 15.42% of the CD3ε population in the untreated samples was CD4 + CD8+ (Fig. 6 D). There was no significant difference between the average percentage of CD4 + CD8 + in the treated and untreated samples (p = 0.1250; unpaired t -test) (Fig. 6 C). Together, these results suggest that treatment with DPX-Survivac, low dose CPA, and anti-PD-1 increases the presence of CD45 + immune cells in ascitic fluid and may increase CD4+/CD8 + T cells but does not have an impact on the presence of other specific immune cell populations. Tumor-infiltrating lymphocyte assay Tumor-infiltrating lymphocytes were assessed using flow cytometry (Fig. 7 ). The tumors of mice treated with DPX-Survivac/CPA and anti-PD-1 appeared to have fewer CD45 + immune cells, CD3 + CD4 + T cells, CD19 + B cells, and granulocytic myeloid-derived suppressor cells (MDSCs) (CD11b + Ly6G + Ly6c neg ; Fig. 7 A, B, D, E) than tumors from untreated mice. Furthermore, there were no significant differences in the percentage of CD3 + CD8 + T cells, transitional MDSCs (CD11b + Ly6G + Ly6c high ), monocytic MDSCs (CD11b + Ly6G neg Ly6c high ), classical or myeloid-derived dendritic cells, macrophages, and F4/80-expressing cells (Fig. 7 C, E, F, G) between untreated and treated mice. However, there was a trend of increased CD3 + CD8 + T cells in treated mice. Overall, the ovarian tumors had few monocytic and transitional MDSCs and very few macrophages or myeloid-derived DCs (Fig. 7 E, F). Discussion The primary objectives of this study were to 1) use molecular imaging to determine the effect of combination therapy with DPX-Survivac, anti-PD-1, and low-dose CPA on tumor progression and 2) use MRI to measure changes in immune cell recruitment to tumors and inguinal lymph nodes as a result of treatment. We also evaluated the cellular composition of ascites and tumor infiltrates at the study endpoint in a preclinical ovarian cancer model. Our primary findings indicated that the combination therapy studied in the project slows ovarian tumor growth, and increases the recruitment of CTLs to tumors and MCs to the DPX-draining lymph node. Late-stage diagnoses account for the high mortality of patients with epithelial ovarian cancer: the 5-year survival rate is less than 50% 23 , with advanced cases having a survival rate of only ~ 30% 4 . Finding effective therapies is imperative for increasing the survival rate of this disease. However, it is challenging to study orthotopic ovarian cancer models in any depth without using imaging to look at the primary tumors located in and around the ovaries. We therefore used anatomical MRI to evaluate the effect of combination therapy with DPX-Survivac, low-dose CPA, and anti-PD-1 on ovarian cancer survival and tumor growth. Our results demonstrated that combination therapy increased the survival of mice by 7% (Supp Fig. 2 ). While there was no statistical significance between the survival of treated and untreated mice, this is likely due to the duration of the study. One limitation of assessing survival was that all remaining mice were euthanized on day 60 post-implant for tissue collection. Future studies will extend to further time points to assess actual survival more accurately. Compared to the untreated group, treated mice had significantly smaller tumors by the end of the study (Fig. 2 ). These results suggested that combination therapy with DPX-Survivac, low-dose CPA, and anti-PD-1 slowed the growth of established ovarian tumors. Due to the latent nature of ovarian cancer, patients are typically diagnosed at stages when the primary tumor has already been established. Therefore, novel treatments must be effective with more advanced cancer models. This clinically relevant treatment schedule replicates a current clinical trial (NCT03836352, clinical trial registration date: 2019-02-07, clinicaltrial.gov) and builds upon previous use in other cancer models 11 , 24 . In addition, slowing the growth of primary tumors may help prolong survival and enhance the efficacy of other treatments, such as debulking surgeries. While this combination shows promise in improving survival and slowing tumor growth, there remain a number of questions about the mechanisms of action of the therapeutics. A primary objective of this study was to evaluate whether MRI immune cell tracking could be used to monitor and quantify the recruitment of adoptively transferred SPIO-labeled immune cells at two different timepoints. While cell tracking of these two cell types has been done previously in a subcutaneous tumor model 12 , this is the first time it has been done in a more clinically relevant orthotopic ovarian model, which can be more difficult due to increased motion artifacts and increased fat in the lower abdomen. These orthotopic models of ovarian cancer, particularly when used in combination with humanized mice with intact immune systems and clinically relevant treatment regimens, are critical for correctly understanding immunological responses. Using MRI, we found that combination therapy increased the recruitment of both SPIO-labeled MCs and SPIO-labeled CTLs to the tumor (Fig. 4 ). For SPIO-labeled CTLs, this increase is likely linked to the slower tumor growth seen in treated mice. Although flow cytometry results did not find a significant difference, likely due to a small N, there was a trend of higher levels of CD8 + T cells in treated mice. Interestingly, untreated mice saw a significant drop in the cellular density of recruited CTLs throughout the study. This is potentially due to an increasingly immunosuppressive environment. As indicated in Fig. 7 , untreated mice had increased numbers of granulocytic myeloid cells, which can be myeloid-derived suppressor cells, a highly suppressive immune cell type. Given the broad range of cells that can be classified as MCs, it is not clear if the increases seen with SPIO-labeled cells represent a more inflammatory or suppressive cell type. Future studies would benefit from further sorting of MCs prior to labeling with SPIO and implanting them to understand this phenotype better. We also quantified cell recruitment to the tumor-draining and DPX-draining inguinal lymph nodes, which are sites of immune cell infiltration and priming. Although we found no significant differences in cell recruitment, we did notice that CTL recruitment to the draining lymph node in untreated mice decreased throughout the study. In contrast, treated mice had similar cell densities throughout. Though this difference was not statistically significant, this may have been due to our limited sample size and an outlier point at day 56 for untreated mice (4/5 untreated mice had no CTLs in the right, or DPX-draining, lymph node at day 56). This data suggested that the combination therapy may have enhanced the recruitment of immune cells, such as CTLs, to the DPX-draining lymph node for priming adaptive immune cells against antigens. The increase of CTLs to the DPX draining lymph node in treated mice may also be linked to changes in the volumetric ratio of the DPX-draining: tumor-draining inguinal lymph node between treated and untreated mice. The average volumetric ratio of the DPX-draining:tumor-draining lymph nodes was consistently increased in mice treated with the combination therapy. In fact, few untreated mice had a DPX-draining:tumor-draining volumetric ratio greater than 1 (Fig. 3 ). These results suggested that DPX-Survivac increased swelling in the DPX-draining lymph node relative to the contralateral lymph node, as the other individual therapies in the combination are systemic. Given that lymph nodes are sites of immune cell priming, the observed increase in size, along with CTL cell recruitment to the DPX-draining lymph node, suggested that DPX-Survivac may increase lymph node size due to immune cell infiltration. Our results were similar to those found in a study 25 , 26 that described the inguinal lymph node volumetric ratio as a potential biomarker for successful therapy with DPX. Using a C3 tumor model, they 25 , 26 found that treatment with a DPX peptide-based therapy increased the volumetric ratio of the DPX-draining lymph node to the tumor-draining lymph node. Furthermore, this volume increase was associated with better outcomes, namely, decreased tumor growth. Weir et al. 15,17 assessed the tumor infiltration of antigen-specific (R9F-specific) CTLs in the C3 cervical tumor model using a similar combination therapy: anti-PD-1, DPX-R9F, and low-dose CPA. The study found that combination therapy with all three agents enhanced the infiltration of R9F-specific CTLs to tumors compared to treatment with DPX-R9F and low-dose CPA alone, using flow cytometry. Similarly, we found that the tumor infiltration of CTLs was increased when mice were treated with the three therapies combined. Upon analysis of the ascitic fluid, mice treated with the combination therapy had consistently higher percentages of CD45 + cells (leukocytes) (Fig. 6 ). The remaining cells were believed to be free-floating tumor cells. In ovarian cancer, malignant ascites contribute to transcoelomic metastasis (metastasis through the peritoneal cavity) by providing primary tumor cells with a medium for dissemination 4 , 27 , 28 . Therefore, the increased percentage of CD45 + cells observed in ascites from treated mice indicates fewer tumor cells in the ascitic fluid. Conversely, the increased presence of tumor cells in the ascites of untreated mice suggests that these mice are more likely to have metastases. Because the presence of tumor cells within ascites is associated with poor prognosis, this could serve as a potential biomarker for the efficacy of this combination therapy to reduce tumor burdens. Future studies would benefit from imaging at longer timepoints and monitoring regions like the lungs for potential metastases. We also noted the presence of a CD4 + CD8+ (double-positive; DP) T cell population in the ascitic fluid of both treated and untreated mice (Fig. 6 ). While untreated mice had consistently decreased proportions of DP T cells, some mice in the treated group had an increased percentage of DP T cells (Fig. 6 ). DP T cells have been reported in healthy and diseased individuals 29 . The roles of this unconventional T cell population are not fully understood, with conflicting reports describing cytotoxic or suppressive roles for these cells 30 . Several studies have reported the role of DP T cells in mediating the tumor response and favouring immune escape in many cancer types, such as urological cancer, renal carcinoma, metastatic colorectal cancer, melanomas, and breast cancer lesions 29 , 31 – 33 . Menard et al. 32 have observed in renal carcinoma patients that DP T cells express high levels of PD-1, which will make them a suitable target for checkpoint inhibitor therapies. This would explain the favorable prognosis in mice treated with combination therapy by restricting the immunosuppressive role or enhancing the cytotoxicity of this cell population. Conclusion Collectively, our results demonstrated that molecular imaging allowed the study of a combination therapy with DPX-Survivac, anti-PD-1, and low-dose CPA in epithelial ovarian cancer, demonstrating that the combination decreases the tumor burden of mice. As evidenced by cell tracking, the combination therapy may exert its effects by inhibiting tumor-mediated immunosuppression and subsequently increasing the infiltration of cytotoxic T lymphocytes into the primary tumor. Additionally, the increased percentage of immune cells shown in the ascitic fluid may indicate the combination therapy's efficacy against ovarian tumors. This study demonstrates that immune cell tracking can be used to probe the longitudinal mechanics of SPIO-labeled cells in orthotopic cancer models. In future work, we plan on investigating more immune cell subtypes, particularly subtypes of MLCs such as monocytes, granulocytes, and myeloid-derived suppressor cells, in single immunotherapies and at more timepoints, to understand individual therapeutic mechanisms of action better. Abbreviations Magnetic resonance imaging (MRI), Cyclophosphamide (CPA), programmed cell death-1 (PD-1), T cell receptor (TCR), PD-1 ligand (PD-L1), monoclonal antibodies (mAb), human papillomavirus (HPV), regions of interest (ROI), superparamagnetic iron oxide (SPIO), natural killer (NK), HLA-A2.I-HLA-DRI-transgenic (HHD), dimethyl sulfoxide (DMSO), DMEM (Dulbecco's Modification of Eagle's Medium), Phosphate buffered solution (PBS), Detailed clinical examinations (DCE), Mouse ovarian surface epithelial (MOSE), Cytotoxic t lymphocyte (CTL), Myeloid cells (MC), Hank’s balanced salt solution (HBSS), Red blood cell lysis (RBC), Paraformaldehyde (PFA), Optical cutting temperature (OCT), Tris buffered saline (TBS), Bovine serum albumin (BSA), Balanced steady state free precession (bSSFP), Repetition time (TR), Echo train length (ETL), Echo spacing (ESP), Field of View (FOV), Lymph node (LN), Analysis of variance (ANOVA), myeloid-derived suppressor cells (MDSCs) Declarations Financial Support KB and MS would like to acknowledge support from the BHCRI New Investigator Program. Conflict of Interest Disclosure: At the time of this study MLT, JTG, BD, AM, KBo, AW, GW, AMJ, OH and MS were employees of IMV Inc. KDB had a research contract with IMV on studying DPX-Svv in ovarian cancer. Competing Interests At the time of this study MLT, JTG, BD, AM, KBo, AW, GW, AMJ, OH and MS were employees of IMV Inc. KDB had a research contract with IMV on studying DPX-Svv in ovarian cancer. Funding KB and MM would like to acknowledge funding from the Beatrice Hunter Cancer Research Institute (BHCRI) via a New Investigator Award, and KB would like to acknowledge funding from an NSERC Discovery Grant. Author Contribution Experiment planning and oversight was done by KDB, MS, GW, OH and AM-J. Data acquisition was done by CSS, MLT, HW, VG, AN, CD, BDa and AV-L. Data analysis was done by CSS, HW, JTG, BDi and KDB. Animal procedures were done by CD, AM, AW and KB. Cells were provided by BV and modified by OH. Manuscript writing was done by JG, CSS, BD and KDB. Manuscript was reviewed by all authors. Acknowledgement Flow Cytometry acquisition, Immunohistochemistry preparation, and microscopy were done at Dalhousie University Core Facilities. KB and MM would like to acknowledge funding from the Beatrice Hunter Cancer Research Institute (BHCRI) via a New Investigator Award, and KB would like to acknowledge funding from an NSERC Discovery Grant. Data Availability Data will be made available upon request to the corresponding author KDB ( [ [email protected] ](mailto: [email protected] ) ). References Vetter MH, Hays JL. Use of Targeted Therapeutics in Epithelial Ovarian Cancer: A Review of Current Literature and Future Directions. Clin Ther . 