MAIT cells accumulate in ovarian cancer-elicited ascites where they retain their capacity to respond to MR1 ligands and cytokine cues.

The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.

Magnetic microspheres can be used for magnetic particle imaging of cancer cells arrested in the mouse brain.

PURPOSE: Magnetic particle imaging (MPI) is a new imaging modality that sensitively and specifically detects superparamagnetic iron oxide nanoparticles (SPIOs). MRI cell tracking with SPIOs has very high sensitivity, but low specificity and quantification is difficult. MPI could overcome these limitations. There are no reports of micron-sized iron oxide particles (MPIO) for cell tracking by MPI. Therefore, the goal was to evaluate if MPIO can be used for in vivo detection and quantification of cancer cells distributed in the mouse brain by MPI. METHODS: In the first experiment mice were injected with either 2.5 × 105 or 5.0 × 105 MPIO-labeled cancer cells and MPI was performed ex vivo. In a second experiment, mice received either 2.5 × 105 or 5.0 × 104 MPIO-labeled cells and MPI was performed in vivo. In a third experiment, mice were injected with 5.0 × 104 cells, labeled with either MPIO or ferucarbotran, and MPI was performed in vivo. RESULTS: MPIO-labeled cells were visible in all MPI images of the mouse brain. The MPI signal and iron content measurements were greater for brains of mice that were injected with higher numbers of MPIO-labeled cells. Ferucarbotran-labeled cells were not detected in the brain by MPI. CONCLUSION: This is the first example of the use of MPIO for cell tracking with MPI. With an intracardiac cell injection, ~15% of cells will arrest in the brain vasculature. For our lowest cell injection of 5.0 × 104 cells, this was ~10 000 cells, distributed throughout the brain.

Preclinical 19F MRI cell tracking at 3 Tesla.

PURPOSE: To develop methods for fluorine-19 (19F) MRI cell tracking in mice on a 3 Tesla clinical scanner. Compared to iron-based cell tracking, 19F MRI has lower sensitivity and, consequently, preclinical 19F cell tracking has only been performed at relatively high magnetic field strengths (> 3 T). Here, we focus on using 19F MRI to detect macrophages in tumors; macrophage density is an indication of tumor aggressiveness and, therefore, 19F MRI could be used as an imaging biomarker. METHODS: Perfluorocarbon (PFC)-labeled macrophages were imaged at 3 T and NMR spectroscopy was performed to validate 19F spin quantification. In vivo 19F MRI was performed on tumor-bearing mice, post-PFC at both 9.4 T and 3 T. 3 T MRI utilized varying NEX and 19F images were analyzed two different ways for 19F quantification. RESULTS: As few as 25,000 cells could be detected as cell pellets at 3 T. 19F quantification in cell pellets by 3 T MRI agreed with NMR spectroscopy. 19F signal was observed in the liver, spleen and tumor in all mice at 9.4 T and 3 T and there was no significant difference in 19F spin quantification. CONCLUSION: This study demonstrates the ability to detect and quantify 19F signal in murine tumors using 19F MRI at 3 T.

NRF1 motif sequence-enriched genes involved in ER/PR -ve HER2 +ve breast cancer signaling pathways.

