Use of Magnetotactic Bacteria as an MRI Contrast Agent for In Vivo Tracking of Adoptively Transferred Immune Cells.

PURPOSE: In vivo immune cell tracking using MRI can be a valuable tool for studying the mechanisms underlying successful cancer therapies. Current cell labeling methods using superparamagnetic iron oxide (SPIO) lack the persistence to track the fate and location of transplanted cells long-term. Magnetospirillum magneticum is a commercially available, iron-producing bacterium that can be taken up by and live harmoniously within mammalian cells as magneto-endosymbionts (MEs). MEs have shown promise as labeling agents for in vivo stem and cancer cell tracking but have yet to be evaluated in immune cells. This pilot study examined ME labeling in myeloid-derived suppressor cells (MDSCs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) and its effects on cell purity, function, and MRI contrast. PROCEDURES: MDSCs, CTLs, and DCs were incubated with MEs at various ME labeling ratios (MLR), and various biological metrics and iron uptake were assessed. For in vivo imaging, MDSCs were labeled overnight with either MEs or SPIO (Molday ION Rhodamine B) and injected into C3 tumor-bearing mice via tail vein injection 24 days post-implant and scanned daily with MRI for 1 week to assess cellular quantification. RESULTS: Following incubations, MDSCs contained > 0.6 pg Fe/cell. CTLs achieved Fe loading of < 0.5 pg/cell, and DCs achieved Fe loading of ~ 1.4 pg/cell. The suppressive functionality of MDSCs at 1000 MLR was not affected by ME labeling but was affected at 2000 MLR. Markers of CTL dysfunction were not markedly affected by ME labeling nor were DC markers. In vivo data demonstrated that the MDSCs labeled with MEs generated sufficient contrast to be detectable using TurboSPI, similar to SPIO-labeled cells. CONCLUSIONS: Cells can be labeled with sufficient numbers of MEs to be detectable with MRI without compromising cell viability. Care must be taken at higher concentrations of MEs, which may affect some cell types' functional activity and/or morphology. Immune cells with minimal phagocytic behavior have much lower iron content per cell after incubation with MEs vs SPIO; however, MEs can successfully be used as a contrast agent for phagocytic immune cells.

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.

DPX is a unique T cell activating formulation that generates robust immune responses (both clinically and preclinically) which can be tailored to various cancers via the use of tumor-specific antigens and adjuvants. While DPX-based immunotherapies may act complementary with checkpoint inhibitors, combination therapy is not always easily predictable based on individual therapeutic responses. Optimizing these combinations can be improved by understanding the mechanism of action underlying the individual therapies. Magnetic Resonance Imaging (MRI) allows tracking of cells labeled with superparamagnetic iron oxide (SPIO), which can yield valuable information about the localization of crucial immune cell subsets. In this work, we evaluated the use of a multi-echo, single point MRI pulse sequence, TurboSPI, for tracking and quantifying cytotoxic T lymphocytes (CTLs) and myeloid lineage cells (MLCs). In a subcutaneous cervical cancer model (C3) we compared untreated mice to mice treated with either a single therapy (anti-PD-1 or DPX-R9F) or a combination of both therapies. We were able to detect, using TurboSPI, significant increases in CTL recruitment dynamics in response to combination therapy. We also observed differences in MLC recruitment to therapy-draining (DPX-R9F) lymph nodes in response to treatment with DPX-R9F (alone or in combination with anti-PD-1). We demonstrated that the therapies presented herein induced time-varying changes in cell recruitment. This work establishes that these quantitative molecular MRI techniques can be expanded to study a number of cancer and immunotherapy combinations to improve our understanding of longitudinal immunological changes and mechanisms of action.

Assessment of retinal ganglion cell damage in glaucomatous optic neuropathy: Axon transport, injury and soma loss.