2018;40(3):361–371. doi: 10.1016/j.clinthera.2018.01.012 Lawson-Michod KA, Watt MH, Grieshober L, et al. Pathways to ovarian cancer diagnosis: a qualitative study. BMC Womens Health . 2022;22(1):430. doi: 10.1186/s12905-022-02016-1 Penet MF, Krishnamachary B, Wildes FB, et al. Ascites Volumes and the Ovarian Cancer Microenvironment. Front Oncol . 2018;8. doi: 10.3389/fonc.2018.00595 Ahmed N, Stenvers KL. Getting to Know Ovarian Cancer Ascites: Opportunities for Targeted Therapy-Based Translational Research. Front Oncol . 2013;3. doi: 10.3389/fonc.2013.00256 Suarez-Almazor ME, Kim ST, Abdel‐Wahab N, Diab A. Review: Immune‐Related Adverse Events With Use of Checkpoint Inhibitors for Immunotherapy of Cancer. Arthritis & Rheumatology . 2017;69(4):687–699. doi: 10.1002/art.40043 Tang Q, Chen Y, Li X, et al. The role of PD-1/PD-L1 and application of immune-checkpoint inhibitors in human cancers. Front Immunol . 2022;13:964442. doi: 10.3389/fimmu.2022.964442 Weiss L, Huemer F, Mlineritsch B, Greil R. Immune checkpoint blockade in ovarian cancer. memo - Magazine of European Medical Oncology . 2016;9(2):82–84. doi: 10.1007/s12254-016-0267-3 Li X, Hu W, Zheng X, et al. Emerging immune checkpoints for cancer therapy. Acta Oncol (Madr) . 2015;54(10):1706–1713. doi: 10.3109/0284186X.2015.1071918 Seidel JA, Otsuka A, Kabashima K. Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations. Front Oncol . 2018;8. doi: 10.3389/fonc.2018.00086 Nowicki TS, Hu-Lieskovan S, Ribas A. Mechanisms of Resistance to PD-1 and PD-L1 Blockade. The Cancer Journal . 2018;24(1):47–53. doi: 10.1097/PPO.0000000000000303 Berinstein NL, Karkada M, Oza AM, et al. Survivin-targeted immunotherapy drives robust polyfunctional T cell generation and differentiation in advanced ovarian cancer patients. Oncoimmunology . 2015;4(8):e1026529. doi: 10.1080/2162402X.2015.1026529 Tremblay ML, O’Brien-Moran Z, Rioux JA, et al. Quantitative MRI cell tracking of immune cell recruitment to tumors and draining lymph nodes in response to anti-PD-1 and a DPX-based immunotherapy. Oncoimmunology . 2020;9(1). doi: 10.1080/2162402X.2020.1851539 Dorigo O, Fiset S, MacDonald LD, et al. DPX-Survivac, a novel T-cell immunotherapy, to induce robust T-cell responses in advanced ovarian cancer. Journal of Clinical Oncology . 2020;38(5_suppl):6–6. doi: 10.1200/JCO.2020.38.5_suppl.6 Weir G, Hrytsenko O, Quinton T, et al. Abstract 4903: Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells. Cancer Res . 2016;76(14_Supplement):4903–4903. doi: 10.1158/1538-7445.AM2016-4903 Weir GM, Hrytsenko O, Stanford MM, et al. Metronomic cyclophosphamide enhances HPV16E7 peptide vaccine induced antigen-specific and cytotoxic T-cell mediated antitumor immune response. Oncoimmunology . 2014;3(8):e953407. doi: 10.4161/21624011.2014.953407 Scurr M, Pembroke T, Bloom A, et al. Low-Dose Cyclophosphamide Induces Antitumor T-Cell Responses, which Associate with Survival in Metastatic Colorectal Cancer. Clinical Cancer Research . 2017;23(22):6771–6780. doi: 10.1158/1078-0432.CCR-17-0895 Weir GM, Hrytsenko O, Quinton T, Berinstein NL, Stanford MM, Mansour M. Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide. J Immunother Cancer . 2016;4(1):68. doi: 10.1186/s40425-016-0169-2 Tremblay ML, Davis C, Bowen C V., et al. Using MRI cell tracking to monitor immune cell recruitment in response to a peptide-based cancer vaccine. Magn Reson Med . 2018;80(1):304–316. doi: 10.1002/mrm.27018 D Brewer K, Stanley O, Davis C, et al. Tracking SPIO-Labeled Effector & Regulatory Cell Migration with MRI. In: Proceedings of the 21st Annual Meeting of the International Society of the Magnetic Resonance in Medicine (ISMRM) .; 2013. Oude Engberink RD, Blezer ELA, Hoff EI, et al. MRI of Monocyte Infiltration in an Animal Model of Neuroinflammation Using SPIO-Labeled Monocytes or Free USPIO. Journal of Cerebral Blood Flow & Metabolism . 2008;28(4):841–851. doi: 10.1038/sj.jcbfm.9600580 Nuschke A, Sobey-Skelton C, Dawod B, et al. Use of Magnetotactic Bacteria as an MRI Contrast Agent for In Vivo Tracking of Adoptively Transferred Immune Cells. Mol Imaging Biol . 2023;25(5):844–856. doi: 10.1007/S11307-023-01849-Y Rioux JA, Brewer KD, Beyea SD, Bowen C V. Quantification of superparamagnetic iron oxide with large dynamic range using TurboSPI. Journal of Magnetic Resonance . 2012;216:152–160. doi: 10.1016/j.jmr.2012.01.017 Wu J, Sun H, Yang L, et al. Improved survival in ovarian cancer, with widening survival gaps of races and socioeconomic status: a period analysis, 1983–2012. J Cancer . 2018;9(19):3548–3556. doi: 10.7150/jca.26300 Berinstein NL, Karkada M, Morse MA, et al. First-in-man application of a novel therapeutic cancer vaccine formulation with the capacity to induce multi-functional T cell responses in ovarian, breast and prostate cancer patients. J Transl Med . 2012;10(1):156. doi: 10.1186/1479-5876-10-156 Brewer KD, Lake K, Pelot N, et al. Clearance of depot vaccine SPIO-labeled antigen and substrate visualized using MRI. Vaccine . 2014;32(51):6956–6962. doi: 10.1016/j.vaccine.2014.10.058 Brewer KD, DeBay DR, Dude I, et al. Using lymph node swelling as a potential biomarker for successful vaccination. Oncotarget . 2016;7(24):35655–35669. doi: 10.18632/oncotarget.9580 Latifi A, Luwor RB, Bilandzic M, et al. Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors. Hawkins SM, ed. PLoS One . 2012;7(10):e46858. doi: 10.1371/journal.pone.0046858 Kipps E, Tan DSP, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer . 2013;13(4):273–282. doi: 10.1038/nrc3432 Desfrançois J, Moreau-Aubry A, Vignard V, et al. Double Positive CD4CD8 αβ T Cells: A New Tumor-Reactive Population in Human Melanomas. Lowenstein PR, ed. PLoS One . 2010;5(1):e8437. doi: 10.1371/journal.pone.0008437 Overgaard NH, Jung JW, Steptoe RJ, Wells JW. CD4+/CD8 + double-positive T cells: more than just a developmental stage? J Leukoc Biol . 2015;97(1):31–38. doi: 10.1189/jlb.1RU0814-382 Bohner P, Chevalier MF, Cesson V, et al. Double Positive CD4 + CD8 + T Cells Are Enriched in Urological Cancers and Favor T Helper-2 Polarization. Front Immunol . 2019;10. doi: 10.3389/fimmu.2019.00622 Menard LC, Fischer P, Kakrecha B, et al. Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4 + CD8 + T Cells With Expression of Exhaustion Markers. Front Immunol . 2018;9. doi: 10.3389/fimmu.2018.02728 Sarrabayrouse G, Corvaisier M, Ouisse LH, et al. Tumor-reactive CD4 + CD8αβ + CD103 + αβT cells: A prevalent tumor-reactive T-cell subset in metastatic colorectal cancers. Int J Cancer . 2011;128(12):2923–2932. doi: 10.1002/ijc.25640 Additional Declarations Competing interest reported. At the time of this study MLT, JTG, BD, AM, KBo, AW, GW, AMJ, OH and MS were employees of IMV Inc. KDB had a research contract with IMV on studying DPX-Svv in ovarian cancer. Supplementary Files SupplementaryMaterialfinal.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 Nov, 2025 Reviews received at journal 04 Nov, 2025 Reviews received at journal 02 Nov, 2025 Reviews received at journal 02 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviewers agreed at journal 17 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers agreed at journal 13 Oct, 2025 Reviewers agreed at journal 12 Oct, 2025 Reviewers agreed at journal 10 Oct, 2025 Reviewers invited by journal 10 Oct, 2025 Editor assigned by journal 10 Oct, 2025 Submission checks completed at journal 07 Oct, 2025 First submitted to journal 25 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7714918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":533561285,"identity":"126469f2-ac51-416d-9aa0-1d5710f5ac93","order_by":0,"name":"Jessica T. Gosse","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"T.","lastName":"Gosse","suffix":""},{"id":533561286,"identity":"99718643-05ce-480f-9a6d-9c79972a4415","order_by":1,"name":"Caitrin Sobey Skelton","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Caitrin","middleName":"Sobey","lastName":"Skelton","suffix":""},{"id":533561287,"identity":"3e4f7fe0-2e59-4dde-a427-032f73f6d550","order_by":2,"name":"Marie-Laurence Tremblay","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Marie-Laurence","middleName":"","lastName":"Tremblay","suffix":""},{"id":533561288,"identity":"1fe27e71-8ef2-4324-86d2-e0201551b5ff","order_by":3,"name":"Hailey Wyatt","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Hailey","middleName":"","lastName":"Wyatt","suffix":""},{"id":533561289,"identity":"ca8b32bd-b470-42ec-b2c8-11750965d9d5","order_by":4,"name":"Victoria Gonzalez","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"","lastName":"Gonzalez","suffix":""},{"id":533561290,"identity":"2766b25f-8269-42d8-b41a-4b784642d503","order_by":5,"name":"Bassel Dawod","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Bassel","middleName":"","lastName":"Dawod","suffix":""},{"id":533561291,"identity":"1ce37a5a-aad2-4b48-905e-18ab7c1aa307","order_by":6,"name":"Andrea Nuschke","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Nuschke","suffix":""},{"id":533561292,"identity":"cbabf3ae-8333-427d-a68f-46432a23c253","order_by":7,"name":"Christa Davis","email":"","orcid":"","institution":"IWK Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Christa","middleName":"","lastName":"Davis","suffix":""},{"id":533561293,"identity":"d2b9ea7d-8abf-473e-a1e2-3eedbaa9bf0f","order_by":8,"name":"Brennan Dirk","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Brennan","middleName":"","lastName":"Dirk","suffix":""},{"id":533561294,"identity":"bdaae459-b12e-46b6-8e11-57dc3b0fe473","order_by":9,"name":"Ava Vila-Leahey","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Ava","middleName":"","lastName":"Vila-Leahey","suffix":""},{"id":533561295,"identity":"e3d9ac62-97e1-43a5-9c94-65e454f0b8c3","order_by":10,"name":"Alecia Mackay","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Alecia","middleName":"","lastName":"Mackay","suffix":""},{"id":533561296,"identity":"98849ed5-223b-4099-9353-990ae61a1835","order_by":11,"name":"Kim Bobbitt","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Kim","middleName":"","lastName":"Bobbitt","suffix":""},{"id":533561297,"identity":"55d67769-e802-4135-842f-00c32c347525","order_by":12,"name":"Andrea West","email":"","orcid":"","institution":"Dalhousie University","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"West","suffix":""},{"id":533561298,"identity":"910fca75-3564-47e0-8b28-c44b5a783db0","order_by":13,"name":"Barbara Vanderhyden","email":"","orcid":"","institution":"Ottawa Hospital Research Institution","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Vanderhyden","suffix":""},{"id":533561299,"identity":"abb659c7-04c1-45d1-9bb8-eb91be6d3a24","order_by":14,"name":"Genevieve Weir","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Genevieve","middleName":"","lastName":"Weir","suffix":""},{"id":533561300,"identity":"d5b77a7d-8299-47fa-a5c5-f32dde105503","order_by":15,"name":"Alexandra Merkx-Jacques","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Merkx-Jacques","suffix":""},{"id":533561301,"identity":"e1dd25fb-d049-4edb-bd2d-549508fae090","order_by":16,"name":"Marianne Stanford","email":"","orcid":"","institution":"Dalhousie University","correspondingAuthor":false,"prefix":"","firstName":"Marianne","middleName":"","lastName":"Stanford","suffix":""},{"id":533561302,"identity":"d9b7a73d-6af3-4065-955a-db554d24cfdc","order_by":17,"name":"Olga Hrytsenko","email":"","orcid":"","institution":"IMV Inc","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"","lastName":"Hrytsenko","suffix":""},{"id":533561303,"identity":"634f48ca-32ba-4867-8646-1c717675c193","order_by":18,"name":"Kimberly D. Brewer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACAwiVIMdwgLmBNC3GDAcYSdSS2EC0FnPpw8ckPu5JS+87frCB4UcNEVos+9LSJGc8y8mdeSaxgbHnGDEOO8NjdpvnQEXuhgOJDcwMbERp4f92+8+BinSD8w+BWv4RZwvbbYYDOQkGN4C2MLYRocWyh838Z8+BNMOZNx42HOztI0KLOQ/zY4MfB5Ll+c4nH3zw4xsRWlDAAVI1jIJRMApGwSjAAQDgWj1N1qbJ2QAAAABJRU5ErkJggg==","orcid":"","institution":"IWK Health Centre","correspondingAuthor":true,"prefix":"","firstName":"Kimberly","middleName":"D.","lastName":"Brewer","suffix":""}],"badges":[],"createdAt":"2025-09-25 16:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7714918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7714918/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94396719,"identity":"74920263-c8fe-4a08-84d6-aa22de4ed41e","added_by":"auto","created_at":"2025-10-27 