Nuclear respiratory factor 1 (NRF1) transcription factor has recently been shown to control breast cancer progression. However, mechanistic aspects by which NRF1 may contribute to susceptibility to different breast tumor subtypes are still not fully understood. Since transcriptional control of NRF1 seems to be dependent on epidermal growth factor receptor signaling, herein, we investigated the role of NRF1 in estrogen receptor/progesterone receptor negative, but human epidermal growth factor receptor 2-positive (ER/PR -ve HER2 +ve) breast cancer. We found that both mRNA and protein levels of NRF1 and its transcriptional activity were significantly higher in ER/PR -ve HER2 +ve breast cancer samples compared to normal breast tissues. This was consistent with our observation of higher NRF1 protein expression in the experimental model of HER2+ breast cancer brain metastasis. To identify network-based pathways involved in the susceptibility to the ER/PR -ve HER2 +ve breast cancer subtype, the NRF1 transcriptional regulatory genome-wide landscape was analyzed using the approach consisting of a systematic integration of ChIP DNA-seq, RNA-Microarray, NRF1 protein-DNA motif binding, signal pathway analysis, and Bayesian machine learning. Our findings show that a high percentage of known HER2+ breast cancer susceptibility genes, including EGFR, IGFR, and E2F1, are under transcriptional control of NRF1. Promoters of several genes from the KEGG HER2+ breast cancer pathway and 11 signaling pathways linked to 6 hallmarks of cancer contain the NRF1 motif. By pathway analysis, key breast cancer hallmark genes of epithelial-mesenchymal transition, stemness, cell apoptosis, cell cycle regulation, chromosomal integrity, and DNA damage/repair were highly enriched with NRF1 motifs. In addition, we found using Bayesian network-based machine learning that 30 NRF1 motif-enriched genes including growth factor receptors-FGFR1, IGF1R; E2Fs transcription factor family-E2F1, E2F3; MAPK pathway-SHC2, GRB2, MAPK1; PI3K-AKT-mTOR signaling pathway-PIK3CD, PIK3R1, PIK3R3, RPS6KB2; WNT signaling pathway-WNT7B, DLV1, DLV2, GSK3B, NRF1, and DDB2, known for its role in DNA repair and involvement in early events associated with metastatic progression of breast cancer cells, were associated with HER2-amplified breast cancer. Machine learning search further revealed that the likelihood of HER2-positive breast cancer is almost 100% in a patient with the high NRF1 expression combined with expression patterns of high E2F3, GSK3B, and MAPK1, low or no change in E2F1 and FGFR1, and high or no change in PIK3R3. In summary, our findings suggest novel roles of NRF1 and its regulatory networks in susceptibility to the ER/PR -ve HER2 +ve aggressive breast cancer subtype. Clinical confirmation of our machine learned Bayesian networks will have significant impact on our understanding of the role of NRF1 as a valuable biomarker for breast cancer diagnosis and prognosis as well as provide strong rationale for future studies to develop NRF1 signaling-based therapeutics to target HER2+ breast cancer.

Cranial irradiation increases tumor growth in experimental breast cancer brain metastasis.

Whole-brain radiotherapy is the standard of care for patients with breast cancer with multiple brain metastases and, although this treatment has been essential in the management of existing brain tumors, there are many known negative consequences associated with the irradiation of normal brain tissue. In our study, we used in vivo magnetic resonance imaging analysis to investigate the influence of radiotherapy-induced damage of healthy brain on the arrest and growth of metastatic breast cancer cells in a mouse model of breast cancer brain metastasis. We observed that irradiated, but otherwise healthy, neural tissue had an increased propensity to support metastatic growth compared with never-irradiated controls. The elucidation of the impact of irradiation on normal neural tissue could have implications in clinical patient management, particularly in patients with residual systemic disease or with residual radio-resistant brain cancer.

Application of dual 19 F and iron cellular MRI agents to track the infiltration of immune cells to the site of a rejected stem cell transplant.

PURPOSE: Cellular MRI) was used to detect implanted human mesenchymal stem cells (hMSCs) and the resulting macrophage infiltration that occurs in response to xenotransplantation. METHODS: Human mesenchymal stem cells were prelabeled with a fluorine-19 (19 F) agent prior to implantation, allowing for their visualization and quantification over time. Following implantation of 1 × 10619 F-labeled hMSCs into the mouse hind limb, longitudinal imaging was performed to monitor the stem cell graft. Macrophages were labeled in situ by the intravenous administration of an ultrasmall superparamagentic iron oxide (USPIO), allowing for tracking of the inflammatory response. RESULTS: Quantification of 19 F MRI on day 0 agreed with the implanted number of cells, and 19 F signal decreased over time. By day 14, only 22% ± 11% of the original 19 F signal remained. In a second group, USPIO were administered intravenously after implantation of 19 F-labeled hMSCs. When imaged on day 2, a significant decrease in 19 F signal was observed compared to the first group alongside a large signal void region in the corresponding proton images. Immunohistochemistry confirmed the presence of iron-labeled macrophages in the stem cell tract. CONCLUSION: A dual-labeling technique was used to noninvasively track two distinct cell populations simultaneously. This information could be used to provide additional insight into the cause of graft failure. Magn Reson Med 78:713-720, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

MRI surveillance of cancer cell fate in a brain metastasis model after early radiotherapy.