Glaucoma is a disease characterized by progressive axonal pathology and death of retinal ganglion cells (RGCs), which causes structural changes in the optic nerve head and irreversible vision loss. Several experimental models of glaucomatous optic neuropathy (GON) have been developed, primarily in non-human primates and, more recently and commonly, in rodents. These models provide important research tools to study the mechanisms underlying glaucomatous damage. Moreover, experimental GON provides the ability to quantify and monitor risk factors leading to RGC loss such as the level of intraocular pressure, axonal health and the RGC population. Using these experimental models we are able to gain a better understanding of GON, which allows for the development of potential neuroprotective strategies. Here we review the advantages and disadvantages of the relevant and most often utilized methods for evaluating axonal degeneration and RGC loss in GON. Axonal pathology in GON includes functional disruption of axonal transport (AT) and structural degeneration. Horseradish peroxidase (HRP), rhodamine-B-isothiocyanate (RITC) and cholera toxin-B (CTB) fluorescent conjugates have proven to be effective reporters of AT. Also, immunohistochemistry (IHC) for endogenous AT-associated proteins is often used as an indicator of AT function. Similarly, structural degeneration of axons in GON can be investigated via changes in the activity and expression of key axonal enzymes and structural proteins. Assessment of axonal degeneration can be measured by direct quantification of axons, qualitative grading, or a combination of both methods. RGC loss is the most frequently quantified variable in studies of experimental GON. Retrograde tracers can be used to quantify RGC populations in rodents via application to the superior colliculus (SC). In addition, in situ IHC for RGC-specific proteins is a common method of RGC quantification used in many studies. Recently, transgenic mouse models that express fluorescent proteins under the Thy-1 promoter have been examined for their potential to provide specific and selective labeling of RGCs for the study of GON. While these methods represent important advances in assessing the structural and functional integrity of RGCs, each has its advantages and disadvantages; together they provide an extensive toolbox for the study of GON.

Longitudinal in vivo imaging of retinal ganglion cells and retinal thickness changes following optic nerve injury in mice.

BACKGROUND: Retinal ganglion cells (RGCs) die in sight-threatening eye diseases. Imaging RGCs in humans is not currently possible and proof of principle in experimental models is fundamental for future development. Our objective was to quantify RGC density and retinal thickness following optic nerve transection in transgenic mice expressing cyan fluorescent protein (CFP) under control of the Thy1 promoter, expressed by RGCs and other neurons. METHODOLOGY/PRINCIPAL FINDINGS: A modified confocal scanning laser ophthalmoscopy (CSLO)/spectral-domain optical coherence tomography (SD-OCT) camera was used to image and quantify CFP+ cells in mice from the B6.Cg-Tg(Thy1-CFP)23Jrs/J line. SD-OCT circle (1 B-scan), raster (37 B-scans) and radial (24 B-scans) scans of the retina were also obtained. CSLO was performed at baseline (n = 11) and 3 (n = 11), 5 (n = 4), 7 (n = 10), 10 (n = 6), 14 (n = 7) and 21 (n = 5) days post-transection, while SD-OCT was performed at baseline and 7, 14 and 35 days (n = 9) post-transection. Longitudinal change in CFP+ cell density and retinal thickness were computed. Compared to baseline, the mean (SD) percentage CFP+ cells remaining at 3, 5, 7, 10, 14 and 21 days post-transection was 86 (9)%, 63 (11)%, 45 (11)%, 31 (9)%, 20 (9)% and 8 (4)%, respectively. Compared to baseline, the mean (SD) retinal thickness at 7 days post-transection was 97 (3)%, 98 (2)% and 97 (4)% for the circle, raster and radial scans, respectively. The corresponding figures at 14 and 35 days post-transection were 96 (3)%, 97 (2)% and 95 (3)%; and 93 (3)%, 94 (3)% and 92 (3)%. CONCLUSIONS/SIGNIFICANCE: Longitudinal imaging showed an exponential decline in CFP+ cell density and a small (≤8%) reduction in SD-OCT measured retinal thickness post-transection. SD-OCT is a promising tool for detecting structural changes in experimental optic neuropathy. These results represent an important step towards translation for clinical use.

Organization and metamorphosis of glia in the Drosophila visual system.

The visual system of Drosophila is an excellent model for determining the interactions that direct the differentiation of the nervous system's many unique cell types. Glia are essential not only in the development of the nervous system, but also in the function of those neurons with which they become associated in the adult. Given their role in visual system development and adult function we need to both accurately and reliably identify the different subtypes of glia, and to relate the glial subtypes in the larval brain to those previously described for the adult. We viewed driver expression in subsets of larval eye disc glia through the earliest stages of pupal development to reveal the counterparts of these cells in the adult. Two populations of glia exist in the lamina, the first neuropil of the adult optic lobe: those that arise from precursors in the eye-disc/optic stalk and those that arise from precursors in the brain. In both cases, a single larval source gives rise to at least three different types of adult glia. Furthermore, analysis of glial cell types in the second neuropil, the medulla, has identified at least four types of astrocyte-like (reticular) glia. Our clarification of the lamina's adult glia and identification of their larval origins, particularly the respective eye disc and larval brain contributions, begin to define developmental interactions which establish the different subtypes of glia.