13:56:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8757420,"visible":true,"origin":"","legend":"","description":"","filename":"NPJOvarianCancerManuscriptv4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/be49b943622fea2c743273c4.docx"},{"id":94397439,"identity":"70abb2e2-bffe-41a8-ad52-5c5fc267917a","added_by":"auto","created_at":"2025-10-27 13:56:40","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18073,"visible":true,"origin":"","legend":"","description":"","filename":"c42945f8dd474e048033f7b4ee1d5617.json","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/2cd50ac74261655f68ae3f6f.json"},{"id":94398064,"identity":"c4fb451f-44dd-4660-8013-80b8314ccca0","added_by":"auto","created_at":"2025-10-27 13:56:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3311874,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/3e75c652b02a067a0dcfca08.docx"},{"id":94398735,"identity":"cc64352f-352f-4c1d-bbf6-2ccc655ac2ed","added_by":"auto","created_at":"2025-10-27 13:57:12","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127279,"visible":true,"origin":"","legend":"","description":"","filename":"c42945f8dd474e048033f7b4ee1d56171enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/e91add2c07f2147a86c7a63e.xml"},{"id":94397277,"identity":"c010a2da-515d-486a-b45d-c167f1a7d47f","added_by":"auto","created_at":"2025-10-27 13:56:35","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5271838,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/46bd3d8630023d5180b5d38d.jpeg"},{"id":94399281,"identity":"23645ad4-7472-40b9-92c4-33837abb647c","added_by":"auto","created_at":"2025-10-27 13:57:26","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":900816,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/d7ddf6100e99b7aece953767.jpeg"},{"id":94397790,"identity":"9b6a1243-372d-4285-8e4e-9036004cc031","added_by":"auto","created_at":"2025-10-27 13:56:48","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":194786,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/9c24214e3258a71a3f54ed7d.jpeg"},{"id":94399452,"identity":"fe94989d-c521-4c3b-bfa4-9f1656b1d652","added_by":"auto","created_at":"2025-10-27 13:57:33","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":430978,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/5ad64ceeda5f0b39e9d3169b.jpeg"},{"id":94397887,"identity":"51951da7-96ac-4d0c-8bf5-5ba908c29bfe","added_by":"auto","created_at":"2025-10-27 13:56:52","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":648748,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/507f7dcd345e78d59bf57eac.jpeg"},{"id":94397136,"identity":"b45394c3-f17e-48a2-815d-3c600238ebb8","added_by":"auto","created_at":"2025-10-27 13:56:29","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":395412,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/0d19acb15c33eb128241637f.jpeg"},{"id":94398227,"identity":"8b18ba96-dda0-4c50-b513-1ecfe5745438","added_by":"auto","created_at":"2025-10-27 13:57:01","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":718928,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/7aae20a0f905590089dc2aac.jpeg"},{"id":94396648,"identity":"f1aee7d9-e7b0-4641-9e5a-fbafce30adb4","added_by":"auto","created_at":"2025-10-27 13:56:08","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48885,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/fd152dc74f843a5b6099259e.png"},{"id":94397542,"identity":"83f07cc7-15f0-4ad9-ab72-8de901f06375","added_by":"auto","created_at":"2025-10-27 13:56:43","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154950,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/b2e4f761025b0fabceea3acc.png"},{"id":94397813,"identity":"009ed072-466a-430e-961c-af624df7d76d","added_by":"auto","created_at":"2025-10-27 13:56:49","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":28571,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/d1eaf3c122f7ef9e1f4f6c4d.png"},{"id":94397301,"identity":"18b187ff-7627-434b-9383-9803fe2772cd","added_by":"auto","created_at":"2025-10-27 13:56:36","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85379,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/795011e08104d8af7cc66532.png"},{"id":94398934,"identity":"4dbffad7-8ee2-4c9b-90ae-55ced9326c7f","added_by":"auto","created_at":"2025-10-27 13:57:16","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125941,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/32fde6e65a9adb7f56eb73ff.png"},{"id":94399111,"identity":"22e82106-a56b-4d1c-8ea3-1e48f70521c1","added_by":"auto","created_at":"2025-10-27 13:57:21","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80757,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/ba23ceedfa1e46d708159d17.png"},{"id":94399142,"identity":"0f8d9bd3-7947-4e50-b90e-db4677896eb8","added_by":"auto","created_at":"2025-10-27 13:57:22","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144880,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/0cb2104330e8b27eb817dd0e.png"},{"id":94398781,"identity":"b0f2c6eb-4eab-479b-8007-29440e824f2c","added_by":"auto","created_at":"2025-10-27 13:57:13","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127208,"visible":true,"origin":"","legend":"","description":"","filename":"c42945f8dd474e048033f7b4ee1d56171structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/4339a2768db8df3870536611.xml"},{"id":94396913,"identity":"73f3b3dc-34e5-4311-a1c4-f46d18a172d6","added_by":"auto","created_at":"2025-10-27 13:56:21","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140020,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/cb08e6217ed2fd267771d1a7.html"},{"id":94396909,"identity":"cdd8ccaa-451d-4167-9a33-10b1c74c8636","added_by":"auto","created_at":"2025-10-27 13:56:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1115289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment and imaging timeline\u003c/strong\u003e. Timeline indicates dates for all treatments and imaging for both donor and recipient mice. MOSE are ovarian cancer cells, CPA is the low-dose cyclophosphamide, MC and CTL are the injected myeloid cells and cytotoxic T lymphocytes, respectively. Donor mice were used exclusively for the isolation of immune cells and therefore did not receive low-dose cyclophosphamide, as internal work revealed that immune cells were too depleted for sufficient yields. Recipient mice received injections of SPIO-labeled immune cells 24 hours prior to MRI scans.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/37826e5ec54f1dc7ed31904d.png"},{"id":94398447,"identity":"61267592-31cd-43b8-8903-7ccfa3e746fa","added_by":"auto","created_at":"2025-10-27 13:57:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3380164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor volumes and growth\u003c/strong\u003e. Tumor volumes were quantified at each time point (days 42, 49, and 56 post-MOSE-implant) by hand-drawing the region of interest. (A) Representative MR images of tumor growth across the three imaging timepoints in a treated and untreated mouse (L: left; R: right). Ovarian tumors are shown outlined in red. (B) Quantified tumor volumes for treated and untreated mice at each time point. (C) Percent tumor growth for mice that were imaged at all three time points. Data were pooled from three separate experiments, shown as average +/- standard error (SEM), n=5-14, Student’s \u003cem\u003et \u003c/em\u003etest, *\u003cem\u003ep \u003c/em\u003e≤ 0.05, **\u003cem\u003ep \u003c/em\u003e≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e ≤ 0.001, ****\u003cem\u003ep ≤ \u003c/em\u003e\u0026nbsp;0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/e99644489dd079bbae2de1ef.png"},{"id":94397585,"identity":"05fd8913-89b3-4ade-b5fa-e53a0159a3f0","added_by":"auto","created_at":"2025-10-27 13:56:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":628467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInguinal lymph node analysis.\u003c/strong\u003e The volumes of the tumor-draining (left) and DPX-draining (right) inguinal lymph nodes were quantified at each time point by hand-drawing the regions of interest. (A) Representative MR images of the inguinal lymph nodes from a treated and untreated mouse on day 42 post MOSE-implant. (B) The volumetric ratio of the DPX-draining to tumor-draining inguinal lymph nodes (i.e., right lymph node volume divided by left lymph node volume). Statistics were done using 2-way ANOVA, n=9-13. Treatment was found to have a significant effect on the volumetric ratio. (C) Lymph node volumetric ratio versus tumor volume for the treated (n = 35) and untreated (n = 30) groups at each time point. Statistics performed using linear regression and the slopes of the lines were found to be significantly different (p\u0026lt;0.05). Average +/- \u0026nbsp;(SEM), *p ≤ 0.05, **p ≤ 0.01. Data were pooled from three separate experiments. L and R indicate left and right, respectively.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/2e42bacf29e6a5038c92a630.png"},{"id":94397754,"identity":"1b1ffd08-3028-478d-bf0e-0b7d726b9fa3","added_by":"auto","created_at":"2025-10-27 13:56:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1079412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of SPIO-labeled cell recruitment to tumors. \u003c/strong\u003eRecruitment of bone marrow-derived myeloid cells (A) and cytotoxic T lymphocytes (B) per mm3 of tumors in both treated and untreated mice. Total cell recruitment was quantified and then divided by tumor volumes for each individual mouse at days 42, 49, and 56 post-MOSE-implant. Data were pooled from three separate experiments, average +/- SEM, n = 3-7, 2way ANOVA, *p ≤ 0.05, **p ≤ 0.01. Treatment was found to have a significant effect on both myeloid cell recruitment (**p \u0026lt; 0.01) and cytotoxic T lymphocyte recruitment (*p \u0026lt; 0.05) to tumors.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/8544b24d92b508deeca00440.png"},{"id":94489218,"identity":"b62c26ba-7232-4dac-a595-95633ae33eab","added_by":"auto","created_at":"2025-10-27 17:03:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1609481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of SPIO-labeled cell recruitment to the inguinal lymph nodes. \u003c/strong\u003eTotal recruitment of myeloid cells (A, B) and cytotoxic T lymphocytes (C, D) to the DPX- and tumor-draining inguinal lymph nodes on days 42, 49, and 56 post-MOSE-implant. Data were pooled from three separate experiments, average +/- SEM, n = 3-8, 2-way ANOVA.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/e7cc51d43021ba95413892fe.png"},{"id":94397604,"identity":"1c0cf3f4-dbb2-4dc2-add4-8175cba98d8c","added_by":"auto","created_at":"2025-10-27 13:56:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1136387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the cellular composition of ascites. \u003c/strong\u003eFlow cytometry was used to assess the cellular composition of ascites fluid collected from both treated and untreated mice. Red blood cells were lysed immediately prior to preparation for flow cytometry. Cells were gated to remove debris, as indicated above. (A) Percentage of leukocytes (CD45+ cells). Treated mice appeared to have consistently higher proportions of CD45+ cells, though not statistically significant. (B) Percentage of F4/80 vs. CD11c populations. (C) Percentage of CD3+ T cells. (D). Percentage of CD4+CD8-, CD4-CD8+, and CD4+CD8+ T cell populations. Average +/- SEM, n = 2-5, statistics by Student t test, *p ≤ 0.05, **p ≤ 0.01.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/dffb946f27679e25327ffecf.png"},{"id":94397615,"identity":"a61d46f0-8a54-4842-bf47-19863ede4002","added_by":"auto","created_at":"2025-10-27 13:56:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1876596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor-infiltrating lymphocyte assay. \u003c/strong\u003eTumors were resected from remaining treated and untreated mice following the last MRI scan. Tumors were digested and processed for flow cytometry to assess the proportions of tumor-infiltrating lymphocytes: (A) CD45+ immune cells. (B) CD4+ T cells. (C) CD8+ T cells. (D) CD19+ B cells. (E) Myeloid-derived suppressor cells and (F-G) dendritic cells and macrophage populations. Average +/- SEM, n = 3 (per treatment group), statistics by Student t test, *p ≤ 0.05, **p ≤ 0.01, ns = not significant, but p \u0026lt; 0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/7e8005bef20984b9876a5c96.png"},{"id":94505721,"identity":"4a4296a0-ee9f-4c2f-bd15-eaa3a6396242","added_by":"auto","created_at":"2025-10-28 16:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11397509,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/2401fb14-e6f1-43e0-9352-e9aea98017d7.pdf"},{"id":94397316,"identity":"6f74247c-b19c-4eca-aa7a-77900356bd4c","added_by":"auto","created_at":"2025-10-27 13:56:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3311874,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7714918/v1/cf7359fe379069de103f239d.docx"}],"financialInterests":"Competing interest reported. At the time of this study MLT, JTG, BD, AM, KBo, AW, GW, AMJ, OH and MS were employees of IMV Inc. KDB had a research contract with IMV on studying DPX-Svv in ovarian cancer.","formattedTitle":"MRI of combination immunotherapy in an epithelial ovarian cancer preclinical model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpithelial ovarian cancer is the most lethal gynecological cancer: the disease is highly aggressive and commonly diagnosed at advanced stages. Most cases are diagnosed at stages III and IV when symptoms have spread to the abdominal area\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, such as ascites and pelvic discomfort. Ascites are often associated with intraperitoneal metastasis caused by cell shedding from the primary tumor\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Late-stage diagnoses are primarily responsible for the global five-year survival rate between 30 and 40%\u003csup\u003e1,4\u003c/sup\u003e. One of the most significant challenges associated with ovarian cancer is that many patients do not respond to current treatment methods. Even if advanced ovarian cancer cases respond to traditional procedures, such as chemotherapy, radiation, and surgery, an estimated 75% of patients will relapse\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn light of these challenges, immunotherapies are a promising class of cancer therapies. Immunotherapies aim to harness and enhance the immune system's natural ability to detect and fight off cancerous cells. Some therapies currently in clinical use include immune checkpoint inhibitors/blockers and peptide-based T cell-activating molecules. Various blockers of checkpoint inhibitors have been FDA-approved and have shown success in clinical trials for skin, lung, and pancreatic cancers\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Immune checkpoint receptors act as \"gatekeepers\" of the immune response, preventing overstimulation of the immune system\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, in tumor microenvironments, this interaction can have a suppressive effect on the activity of T cells, which can result in the exhaustion of their immune function\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Checkpoint blockers work by blocking surface-expressed immune cell checkpoints that regulate the duration and intensity of immune responses.