PURPOSE: Incidence of brain metastasis attributed to breast cancer is increasing and prognosis is poor. It is thought that disseminated dormant cancer cells persist in metastatic organs and may evade treatments, thereby facilitating a mechanism for recurrence. Radiotherapy is used to treat brain metastases clinically, but assessment has been limited to macroscopic tumor volumes detectable by clinical imaging. Here, we use cellular MRI to understand the concurrent responses of metastases and nonproliferative or slowly cycling cancer cells to radiotherapy. METHODS: MRI cell tracking was used to investigate the impact of early cranial irradiation on the fate of individual iron-labeled cancer cells and outgrowth of breast cancer brain metastases in the human MDA-MB-231-BR-HER2 cell model. RESULTS: Early whole-brain radiotherapy significantly reduced the outgrowth of metastases from individual disseminated cancer cells in treated animals compared to controls. However, the numbers of nonproliferative iron-retaining cancer cells in the brain were not significantly different. CONCLUSIONS: Radiotherapy, when given early in cancer progression, is effective in preventing the outgrowth of solitary cancer cells to brain metastases. Future studies of the nonproliferative cancer cells' clonogenic potentials are warranted, given that their persistent presence suggests that they may have evaded treatment. Magn Reson Med 78:1506-1512, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Swift Intrahepatic Accumulation of Granulocytic Myeloid-Derived Suppressor Cells in a Humanized Mouse Model of Toxic Shock Syndrome.

Toxic shock syndrome (TSS) and other superantigen-mediated illnesses are associated with 'systemic' immunosuppression that jeopardizes the host's ability to fight pathogens. Here, we define a novel mechanism of 'local' immunosuppression that may benefit the host. Systemic exposure to staphylococcal enterotoxin B (SEB) rapidly and selectively recruited CD11b(+)Gr-1(high)Ly-6C(+) granulocytic myeloid-derived suppressor cells (MDSCs) to the liver of HLA-DR4 transgenic mice. Hepatic MDSCs inhibited SEB-triggered T cell proliferation in a reactive oxygen species-dependent manner, and ex vivo-generated human MDSCs also similarly attenuated the proliferative response of autologous T cells to SEB. We propose a role for MDSCs in mitigating excessive tissue injury during TSS.

Advances in Neurotrophic Factor and Cell-Based Therapies for Parkinson's Disease: A Mini-Review.

Parkinson's disease (PD) affects an estimated 7-10 million people worldwide and remains without definitive or disease-modifying treatment. There have been many recent developments in cell-based therapy (CBT) to replace lost circuitry and provide chronic biological sources of therapeutic agents to the PD-affected brain. Early neural transplantation studies underscored the challenges of immune compatibility, graft integration and the need for renewable, autologous graft sources. Neurotrophic factors (NTFs) offer a potential class of cytoprotective pharmacotherapeutics that may complement dopamine (DA) replacement and CBT strategies in PD. Chronic NTF delivery may be an integral goal of CBT, with grafts consisting of autologous drug-producing (e.g., DA, NTF) cells that are capable of integration and function in the host brain. In this mini-review, we outline the past experience and recent advances in NTF technology and CBT as promising and integrated approaches for the treatment of PD.

Longitudinal anatomical and metabolic MRI characterization of orthotopic xenograft prostate tumors in nude mice.

PURPOSE: To assess anatomic and functional magnetic resonance imaging (MRI) for monitoring of tumor volume and metabolism of orthotopic xenograft prostate cancer tumors. MATERIALS AND METHODS: Human-derived PC-3M cells were implanted into the prostate in 22 nude mice. Tumor volume and MRI appearance were monitored for up to 29 days. Histology was performed to detect metastases. Hyperpolarized [1-(13) C]pyruvate MRI was used to measure tumor metabolism on day 22. RESULTS: Tumors were visible by MRI 9 days after tumor cell implantation. Tumor volume increased to 720 ± 190 mm(3) on day 29 of imaging. Metastasis was seen in the iliac lymph nodes at all timepoints, and in more distant lymph nodes at later timepoints, but was not detectable by MRI. Regions with low pyruvate uptake corresponded to regions with necrosis and had a higher lactate/pyruvate ratio (0.98 ± 0.4 vs. 1.6 ± 1.1). CONCLUSION: MRI using the balanced steady-state free precession (bSSFP) sequence can be used to monitor tumor growth in orthotopic PC-3M tumors as early as 9 days post-injection. Hyperpolarized pyruvate MRI has potential to assess tumor metabolism and necrosis.

Examination of the newborn: the key skills. Part 1. The eye.