\u003c/p\u003e\u003cp\u003eOne significant checkpoint inhibitor protein is the programmed cell death-1 (PD-1) receptor. PD-1 receptors are upregulated on the surface of T cells following activation and modulate cell function, survival, and proliferation upon ligand binding\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Ligand binding to PD-1 will induce a signaling cascade that ultimately inactivates T cell receptor (TCR) associated proteins essential for T cell activation. Many malignant cell types have evolved the ability to subvert host immune responses by expressing a PD-1 ligand (PD-L1)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Upon binding to PD-1, PD-L1 inhibits the ability of cytotoxic T cells to target and attack tumor cells. Immunotherapeutic checkpoint inhibitor blockers, such as anti-PD-1, are monoclonal antibodies (mAb) that bind and block the interaction between PD-1 and PD-L1, enhancing T cell antitumor activity by preventing PD-L1 tumor-mediated shutoff of T cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePeptide-based therapies enhance the immune system's ability to recognize and respond to specific tumor antigens. The novel immunotherapy DPX\u0026trade; (IMV Inc., Halifax, NS, Canada) is a specialized formulation that stimulates strong and specific immune responses\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. DPX's oil-based, water-free formulation enhances immune responses by providing the immune system with prolonged exposure to antigens\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Specifically, DPX-Survivac combines the DPX formulation and a mixture of survivin peptides. Survivin, a protein necessary for inhibiting apoptosis, is highly expressed in many cancer types with solid tumors, including ovarian cancer\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. DPX-Survivac therefore enhances antitumor immune responses by stimulating host immune systems\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOne of the most significant limitations of single-agent therapies is the increased likelihood of acquired therapeutic resistance in malignant cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Combination therapies avoid acquiring resistance by enhancing antitumor activity through multiple mechanisms\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. For example, positive outcomes have been demonstrated in ovarian cancer patients treated with a combination of DPX-Survivac and low-dose cyclophosphamide (CPA)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Low-dose CPA is a chemotherapeutic agent regimen that has been shown to selectively reduce the number of regulatory T cells and subsequently enhance targeted immune responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStudies demonstrated that low-dose CPA enhanced antigen-specific immune responses induced by DPX-Survivac in ovarian cancer patients\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, Weir et al.\u003csup\u003e17\u003c/sup\u003e found that triple combination therapy with DPX, low-dose CPA, and anti-PD-1 provided better long-term control of established tumors via slower growth in a derived preclinical model of human papillomavirus (HPV) cancer compared to treatment with anti-PD-1 alone. Given these results, this triple combination therapy may also be beneficial for epithelial ovarian cancer.\u003c/p\u003e\u003cp\u003eEvaluating the efficacy of novel combination therapies like this triple combination therapy in preclinical models is vital for their translation into a clinical setting. Tumor progression and cell recruitment can be tracked using magnetic resonance imaging (MRI). Once acquired, anatomical MR images can be used to identify the volume and properties of specific regions of interest (ROI), such as tumors and lymph nodes. Quantification and recruitment of cells to particular ROIs can be performed using the MRI pulse sequence, TurboSPI\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. TurboSPI is used to create R\u003csub\u003e2\u003c/sub\u003e* maps that can be overlaid onto anatomical images and is particularly sensitive to the field inhomogeneities caused by the encapsulation of superparamagnetic iron oxide (SPIO) within cells. SPIO nanoparticles can be used to label a wide variety of immune cells, including dendritic cells, T cells, and natural killer (NK) cells, amongst others\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Our lab has previously demonstrated the use of TurboSPI to semi-quantitatively evaluate SPIO-labeled T cells and myeloid-derived suppressor cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. MRI and molecular imaging in general have proven to be particularly useful in better understanding the roles of treatment and interaction with the immune system on tumor growth and individual outcomes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe objectives of this study were to use MRI in combination with biological assays to (1) study immune cell recruitment in an orthotopic preclinical model of ovarian cancer and (2) evaluate the effects of combination therapy on survival and tumour progression. We hypothesized that mice treated with DPX-Survivac, anti-PD-1, and low-dose CPA would have better tumor control (i.e., slower tumour growth) and increase immune cell recruitment to tumors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eFemale humanized transgenic mice (HLA-A2.I-HLA-DRI-transgenic (HHD) H-2 class I/II knockout), 6\u0026ndash;23 weeks old, were obtained from Charles River Laboratory (France) and bred in-house. All mice were housed in filter-top cages and were provided food and water \u003cem\u003ead-libitum\u003c/em\u003e. Recipient mice were either untreated (n\u0026thinsp;=\u0026thinsp;10) or treated (n\u0026thinsp;=\u0026thinsp;14) with DPX-Survivac, anti-PD-1, and low-dose CPA. Experiments were conducted under ethics protocols approved by the University Committee on Laboratory Animals at Dalhousie University, Halifax, N.S., Canada.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCancer cell line and implant\u003c/h3\u003e\n\u003cp\u003eMouse ovarian surface epithelial (MOSE) cancer cells were provided by Dr. Vanderhyden and were genetically modified in-house to express the survivin epitope and cryopreserved in Calf Bovine Serum with 10% dimethyl sulfoxide (DMSO). Cells were thawed and cultured in DMEM (Dulbecco's Modification of Eagle's Medium; Corning, Corning NY) supplemented with 100 Units/mL Penicillin, 100\u0026micro;g/mL streptomycin (Gibco, Burlington, ON), and 10% Calf Bovine Serum (Hyclone) and grown at 37\u0026deg;C in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. After the first passage, cells were selected for survivin by adding 10 \u0026micro;g/mL of puromycin. On intrabursal surgery day, cells were resuspended at 5x10\u003csup\u003e6\u003c/sup\u003e cells/mL in 1X PBS (Phosphate Buffered Solution; Corning), and 1x10\u003csup\u003e4\u003c/sup\u003e MOSE cells were injected into the left ovary.\u003c/p\u003e\u003cp\u003eAnimals received frequent detailed clinical examinations (DCEs) until the pre-surgery weight was reached. Once mice regained weight (approximately 3 weeks post-surgery), DCEs were conducted twice/week. Tumors were monitored throughout the study by palpating the abdomen. Mice were terminated if they lost 15% of their pre-surgery weight, presented severe ascites or ulcerations, or showed signs of pain and lethargy. Implant and treatment timelines can be found in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDPX-Survivac Treatment\u003c/h3\u003e\n\u003cp\u003eDPX-Survivac was prepared at IMV Inc. using their proprietary DPX formulation described elsewhere\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Detailed methodology can be found in the Supplementary Materials. Doses of 50 \u0026micro;L were given subcutaneously to each mouse on days 28 and 49 post-MOSE implants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The dosing schedule for all treatments was chosen based on previous work done by Weir et al.\u003csup\u003e17\u003c/sup\u003e demonstrating that this dosing combination and schedule worked well in an HPV model.\u003c/p\u003e\u003cp\u003eCyclophosphamide (CPA; MilliporeSigma, Oakville, ON, Canada) was reconstituted in PBS and delivered to mice in drinking water at 0.133 mg/mL for seven consecutive days, beginning on days 14, 28, and 42 post-MOSE implants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mice received 20 mg/kg/day, assuming a 20 g mouse consumes 3 mL water/day.\u003c/p\u003e\u003cp\u003eAnti-PD-1 (clone RPM1-14; BioXCell; West Lebanon, NH, USA) was diluted to 2 mg/mL in 1x PBS. Treated donor mice received six intraperitoneal injections of anti-PD-1 at 200 \u0026micro;g per dose on days 21, 24, 27, 42, 45, and 48 post-MOSE implants. Treated recipient mice received injections on days 28, 31, 34, 49, 52, and 55 post-MOSE implants. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCD8 Cytotoxic T Lymphocyte (CTL) Isolation \u0026 Labeling\u003c/h3\u003e\n\u003cp\u003eInguinal, axial, brachial, and mesenteric lymph nodes were collected from disease-matched and treatment-matched donor mice for isolation of CD8\u0026thinsp;+\u0026thinsp;T cells. CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated from lymph nodes using the EasySep\u0026trade; Mouse CD8\u003csup\u003e+\u003c/sup\u003e T cell Isolation Kit (Stemcell Technologies, Cambridge, MA, United States). Cells were suspended at 5 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL in complete RPMI (cRPMI) media (RPMI 1640 (Corning), 10% FBS (Hyclone), 1% penicillin/streptomycin (Gibco), and 55\u0026micro;M β-mercaptoethanol (Gibco)) and incubated in a CD3-coated cell culture plate with human IL-2 (20 U/mL), mouse IL-12 (100 ng/mL), hamster anti-mouse CD28 (1 \u0026micro;g/mL), and Gentamicin (5 \u0026micro;g/mL). Cells were monitored and kept at a density of 0.5 to 1 x 10\u003csup\u003e6\u003c/sup\u003e. Fresh cRPMI with IL-2 (20 U/mL) was added as required. Four days following CTL isolation, antigen-presenting cells (APC) were isolated from the spleens of disease- and treatment-matched donor mice. Splenocytes were incubated with LPS (10 \u0026micro;g/mL) in media (DMEM\u0026thinsp;+\u0026thinsp;10% FBS\u0026thinsp;+\u0026thinsp;55\u0026micro;M β-mercaptoethanol\u0026thinsp;+\u0026thinsp;1% Penicillin-Streptomycin\u0026thinsp;+\u0026thinsp;1% L-glutamine) for 48 hours. Non-adherent cells were then treated with mitomycin-c (50 \u0026micro;g/mL) for 20 mins, washed, and added to the CTL culture at a ratio of 1:6 (APC: CTL) with survivin peptides (10 \u0026micro;g/mL). Following a 48-hour incubation with the APCs, a sample of cells was collected to assess cell purity via flow cytometry, and the other cells were used for \u003cem\u003ein vivo\u003c/em\u003e studies. Activated CTLs were collected, washed, and incubated with SPIO-Rhodamine B Molday ION (0.075 mg/mL; Biopal Inc., Worchester, MA, USA) and IL-2 (100 U/mL) at 4 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL in cRPMI for 22\u0026ndash;24 hours. Cell viability of CTLs post SPIO labeling was found to be \u0026gt;\u0026thinsp;90%. Previous work done in \u003csup\u003e21\u003c/sup\u003e demonstrated that labeling CTLs using this methodology does not affect the cytotoxicity ability of CTLs. A subset of labeled CTLs were also removed for flow cytometry analysis to assess the effects of labeling on functionality.\u003c/p\u003e\n\u003ch3\u003eMyeloid Cell (MC) Isolation \u0026 Labeling\u003c/h3\u003e\n\u003cp\u003eBone marrow was collected from the femurs and tibias of disease- and treatment-matched donor mice for isolation of myeloid cells. Red blood cells were lysed with 1x RBC lysis buffer (Tonbo Biosciences, San Diego, CA, USA). Three million cells were incubated in Petri dishes with 10 mL of media containing RPMI, 10% FBS, 1% penicillin-streptomycin, 20 mM HEPES (MilliporeSigma), and 20 ng/mL of granulocyte monocyte-colony stimulating factor for 72 hours (GM-CSF; Peprotech, Rocky Hill, NJ, USA). After incubation, 10 mL of media with 20 ng/mL of GM-CSF was added to each plate. Following another 72 hours, cells were collected, centrifuged, resuspended in fresh media with 20 ng/mL of GM-CSF, and returned to the plates. The next day, cells were stimulated overnight with survivin peptide SurA2.M (20 \u0026micro;g/mL). Forty-eight hours later, cells were collected, resuspended at 4 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL, and a sample of cells was assessed for cell purity using flow cytometry, with remaining cells being used for \u003cem\u003ein vivo\u003c/em\u003e studies. The purified myeloid cells were incubated with 0.030 mg/mL of SPIO-Rhodamine B Molday ION for 18\u0026ndash;20 hours. Cell viability of myeloid cells (MCs) post SPIO labeling was found to be \u0026gt;\u0026thinsp;90%. A subset of labeled MCs were also removed for flow cytometry analysis to assess the effects of labeling on functionality.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell Injection \u0026amp; Preparation\u003c/h2\u003e\u003cp\u003eSPIO-labeled cells (CTLs or MCs) were collected, washed twice with 1x PBS, twice with HBSS++ (Hank's Balanced Salt Solution; Corning), and then resuspended in HBSS\u0026thinsp;+\u0026thinsp;+\u0026thinsp;with 20 mM HEPES at 5 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL (MCs) and 25 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL (CTLs). All mice received 200 \u0026micro;L of CTLs or MCs through intravenous tail vein injections. Cells were injected on days 41, 48, and 55 post-implant (24 hours before MRI). Iron loading was assessed in the remaining cells using a Prussian blue assay\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e; cells were lysed overnight in 100 \u0026micro;L of 1M HCl, and then 100 \u0026micro;L of K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e was added to each sample. The absorbance (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\lambda\\:\\)\u003c/span\u003e\u003c/span\u003e = 620 nm) was recorded on SpectraMax i3 (Molecular Devices, San Jose, CA, USA) and compared to a standardized \u003cem\u003ein-vitro\u003c/em\u003e calibration curve.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTumor Dissociation for Tumor-Infiltrating Lymphocyte Assay\u003c/h3\u003e\n\u003cp\u003eTumors were resected from mice following their final MRI scans. In Petri dishes, the tumor was chopped into smaller pieces with a scalpel and then incubated in digestion buffer [1 mg/mL collagenase type 1 (Gibco)\u0026thinsp;+\u0026thinsp;0.1 mg/mL DNase I (MilliporeSigma)\u0026thinsp;+\u0026thinsp;5% FB Essence in HBSS++] at 37\u0026deg;C for 30 min. Samples were then filtered through a 70 \u0026micro;m strainer with separation buffer [2% FB essence\u0026thinsp;+\u0026thinsp;1mM EDTA in 1x PBS (Gibco)]. Red blood cell (RBC) lysis was performed on the suspension as required. Cells were washed with 1x PBS and used for flow cytometry.\u003c/p\u003e\n\u003ch3\u003eAscites Sample Preparation\u003c/h3\u003e\n\u003cp\u003eAt the endpoint, mice were euthanized, and ascites fluid was collected using a 25G needle and a 5 mL syringe. Red blood cells were lysed with an equal volume of 1x RBC lysis buffer, and samples were centrifuged to collect cells. These remaining cells were washed thoroughly with 1x PBS and used for flow cytometry.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFlow Cytometry\u003c/h2\u003e\u003cp\u003eCell samples were blocked in 5% normal rat serum (NRS) for 10 min and then incubated with antibody cocktails at 4\u0026deg;C for 20 min (Supplementary Tables\u0026nbsp;1). Samples requiring intracellular staining were permeabilized with permeabilization buffer and stained with the intracellular antibody for 40\u0026ndash;50 min at 4\u0026deg;C. The same staining procedure was followed as in \u003csup\u003e21\u003c/sup\u003e. After staining, samples were fixed with 4% paraformaldehyde (PFA). OneComp ebeads (eBioscience) were used for controls. Data were acquired with a FACS Celesta or FACS Canto II equipped with FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA) at the Dalhousie University Flow Cytometry Core Facility. Samples were analyzed using FlowJo v10.6.2 (Vicro, Torrance, CA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eSamples (spleen, tumor, and lymph nodes) were frozen immediately after termination in an Optical cutting temperature (OCT; Fisher)/Sucrose (1:1) solution, and stored in a -80\u003csup\u003eo\u003c/sup\u003eC freezer until sectioning. Samples were taken to the Dalhousie immunohistochemistry core for processing; tissues were sectioned on the cryostat and placed on slides. Slides were then fixed in -20\u003csup\u003eo\u003c/sup\u003eC cold acetone for 2 min, dried, and stored in a -20\u003csup\u003eo\u003c/sup\u003eC freezer until staining. For IHC staining, slides were brought to RT, dried, and fixed in cold acetone for 10 min and air-dried again for 30 min. They were then washed in a Tris buffered saline (TBS)/Bovine serum albumin (BSA) wash and blocked with 20% horse serum for 1 hour. They were rinsed again in the TBS/BSA solution before staining with the Avidin-Biotin Vector kit for 15 min. The samples were stained with the biotinylated primary antibodies overnight (CD8-biotin for the CD8 T cells and CD11-biotin for the MCs, 1:50 dilution). Samples were washed with the TBS/BSA and stained with the Avidin-Alexa Fluor 633 fluorophore (1:200) for one hour, washed with TBS/BSA and TBS alone. Slides were mounted with antifade mounting media with 4',6-diamidino-2-phenylindole (DAPI) and visualized on the Zeiss LSM 710 (upright) laser-scanning confocal microscope at the Dalhousie CDMI core facility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMRI Acquisition\u003c/h2\u003e\u003cp\u003eMice were imaged with MRI using a 3T preclinical Agilent MRI (Varian Inc., Santa Clara, CA, USA). The MRI contained a 21-cm inner diameter gradient coil (200 mT/m; Magnex Scientific, Oxford, UK) and was interfaced with a Varian DD Console (Varian Inc.). Mice were anesthetized and secured in an animal holder immediately before imaging. Temperature and respiration rates were monitored throughout imaging using a rectal probe and breathing monitor.\u003c/p\u003e\u003cp\u003eAnatomical images were acquired using a balanced steady-state free precession (bSSFP) pulse sequence. The bSSFP parameters were set at a repetition time of 8ms, echo time of 4 ms, and a flip angle of 30\u0026deg;. The field of view (FOV), 256 x 170 x 170 matrix, was set at an isotropic resolution of 200 \u0026micro;m and was centred over the torso. The TurboSPI parameters were set with a FOV of 32 x 32 x 32 mm and a slab size of 30 mm\u003csup\u003e12,22\u003c/sup\u003e. The repetition time (TR) was 250 ms, the echo train length (ETL) was 8, and the echo spacing (ESP) was 10 ms. Mice were scanned on days 42, 49, and 56 post-implant, approximately 24h after SPIO-labeled cell injections.\u003c/p\u003e\u003cp\u003e\u003cem\u003eImaging Analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMRI Images were loaded in VivoQuant (InVicro, Ma, US), and regions of interest (ROI) were drawn on the tumor and lymph nodes using the bSSFP image for each imaging time point as in \u003csup\u003e12\u003c/sup\u003e. Cell density in tumors and lymph nodes was obtained by extracting frequency histograms of the R\u003csub\u003e2\u003c/sub\u003e* signal from the ROIs, and converted from R\u003csub\u003e2\u003c/sub\u003e* values per voxel to cell density per mm\u003csup\u003e3\u003c/sup\u003e using the calibration curve for either CTLs or MCs. Each voxel was then summed over the ROI, resulting in total cell density for each tumor and lymph node ROI (same methods as \u003csup\u003e12,18\u003c/sup\u003e). Data were then imported into GraphPad Prism 8 (San Diego, CA, USA) for statistical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eTo compare the results between the two treatment groups, we used a student's \u003cem\u003et\u003c/em\u003e-test with Bonferroni correction for multiple comparisons, and to evaluate group-level results across both time and treatment groups, we used a two-way ANOVA. Significance is designated as *\u003cem\u003ep\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.05, **\u003cem\u003ep\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.01, ***\u003cem\u003ep\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eImaging demonstrates the success of triple combination therapy\u003c/h2\u003e\u003cp\u003eThe average tumor volume of mice treated with DPX-Survivac, anti-PD-1, and CPA was smaller than the average tumor volume of untreated mice at all three imaging timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). There was a statistically significant difference in tumor volume between groups on day 56 post-implant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, the tumor growth percentage was calculated for all mice imaged across the three-time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Percent tumor growth between days 42\u0026ndash;56 post-implant in untreated mice was significantly increased compared to the treated group (335% increase vs 93% increase, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Student's \u003cem\u003et-\u003c/em\u003etest, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Taken together, these results suggested that a combination therapy of DPX-Survivac, anti-PD-1, and CPA slows the growth of primary tumors, resulting in reduced tumor volumes. However, there was no significant change in survival due to treatment (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition to quantifying primary tumor volumes, the volumes of both inguinal lymph nodes were quantified at each imaging time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, quantitative values in Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The average volume of the DPX-draining LN (i.e., the left inguinal lymph node) trended higher than the tumor-draining LN (i.e., the right inguinal lymph node) within the treated group. We then calculated the volumetric ratio between DPX-draining and tumor-draining lymph nodes by dividing the volume of the DPX-draining LN by the tumor-draining LN. Therefore, a ratio\u0026thinsp;\u0026gt;\u0026thinsp;1 indicated increased swelling in the DPX-draining LN relative to the tumor-draining LN. At each imaging time point, the average volumetric ratio was \u0026gt;\u0026thinsp;1 in the treated group, whereas it was \u0026lt;\u0026thinsp;1 in the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Using a two-way ANOVA, we found there were significant group-level differences due to treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs DPX-draining lymph nodes were generally more swollen than the tumor-draining LNs, we then assessed whether increased swelling correlated with tumor volume. The lymph node volumetric ratio of the treated animals negatively correlated with tumor volume, whereas the opposite was observed in the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We found there was a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the slope of the untreated and treated mice. These results suggested that DPX-Survivac induces lymph node swelling in the DPX-draining lymph node, which correlates with a smaller tumor volume.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCell culture phenotyping and SPIO labelling\u003c/h2\u003e\u003cp\u003eFlow cytometry was used on samples not tagged with SPIO to assess the purity of cell cultures. Cultures of marrow-derived myeloid cells from treated mice appeared to have a higher proportion of macrophages and fewer monocytes and dendritic cells. Still, they were not significantly different than those in untreated mice (data not shown). Furthermore, the percentage of myeloid cells cultured from treated mice appeared to be higher than that of untreated mice expressing MHCII. There were no evident differences between treated and untreated cultures in the CD11b/CD11c subsets of macrophages, monocytes, and dendritic cells. Cultured CTLs were very pure: \u0026gt;90% of CD3\u003csup\u003e+\u003c/sup\u003e cells were CD8\u003csup\u003e+\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;3A). There were no differences in the expression of PD-1, CTLA-4, or TIM3 between CTLs cultured from treated and untreated mice (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, CTLs cultured from untreated mice appeared to express higher levels of Ki67 (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eUptake of SPIO by labeled CTLs and MCs was evaluated using a Prussian Blue assay measured on a spectrophotometer, validated against a known concentration curve\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. CTLs were found to have approximately 4pg of iron/cell, and MCs were found to have approximately 7pg of iron/cell. Viability for all labeled cells was \u0026gt;\u0026thinsp;90%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eSemi-quantitative analysis of SPIO-labeled cells in the tumor and inguinal lymph nodes\u003c/h2\u003e\u003cp\u003eTo quantify the recruitment of SPIO-labeled MCs and CTLs, R\u003csub\u003e2\u003c/sub\u003e* maps were generated with the TurboSPI MRI pulse sequence and overlaid onto anatomical MR images. R\u003csub\u003e2\u003c/sub\u003e* values within the specific ROI were converted into the number of SPIO-labeled cells per mm\u003csup\u003e3\u003c/sup\u003e of ROI using R\u003csub\u003e2\u003c/sub\u003e* relaxivity curves generated from cell phantoms made with SPIO-labeled MCs and CTLs (same isolation and labeling procedures as \u003cem\u003ein vivo\u003c/em\u003e studies).