Midwives are increasingly performing the examination of the newborn. In the first of a four-part series, this article considers the importance of the eye examination in the screening process. The significance of history taking, knowledge of risk factors and the detection of the red reflex will be explored. The necessity for early detection of retinoblastoma, congenital cataracts and glaucoma, and the prerequisite referral pathways that the Newborn infant physical examination (NIPE) requires will also be highlighted.

Part 2: The cardiovascular system and congenital heart disease.

Midwives are increasingly performing the examination of the newborn. In the second of a four-part series, this article considers the importance of the cardiovascular examination in the screening process. The significance of history taking, knowledge of risk factors and auscultation of the heart will be explored. The necessity for early detection and treatment of congenital cardiac abnormalities, along with the prerequisite referral pathways that the Newborn infant physical examination (NIPE) requires will also be highlighted.

Examination of the newborn: the key skills--Part 4 the testes.

Midwives are increasingly performing the examination of the newborn. In the final of this four-part series, we consider the importance of the examination of the testes in the screening process. The significance of history taking, knowledge of risk factors and the testes examination will be explored, along with referral pathways that the National Screening Committee requires, as will be highlighted. The impact of late detection and poor referral of undescended testes on the lives of children and adults will also be considered.

Examination of the newborn: the key skills. Part 3: the hips.

Midwives are increasingly performing the examination of the newborn. his article considers the importance of the examination of the hips in the screening process. The significance of history taking, knowledge of risk factors and the hip examination will be explored. The necessity for early detection and treatment of hip abnormalities, along with referral pathways that the National Screening Committee quires will be highlighted. The impact of late detection of developmental dysplasia of the hip (DDH) on the lives of families and children will also be considered.

Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI.

Gamma delta T cells (GDTc) comprise a small subset of cytolytic T cells shown to kill malignant cells in vitro and in vivo. We have developed a novel protocol to expand GDTc from human blood whereby GDTc were initially expanded in the presence of alpha beta T cells (ABTc) that were then depleted prior to use. We achieved clinically relevant expansions of up to 18,485-fold total GDTc, with 18,849-fold expansion of the Vδ1 GDTc subset over 21 days. ABTc depletion yielded 88.1 ± 4.2 % GDTc purity, and GDTc continued to expand after separation. Immunophenotyping revealed that expanded GDTc were mostly CD27-CD45RA- and CD27-CD45RA+ effector memory cells. GDTc cytotoxicity against PC-3M prostate cancer, U87 glioblastoma and EM-2 leukemia cells was confirmed. Both expanded Vδ1 and Vδ2 GDTc were cytotoxic to PC-3M in a T cell antigen receptor- and CD18-dependent manner. We are the first to label GDTc with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for cellular MRI. Using protamine sulfate and magnetofection, we achieved up to 40 % labeling with clinically approved Feraheme (Ferumoxytol), as determined by enumeration of Perls' Prussian blue-stained cytospins. Electron microscopy at 2,800× magnification verified the presence of internalized clusters of iron oxide; however, high iron uptake correlated negatively with cell viability. We found improved USPIO uptake later in culture. MRI of GDTc in agarose phantoms was performed at 3 Tesla. The signal-to-noise ratios for unlabeled and labeled cells were 56 and 21, respectively. Thus, Feraheme-labeled GDTc could be readily detected in vitro via MRI.

MRI tracking of transplanted iron-labeled mesenchymal stromal cells in an immune-compromised mouse model of critical limb ischemia.

Peripheral arterial disease is a clinical problem in which mesenchymal stromal cell (MSC) transplantation may offer substantial benefit by promoting the generation of new blood vessels and improving limb ischemia and wound healing via their potent paracrine activities. MRI allows for the noninvasive tracking of cells over time using iron oxide contrast agents to label cells before they are injected or transplanted. However, a major limitation of the tracking of iron oxide-labeled cells with MRI is the possibility that dead or dying cells will transfer the iron oxide label to local bystander macrophages, making it very difficult to distinguish between viable transplanted cells and endogenous macrophages in the images. In this study, a severely immune-compromised mouse, with limited macrophage activity, was investigated to examine cell tracking in a system in which bystander cell uptake of dead, iron-labeled cells or free iron particles was minimized. MRI was used to track the fate of MSCs over 21 days after their intramuscular transplantation in mice with a femoral artery ligation. In all mice, a region of signal loss was observed at the injection site and the volume of signal hypointensity diminished over time. Fluorescence and light microscopy showed that iron-positive MSCs persisted at the transplant site and often appeared to be integrated in perivascular niches. This was compared with MSC transplantation in immune-competent mice with femoral artery ligation. In these mice, the regions of signal loss caused by iron-labeled MSC cleared more slowly, and histology revealed iron particles trapped at the site of cell transplantation and associated with areas of inflammation.