\u003c/p\u003e\u003cp\u003eTreatment with DPX-Survivac, low dose CPA, and anti-PD-1 significantly increased MC recruitment to the tumor, as measured by MCs per mm\u003csup\u003e3\u003c/sup\u003e of tumor (p\u0026thinsp;=\u0026thinsp;0.0211, two-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Tumor MCs were recruited to the tumors of treated mice at a density of approximately 750 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 42), 590 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 49), and 540 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 56). MCs were recruited to the tumors of untreated mice at a density of 185 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 42), 40 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 49), and 55 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 56).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that lymph nodes are immune cell infiltration and priming sites, it was hypothesized that the combination therapy might increase the recruitment of MCs to lymph nodes. However, treatment did not significantly impact MC recruitment to the DPX-draining lymph node (i.e., left LLN, p\u0026thinsp;=\u0026thinsp;0.1598; two-way ANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, treatment did not substantially affect MC recruitment to the tumor-draining lymph node (p\u0026thinsp;=\u0026thinsp;0.8903; two-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCTL recruitment was also quantified using the same methods. Treatment with DPX-Survivac, anti-PD-1 and low dose CPA significantly increased CTL recruitment to the tumor, as measured by CTLs per mm\u003csup\u003e3\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.0155, two-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Average CTL recruitment to the tumors of treated mice was 2500 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 42), 2300 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 49), and 2300 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 56; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Average CTL recruitment to tumors in the untreated group was 2200 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 42), 450 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 49), and 83 cells/mm\u003csup\u003e3\u003c/sup\u003e (day 56; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In untreated mice, only 1 of 5 mice had CTLs present in the tumors on days 49 and 56. These results suggest that treatment with DPX-Survivac, low-dose CPA, and anti-PD-1 increases the recruitment of CTLs to the tumors of treated mice.\u003c/p\u003e\u003cp\u003eWe found that treatment did not significantly impact CTL recruitment to the DPX-draining lymph node (p\u0026thinsp;=\u0026thinsp;0.2405, two-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) or the tumor-draining lymph (p\u0026thinsp;=\u0026thinsp;0.9820, two-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). However, it did appear that the recruitment of CTLs to the DPX-draining lymph node decreased in untreated mice as the study progressed, whereas it remained relatively consistent in treated mice. This resulted in far more CTLs in the DPX-draining lymph node in treated mice at day 56 compared to untreated mice. There was a single outlier mouse in the untreated group, but the other 4 of 5 untreated mice had no detectable CTLs in the DPX-draining lymph node at day 56.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTurboSPI validation\u003c/h2\u003e\u003cp\u003eIHC was used to validate that the injected cells labeled with SPIO were the cells of interest in the terminal tissues. Lymph nodes were utilized as the stains were more homogeneous. Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows lymph nodes from mice that received either CD8\u0026thinsp;+\u0026thinsp;T cells (top) or CD11\u0026thinsp;+\u0026thinsp;myeloid cells (bottom). Immune cells were stained with Alexa Fluor 633 Avidin and biotinylated anti-CD8 (top) or biotinylated anti-CD11 (bottom). The SPIO used had a rhodamine B tag also visible with IHC. All of the rhodamine B positive cells are also CD8 or CD11 positive in the lymph nodes (as indicated by the green arrows), indicating that the cells visualized with MRI are the cells of interest.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of Ascites\u003c/h2\u003e\u003cp\u003eThe total proportion of immune cells in the ascitic fluid was determined using the CD45 marker (common leukocyte antigen). On average, treated mice had a higher proportion of leukocytes than untreated mice, albeit not statistically significant (p\u0026thinsp;=\u0026thinsp;0.1429; unpaired \u003cem\u003et\u003c/em\u003e-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The cellular composition of ascitic fluid collected from treated mice had an average of 97.16% CD45\u0026thinsp;+\u0026thinsp;cells, while the untreated mice had an average of only 83.3% CD45\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Within the CD45\u0026thinsp;+\u0026thinsp;population in ascites from treated mice, an average of 22.02% expressed F4/80, 27.90% expressed CD11c, and 16.76% expressed CD3ε (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In the untreated ascites samples, 34.89% of the CD45\u0026thinsp;+\u0026thinsp;population expressed F4/80, 32.88% expressed CD11c, and 17.39% expressed CD3ε (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). There were no statistical differences in the percentage of F4/80 (p\u0026thinsp;=\u0026thinsp;0.1712), CD11c (p\u0026thinsp;=\u0026thinsp;0.6212), and CD3ε (p\u0026thinsp;=\u0026thinsp;0.9310) populations between treated and untreated samples (unpaired \u003cem\u003et\u003c/em\u003e-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe CD3ε marker encompasses all T cell populations; therefore, we also examined the proportions of CD4 and CD8 cells within the CD3ε population (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). There were more CD4\u0026thinsp;+\u0026thinsp;T cells than CD8\u0026thinsp;+\u0026thinsp;T cells in the ascitic fluid collected from treated and untreated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Interestingly, a population of double-positive T cells also expressed both CD4 and CD8. In the treated ascites samples, an average of 44.37% of the CD3ε population was CD4\u0026thinsp;+\u0026thinsp;CD8+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Only 15.42% of the CD3ε population in the untreated samples was CD4\u0026thinsp;+\u0026thinsp;CD8+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). There was no significant difference between the average percentage of CD4\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;in the treated and untreated samples (p\u0026thinsp;=\u0026thinsp;0.1250; unpaired \u003cem\u003et\u003c/em\u003e-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Together, these results suggest that treatment with DPX-Survivac, low dose CPA, and anti-PD-1 increases the presence of CD45\u0026thinsp;+\u0026thinsp;immune cells in ascitic fluid and may increase CD4+/CD8\u0026thinsp;+\u0026thinsp;T cells but does not have an impact on the presence of other specific immune cell populations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eTumor-infiltrating lymphocyte assay\u003c/h2\u003e\u003cp\u003eTumor-infiltrating lymphocytes were assessed using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The tumors of mice treated with DPX-Survivac/CPA and anti-PD-1 appeared to have fewer CD45\u0026thinsp;+\u0026thinsp;immune cells, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, CD19\u003csup\u003e+\u003c/sup\u003e B cells, and granulocytic myeloid-derived suppressor cells (MDSCs) (CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003eLy6c\u003csup\u003eneg\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B, D, E) than tumors from untreated mice. Furthermore, there were no significant differences in the percentage of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells, transitional MDSCs (CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003eLy6c\u003csup\u003ehigh\u003c/sup\u003e), monocytic MDSCs (CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003eneg\u003c/sup\u003eLy6c\u003csup\u003ehigh\u003c/sup\u003e), classical or myeloid-derived dendritic cells, macrophages, and F4/80-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, E, F, G) between untreated and treated mice. However, there was a trend of increased CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in treated mice. Overall, the ovarian tumors had few monocytic and transitional MDSCs and very few macrophages or myeloid-derived DCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary objectives of this study were to 1) use molecular imaging to determine the effect of combination therapy with DPX-Survivac, anti-PD-1, and low-dose CPA on tumor progression and 2) use MRI to measure changes in immune cell recruitment to tumors and inguinal lymph nodes as a result of treatment. We also evaluated the cellular composition of ascites and tumor infiltrates at the study endpoint in a preclinical ovarian cancer model. Our primary findings indicated that the combination therapy studied in the project slows ovarian tumor growth, and increases the recruitment of CTLs to tumors and MCs to the DPX-draining lymph node.\u003c/p\u003e\u003cp\u003eLate-stage diagnoses account for the high mortality of patients with epithelial ovarian cancer: the 5-year survival rate is less than 50%\u003csup\u003e23\u003c/sup\u003e, with advanced cases having a survival rate of only\u0026thinsp;~\u0026thinsp;30%\u003csup\u003e4\u003c/sup\u003e. Finding effective therapies is imperative for increasing the survival rate of this disease. However, it is challenging to study orthotopic ovarian cancer models in any depth without using imaging to look at the primary tumors located in and around the ovaries. We therefore used anatomical MRI to evaluate the effect of combination therapy with DPX-Survivac, low-dose CPA, and anti-PD-1 on ovarian cancer survival and tumor growth. Our results demonstrated that combination therapy increased the survival of mice by 7% (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). While there was no statistical significance between the survival of treated and untreated mice, this is likely due to the duration of the study. One limitation of assessing survival was that all remaining mice were euthanized on day 60 post-implant for tissue collection. Future studies will extend to further time points to assess actual survival more accurately.\u003c/p\u003e\u003cp\u003eCompared to the untreated group, treated mice had significantly smaller tumors by the end of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results suggested that combination therapy with DPX-Survivac, low-dose CPA, and anti-PD-1 slowed the growth of established ovarian tumors. Due to the latent nature of ovarian cancer, patients are typically diagnosed at stages when the primary tumor has already been established. Therefore, novel treatments must be effective with more advanced cancer models. This clinically relevant treatment schedule replicates a current clinical trial (NCT03836352, clinical trial registration date: 2019-02-07, clinicaltrial.gov) and builds upon previous use in other cancer models \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In addition, slowing the growth of primary tumors may help prolong survival and enhance the efficacy of other treatments, such as debulking surgeries. While this combination shows promise in improving survival and slowing tumor growth, there remain a number of questions about the mechanisms of action of the therapeutics.\u003c/p\u003e\u003cp\u003eA primary objective of this study was to evaluate whether MRI immune cell tracking could be used to monitor and quantify the recruitment of adoptively transferred SPIO-labeled immune cells at two different timepoints. While cell tracking of these two cell types has been done previously in a subcutaneous tumor model\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, this is the first time it has been done in a more clinically relevant orthotopic ovarian model, which can be more difficult due to increased motion artifacts and increased fat in the lower abdomen. These orthotopic models of ovarian cancer, particularly when used in combination with humanized mice with intact immune systems and clinically relevant treatment regimens, are critical for correctly understanding immunological responses.\u003c/p\u003e\u003cp\u003eUsing MRI, we found that combination therapy increased the recruitment of both SPIO-labeled MCs and SPIO-labeled CTLs to the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). For SPIO-labeled CTLs, this increase is likely linked to the slower tumor growth seen in treated mice. Although flow cytometry results did not find a significant difference, likely due to a small N, there was a trend of higher levels of CD8\u0026thinsp;+\u0026thinsp;T cells in treated mice.\u003c/p\u003e\u003cp\u003eInterestingly, untreated mice saw a significant drop in the cellular density of recruited CTLs throughout the study. This is potentially due to an increasingly immunosuppressive environment. As indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, untreated mice had increased numbers of granulocytic myeloid cells, which can be myeloid-derived suppressor cells, a highly suppressive immune cell type. Given the broad range of cells that can be classified as MCs, it is not clear if the increases seen with SPIO-labeled cells represent a more inflammatory or suppressive cell type. Future studies would benefit from further sorting of MCs prior to labeling with SPIO and implanting them to understand this phenotype better.