Cellular MRI as a suitable, sensitive non-invasive modality for correlating in vivo migratory efficiencies of different dendritic cell populations with subsequent immunological outcomes.

The clinical application of dendritic cells (DC) as adjuvants in immunotherapies such as the cell-based cancer vaccine continues to gain interest. The overall efficacy of this emerging immunotherapy, however, remains low. Studies suggest the stage of maturation and activation of ex vivo-prepared DC immediately prior to patient administration is critical to subsequent DC migration in vivo, which ultimately affects overall vaccine efficacy. While it is possible to generate mature and activated DC ex vivo using various stimulatory cocktails, in the case of cancer patients, the qualitative and quantitative assessment of which DC stimulatory cocktail works most effectively to enhance subsequent DC migration in vivo is difficult. Thus, a non-invasive imaging modality capable of monitoring the real-time migration of DC in long-term studies is required. In this paper, we address whether cellular magnetic resonance imaging (MRI) is sufficiently sensitive to quantitatively detect differences in the migratory abilities of two different DC preparations: untreated (resting) versus ex vivo matured in a mouse model. In order to distinguish our ex vivo-generated DC of interest from surrounding tissues in magnetic resonance (MR) images, DC were labeled in vitro with the superparamagnetic iron oxide (SPIO) nanoparticle FeREX®. Characterization of DC phenotype and function following addition of a cytokine maturation cocktail and the toll-like receptor ligand CpG, both in the presence and in the absence of SPIO, were also carried out. Conventional histological techniques were used to verify the quantitative data obtained from MR images. This study provides important information relevant to tracking the in vivo migration of ex vivo-prepared and stimulated DC.

Dual Magnetic Particle Imaging and Akaluc Bioluminescence Imaging for Tracking Cancer Cell Metastasis.

Magnetic particle imaging (MPI) provides hotspot tracking and direct quantification of superparamagnetic iron oxide nanoparticle (SPIO)-labelled cells. Bioluminescence imaging (BLI) with the luciferase reporter gene Akaluc can provide complementary information on cell viability. Thus, we explored combining these technologies to provide a more holistic view of cancer cell fate in mice. Akaluc-expressing 4T1Br5 cells were labelled with the SPIO Synomag-D and injected into the mammary fat pads (MFP) of four nude mice. BLI was performed on days 0, 6 and 13, and MPI was performed on days 1, 8 and 14. Ex vivo histology and fluorescence microscopy of MFP and a potential metastatic site was conducted. The BLI signal in the MFP increased significantly from day 0 to day 13 (p < 0.05), mirroring tumor growth. The MPI signal significantly decreased from day 1 to day 14 (p < 0.05) due to SPIO dilution in proliferating cells. Both modalities detected secondary metastases; however, they were visualized in different anatomical regions. Akaluc BLI complemented MPI cell tracking, allowing for longitudinal measures of cell viability and sensitive detection of distant metastases at different locations. We predict this multimodal imaging approach will help to evaluate novel therapeutics and give a better understanding of metastatic mechanisms.

In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging.

BACKGROUND: Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo postinjection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Magnetic particle imaging (MPI) has emerged as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide (SPIO)-labeled DC. Herein, we describe a popliteal lymph node (pLN)-focused MPI scan to quantify DC in vivo migration accurately and consistently. METHODS: Adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype, then fluorescently labeled and injected into mouse hind footpads (n = 6). Two days later, in vivo DC migration was quantified using whole animal, pLN-focused, and ex vivo pLN MPI scans. RESULTS: No significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed that pLN MPI signal was due to originally injected Ad SPIO+ and SPIO+ DC. CONCLUSION: We overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases and detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable preclinical imaging modality to assess DC-based cancer immunotherapeutic efficacy. RELEVANCE STATEMENT: Tracking the in vivo fate of DC using noninvasive quantifiable magnetic particle imaging can potentially serve as a surrogate marker of therapeutic effectiveness. KEY POINTS: • Adenoviral-transduced and iron oxide-labeled dendritic cells are in vivo migration competent. • Magnetic particle imaging is a suitable modality to quantify in vivo dendritic cell migration. • Magnetic particle imaging focused field of view overcomes dynamic range limitation.