\u003c/p\u003e\u003cp\u003eWe also quantified cell recruitment to the tumor-draining and DPX-draining inguinal lymph nodes, which are sites of immune cell infiltration and priming. Although we found no significant differences in cell recruitment, we did notice that CTL recruitment to the draining lymph node in untreated mice decreased throughout the study. In contrast, treated mice had similar cell densities throughout. Though this difference was not statistically significant, this may have been due to our limited sample size and an outlier point at day 56 for untreated mice (4/5 untreated mice had no CTLs in the right, or DPX-draining, lymph node at day 56). This data suggested that the combination therapy may have enhanced the recruitment of immune cells, such as CTLs, to the DPX-draining lymph node for priming adaptive immune cells against antigens.\u003c/p\u003e\u003cp\u003eThe increase of CTLs to the DPX draining lymph node in treated mice may also be linked to changes in the volumetric ratio of the DPX-draining: tumor-draining inguinal lymph node between treated and untreated mice. The average volumetric ratio of the DPX-draining:tumor-draining lymph nodes was consistently increased in mice treated with the combination therapy. In fact, few untreated mice had a DPX-draining:tumor-draining volumetric ratio greater than 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggested that DPX-Survivac increased swelling in the DPX-draining lymph node relative to the contralateral lymph node, as the other individual therapies in the combination are systemic. Given that lymph nodes are sites of immune cell priming, the observed increase in size, along with CTL cell recruitment to the DPX-draining lymph node, suggested that DPX-Survivac may increase lymph node size due to immune cell infiltration. Our results were similar to those found in a study\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e that described the inguinal lymph node volumetric ratio as a potential biomarker for successful therapy with DPX. Using a C3 tumor model, they\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e found that treatment with a DPX peptide-based therapy increased the volumetric ratio of the DPX-draining lymph node to the tumor-draining lymph node. Furthermore, this volume increase was associated with better outcomes, namely, decreased tumor growth.\u003c/p\u003e\u003cp\u003eWeir et al.\u003csup\u003e15,17\u003c/sup\u003e assessed the tumor infiltration of antigen-specific (R9F-specific) CTLs in the C3 cervical tumor model using a similar combination therapy: anti-PD-1, DPX-R9F, and low-dose CPA. The study found that combination therapy with all three agents enhanced the infiltration of R9F-specific CTLs to tumors compared to treatment with DPX-R9F and low-dose CPA alone, using flow cytometry. Similarly, we found that the tumor infiltration of CTLs was increased when mice were treated with the three therapies combined.\u003c/p\u003e\u003cp\u003eUpon analysis of the ascitic fluid, mice treated with the combination therapy had consistently higher percentages of CD45\u0026thinsp;+\u0026thinsp;cells (leukocytes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The remaining cells were believed to be free-floating tumor cells. In ovarian cancer, malignant ascites contribute to transcoelomic metastasis (metastasis through the peritoneal cavity) by providing primary tumor cells with a medium for dissemination\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, the increased percentage of CD45\u0026thinsp;+\u0026thinsp;cells observed in ascites from treated mice indicates fewer tumor cells in the ascitic fluid. Conversely, the increased presence of tumor cells in the ascites of untreated mice suggests that these mice are more likely to have metastases. Because the presence of tumor cells within ascites is associated with poor prognosis, this could serve as a potential biomarker for the efficacy of this combination therapy to reduce tumor burdens. Future studies would benefit from imaging at longer timepoints and monitoring regions like the lungs for potential metastases.\u003c/p\u003e\u003cp\u003eWe also noted the presence of a CD4\u0026thinsp;+\u0026thinsp;CD8+ (double-positive; DP) T cell population in the ascitic fluid of both treated and untreated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). While untreated mice had consistently decreased proportions of DP T cells, some mice in the treated group had an increased percentage of DP T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). DP T cells have been reported in healthy and diseased individuals\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The roles of this unconventional T cell population are not fully understood, with conflicting reports describing cytotoxic or suppressive roles for these cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Several studies have reported the role of DP T cells in mediating the tumor response and favouring immune escape in many cancer types, such as urological cancer, renal carcinoma, metastatic colorectal cancer, melanomas, and breast cancer lesions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Menard et al.\u003csup\u003e32\u003c/sup\u003e have observed in renal carcinoma patients that DP T cells express high levels of PD-1, which will make them a suitable target for checkpoint inhibitor therapies. This would explain the favorable prognosis in mice treated with combination therapy by restricting the immunosuppressive role or enhancing the cytotoxicity of this cell population.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCollectively, our results demonstrated that molecular imaging allowed the study of a combination therapy with DPX-Survivac, anti-PD-1, and low-dose CPA in epithelial ovarian cancer, demonstrating that the combination decreases the tumor burden of mice. As evidenced by cell tracking, the combination therapy may exert its effects by inhibiting tumor-mediated immunosuppression and subsequently increasing the infiltration of cytotoxic T lymphocytes into the primary tumor. Additionally, the increased percentage of immune cells shown in the ascitic fluid may indicate the combination therapy's efficacy against ovarian tumors. This study demonstrates that immune cell tracking can be used to probe the longitudinal mechanics of SPIO-labeled cells in orthotopic cancer models. In future work, we plan on investigating more immune cell subtypes, particularly subtypes of MLCs such as monocytes, granulocytes, and myeloid-derived suppressor cells, in single immunotherapies and at more timepoints, to understand individual therapeutic mechanisms of action better.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMagnetic resonance imaging (MRI), Cyclophosphamide (CPA), programmed cell death-1 (PD-1), T cell receptor (TCR), PD-1 ligand (PD-L1), monoclonal antibodies (mAb), human papillomavirus (HPV), regions of interest (ROI), superparamagnetic iron oxide (SPIO), natural killer (NK), HLA-A2.I-HLA-DRI-transgenic (HHD), dimethyl sulfoxide (DMSO), DMEM (Dulbecco's Modification of Eagle's Medium), Phosphate buffered solution (PBS), Detailed clinical examinations (DCE), Mouse ovarian surface epithelial (MOSE), Cytotoxic t lymphocyte (CTL), Myeloid cells (MC), Hank’s balanced salt solution (HBSS), Red blood cell lysis (RBC), Paraformaldehyde (PFA), Optical cutting temperature (OCT), Tris buffered saline (TBS), Bovine serum albumin (BSA), Balanced steady state free precession (bSSFP), Repetition time (TR), Echo train length (ETL), Echo spacing (ESP), Field of View (FOV), Lymph node (LN), Analysis of variance (ANOVA), myeloid-derived suppressor cells (MDSCs)\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFinancial Support\u003c/h2\u003e\n\u003cp\u003eKB and MS would like to acknowledge support from the BHCRI New Investigator Program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Disclosure:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the time of this study MLT, JTG, BD, AM, KBo, AW, GW, AMJ, OH and MS were employees of IMV Inc. KDB had a research contract with IMV on studying DPX-Svv in ovarian cancer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the time of this study MLT, JTG, BD, AM, KBo, AW, GW, AMJ, OH and MS were employees of IMV Inc. KDB had a research contract with IMV on studying DPX-Svv in ovarian cancer.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eKB and MM would like to acknowledge funding from the Beatrice Hunter Cancer Research Institute (BHCRI) via a New Investigator Award, and KB would like to acknowledge funding from an NSERC Discovery Grant.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eExperiment planning and oversight was done by KDB, MS, GW, OH and AM-J. Data acquisition was done by CSS, MLT, HW, VG, AN, CD, BDa and AV-L. Data analysis was done by CSS, HW, JTG, BDi and KDB. Animal procedures were done by CD, AM, AW and KB. Cells were provided by BV and modified by OH. Manuscript writing was done by JG, CSS, BD and KDB. Manuscript was reviewed by all authors.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eFlow Cytometry acquisition, Immunohistochemistry preparation, and microscopy were done at Dalhousie University Core Facilities. KB and MM would like to acknowledge funding from the Beatrice Hunter Cancer Research Institute (BHCRI) via a New Investigator Award, and KB would like to acknowledge funding from an NSERC Discovery Grant.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData will be made available upon request to the corresponding author KDB ( [[email protected]](mailto:[email protected]) ).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVetter MH, Hays JL. Use of Targeted Therapeutics in Epithelial Ovarian Cancer: A Review of Current Literature and Future Directions. \u003cem\u003eClin Ther\u003c/em\u003e. 2018;40(3):361\u0026ndash;371. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.clinthera.2018.01.012\u003c/span\u003e\u003cspan address=\"10.1016/j.clinthera.2018.01.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLawson-Michod KA, Watt MH, Grieshober L, et al. Pathways to ovarian cancer diagnosis: a qualitative study. \u003cem\u003eBMC Womens Health\u003c/em\u003e. 2022;22(1):430. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12905-022-02016-1\u003c/span\u003e\u003cspan address=\"10.1186/s12905-022-02016-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePenet MF, Krishnamachary B, Wildes FB, et al. Ascites Volumes and the Ovarian Cancer Microenvironment. \u003cem\u003eFront Oncol\u003c/em\u003e. 2018;8. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2018.00595\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2018.00595\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmed N, Stenvers KL. Getting to Know Ovarian Cancer Ascites: Opportunities for Targeted Therapy-Based Translational Research. \u003cem\u003eFront Oncol\u003c/em\u003e. 2013;3. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2013.00256\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2013.00256\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuarez-Almazor ME, Kim ST, Abdel‐Wahab N, Diab A. Review: Immune‐Related Adverse Events With Use of Checkpoint Inhibitors for Immunotherapy of Cancer. \u003cem\u003eArthritis \u0026amp; Rheumatology\u003c/em\u003e. 2017;69(4):687\u0026ndash;699. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/art.40043\u003c/span\u003e\u003cspan address=\"10.1002/art.40043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang Q, Chen Y, Li X, et al. The role of PD-1/PD-L1 and application of immune-checkpoint inhibitors in human cancers. \u003cem\u003eFront Immunol\u003c/em\u003e. 2022;13:964442. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.964442\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.964442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeiss L, Huemer F, Mlineritsch B, Greil R. Immune checkpoint blockade in ovarian cancer. \u003cem\u003ememo - Magazine of European Medical Oncology\u003c/em\u003e. 2016;9(2):82\u0026ndash;84. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12254-016-0267-3\u003c/span\u003e\u003cspan address=\"10.1007/s12254-016-0267-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi X, Hu W, Zheng X, et al. Emerging immune checkpoints for cancer therapy. \u003cem\u003eActa Oncol (Madr)\u003c/em\u003e. 2015;54(10):1706\u0026ndash;1713. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/0284186X.2015.1071918\u003c/span\u003e\u003cspan address=\"10.3109/0284186X.2015.1071918\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeidel JA, Otsuka A, Kabashima K. Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations. \u003cem\u003eFront Oncol\u003c/em\u003e. 2018;8. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2018.00086\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2018.00086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNowicki TS, Hu-Lieskovan S, Ribas A. Mechanisms of Resistance to PD-1 and PD-L1 Blockade. \u003cem\u003eThe Cancer Journal\u003c/em\u003e. 2018;24(1):47\u0026ndash;53. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/PPO.0000000000000303\u003c/span\u003e\u003cspan address=\"10.1097/PPO.0000000000000303\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerinstein NL, Karkada M, Oza AM, et al. Survivin-targeted immunotherapy drives robust polyfunctional T cell generation and differentiation in advanced ovarian cancer patients. \u003cem\u003eOncoimmunology\u003c/em\u003e. 2015;4(8):e1026529. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/2162402X.2015.1026529\u003c/span\u003e\u003cspan address=\"10.1080/2162402X.2015.1026529\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTremblay ML, O\u0026rsquo;Brien-Moran Z, Rioux JA, et al. Quantitative MRI cell tracking of immune cell recruitment to tumors and draining lymph nodes in response to anti-PD-1 and a DPX-based immunotherapy. \u003cem\u003eOncoimmunology\u003c/em\u003e. 2020;9(1). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/2162402X.2020.1851539\u003c/span\u003e\u003cspan address=\"10.1080/2162402X.2020.1851539\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDorigo O, Fiset S, MacDonald LD, et al. DPX-Survivac, a novel T-cell immunotherapy, to induce robust T-cell responses in advanced ovarian cancer. \u003cem\u003eJournal of Clinical Oncology\u003c/em\u003e. 2020;38(5_suppl):6\u0026ndash;6. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.2020.38.5_suppl.6\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2020.38.5_suppl.6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeir G, Hrytsenko O, Quinton T, et al. Abstract 4903: Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells. \u003cem\u003eCancer Res\u003c/em\u003e. 2016;76(14_Supplement):4903\u0026ndash;4903. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1538-7445.AM2016-4903\u003c/span\u003e\u003cspan address=\"10.1158/1538-7445.AM2016-4903\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeir GM, Hrytsenko O, Stanford MM, et al. Metronomic cyclophosphamide enhances HPV16E7 peptide vaccine induced antigen-specific and cytotoxic T-cell mediated antitumor immune response. \u003cem\u003eOncoimmunology\u003c/em\u003e. 2014;3(8):e953407. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4161/21624011.2014.953407\u003c/span\u003e\u003cspan address=\"10.4161/21624011.2014.953407\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScurr M, Pembroke T, Bloom A, et al. Low-Dose Cyclophosphamide Induces Antitumor T-Cell Responses, which Associate with Survival in Metastatic Colorectal Cancer. \u003cem\u003eClinical Cancer Research\u003c/em\u003e. 2017;23(22):6771\u0026ndash;6780. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.CCR-17-0895\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-17-0895\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeir GM, Hrytsenko O, Quinton T, Berinstein NL, Stanford MM, Mansour M. Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide. \u003cem\u003eJ Immunother Cancer\u003c/em\u003e. 2016;4(1):68. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40425-016-0169-2\u003c/span\u003e\u003cspan address=\"10.1186/s40425-016-0169-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTremblay ML, Davis C, Bowen C V., et al. Using MRI cell tracking to monitor immune cell recruitment in response to a peptide-based cancer vaccine. \u003cem\u003eMagn Reson Med\u003c/em\u003e. 2018;80(1):304\u0026ndash;316. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/mrm.27018\u003c/span\u003e\u003cspan address=\"10.1002/mrm.27018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD Brewer K, Stanley O, Davis C, et al. Tracking SPIO-Labeled Effector \u0026amp; Regulatory Cell Migration with MRI. In: \u003cem\u003eProceedings of the 21st Annual Meeting of the International Society of the Magnetic Resonance in Medicine (ISMRM)\u003c/em\u003e.; 2013.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOude Engberink RD, Blezer ELA, Hoff EI, et al. MRI of Monocyte Infiltration in an Animal Model of Neuroinflammation Using SPIO-Labeled Monocytes or Free USPIO. \u003cem\u003eJournal of Cerebral Blood Flow \u0026amp; Metabolism\u003c/em\u003e. 2008;28(4):841\u0026ndash;851. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/sj.jcbfm.9600580\u003c/span\u003e\u003cspan address=\"10.1038/sj.jcbfm.9600580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNuschke A, Sobey-Skelton C, Dawod B, et al. Use of Magnetotactic Bacteria as an MRI Contrast Agent for In Vivo Tracking of Adoptively Transferred Immune Cells. \u003cem\u003eMol Imaging Biol\u003c/em\u003e. 2023;25(5):844\u0026ndash;856. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/S11307-023-01849-Y\u003c/span\u003e\u003cspan address=\"10.1007/S11307-023-01849-Y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRioux JA, Brewer KD, Beyea SD, Bowen C V. Quantification of superparamagnetic iron oxide with large dynamic range using TurboSPI. \u003cem\u003eJournal of Magnetic Resonance\u003c/em\u003e. 2012;216:152\u0026ndash;160. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmr.2012.01.017\u003c/span\u003e\u003cspan address=\"10.1016/j.jmr.2012.01.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu J, Sun H, Yang L, et al. Improved survival in ovarian cancer, with widening survival gaps of races and socioeconomic status: a period analysis, 1983\u0026ndash;2012. \u003cem\u003eJ Cancer\u003c/em\u003e. 2018;9(19):3548\u0026ndash;3556. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/jca.26300\u003c/span\u003e\u003cspan address=\"10.7150/jca.26300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerinstein NL, Karkada M, Morse MA, et al. First-in-man application of a novel therapeutic cancer vaccine formulation with the capacity to induce multi-functional T cell responses in ovarian, breast and prostate cancer patients. \u003cem\u003eJ Transl Med\u003c/em\u003e. 2012;10(1):156. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1479-5876-10-156\u003c/span\u003e\u003cspan address=\"10.1186/1479-5876-10-156\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrewer KD, Lake K, Pelot N, et al. Clearance of depot vaccine SPIO-labeled antigen and substrate visualized using MRI. \u003cem\u003eVaccine\u003c/em\u003e. 2014;32(51):6956\u0026ndash;6962. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2014.10.058\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2014.10.058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrewer KD, DeBay DR, Dude I, et al. Using lymph node swelling as a potential biomarker for successful vaccination. \u003cem\u003eOncotarget\u003c/em\u003e. 2016;7(24):35655\u0026ndash;35669. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.9580\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.9580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLatifi A, Luwor RB, Bilandzic M, et al. Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors. Hawkins SM, ed. \u003cem\u003ePLoS One\u003c/em\u003e. 2012;7(10):e46858. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0046858\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0046858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKipps E, Tan DSP, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. \u003cem\u003eNat Rev Cancer\u003c/em\u003e. 2013;13(4):273\u0026ndash;282. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrc3432\u003c/span\u003e\u003cspan address=\"10.1038/nrc3432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesfran\u0026ccedil;ois J, Moreau-Aubry A, Vignard V, et al. Double Positive CD4CD8 αβ T Cells: A New Tumor-Reactive Population in Human Melanomas. Lowenstein PR, ed. \u003cem\u003ePLoS One\u003c/em\u003e. 2010;5(1):e8437. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0008437\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0008437\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOvergaard NH, Jung JW, Steptoe RJ, Wells JW. CD4+/CD8\u0026thinsp;+\u0026thinsp;double-positive T cells: more than just a developmental stage? \u003cem\u003eJ Leukoc Biol\u003c/em\u003e. 2015;97(1):31\u0026ndash;38. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1189/jlb.1RU0814-382\u003c/span\u003e\u003cspan address=\"10.1189/jlb.1RU0814-382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBohner P, Chevalier MF, Cesson V, et al. Double Positive CD4\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T Cells Are Enriched in Urological Cancers and Favor T Helper-2 Polarization. \u003cem\u003eFront Immunol\u003c/em\u003e. 2019;10. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2019.00622\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2019.00622\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenard LC, Fischer P, Kakrecha B, et al. Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T Cells With Expression of Exhaustion Markers. \u003cem\u003eFront Immunol\u003c/em\u003e. 2018;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2018.02728\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2018.02728\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarrabayrouse G, Corvaisier M, Ouisse LH, et al. Tumor-reactive CD4\u0026thinsp;+\u0026thinsp;CD8αβ\u0026thinsp;+\u0026thinsp;CD103\u0026thinsp;+\u0026thinsp;αβT cells: A prevalent tumor-reactive T-cell subset in metastatic colorectal cancers. \u003cem\u003eInt J Cancer\u003c/em\u003e. 2011;128(12):2923\u0026ndash;2932. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ijc.25640\u003c/span\u003e\u003cspan address=\"10.1002/ijc.25640\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Magnetic resonance imaging (MRI), immunotherapy, cell tracking, cytotoxic T lymphocytes, ovarian cancer","lastPublishedDoi":"10.21203/rs.3.rs-7714918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7714918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNovel treatments are needed for epithelial ovarian cancer, the most lethal gynecologic malignancy due to late diagnosis, resistance to treatment, and high relapse rate. Immunotherapies such as checkpoint inhibitors (i.e, anti-PD-1) and peptide-based therapies (DPX-Survivac) have strong potential to improve responses. Magnetic resonance imaging (MRI) can be used to track tumor growth and iron-labelled immune cells longitudinally at the individual level. We studied MRI immune cell tracking in response to the combination of DPX-Survivac, anti-PD-1, and an intermittent low dose of Cyclophosphamide (CPA), which has been shown to suppress cancer growth in a preclinical model of ovarian cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eHHD-DR1 mice were orthotopically implanted with mouse ovarian surface epithelial (MOSE) cancer cells. Myeloid and activated CD8\u003csup\u003e+\u003c/sup\u003e cells were isolated from disease- and treatment-matched donor mice, labelled with superparamagnetic iron oxide (SPIO) and intravenously injected on 41, 48, and 55 days post-implant with either type of cells. Mice were scanned using MRI approximately 24h after SPIO-labeled cell injections.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTumor volumes in the treatment group were significantly lower than in the control group as measured by MRI (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The density of SPIO-labelled myeloid and CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumors was higher in the treatment group than in the control group. Furthermore, ascitic fluid in treated mice has a significantly higher frequency of CD45\u003csup\u003e+\u003c/sup\u003e leukocytes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eUsing MRI, our study has shown that this combination treatment can slow down ovarian tumor growth and increase the recruitment of myeloid and CD8\u0026thinsp;+\u0026thinsp;cells to tumors. This study provides insights into how MRI can be used in concert with biological assays to study how immunotherapy and chemotherapy combinations exert their antitumor effects.\u003c/p\u003e","manuscriptTitle":"MRI of combination immunotherapy in an epithelial ovarian cancer preclinical model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 00:40:51","doi":"10.21203/rs.3.rs-7714918/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-12T14:57:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T02:48:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-02T17:44:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-02T09:51:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T21:39:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T18:22:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315777173196621796284135034112749657430","date":"2025-10-17T09:17:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68613656274533100197970541274785520441","date":"2025-10-14T21:06:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191541913591251386327681782570559432405","date":"2025-10-13T13:50:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147317908910829161104128020137192574182","date":"2025-10-12T19:53:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300650464548389712011853100593939627685","date":"2025-10-10T14:47:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T14:08:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T12:27:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-07T11:40:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Imaging","date":"2025-09-25T16:15:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4cdc10b9-03b4-41ef-b7c4-102976838c32","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56730002,"name":"Biological sciences/Cancer"},{"id":56730003,"name":"Biological sciences/Immunology"},{"id":56730004,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-20T09:24:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 00:40:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7714918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7714918","identity":"rs-7714918","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}