Intravital 2-photon Microscopy Research Articles

Proline catabolism is a key factor facilitating Candida albicans pathogenicity.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Leishmania major drives host phagocyte death and cell-to-cell transfer depending on intracellular pathogen proliferation rate.

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.

Tracing causes of tubule atrophy in vivo

Acute kidney injury (AKI) is a known risk factor for chronic kidney disease (CKD). The mechanisms for AKI-CKD transition may involve failed tubule repair and progressive interstitial fibrosis but are incompletely understood. Which events lead to failed tubule repair? Can injury spread over time? And are these processes steered by myofibroblasts and fibrotic tissue remodeling?Here, we combined serial intravital 2-photon microscopy and a novel model of partial ischemia-reperfusion injury (partial IRI), which allowed us to investigate injured tissue alongside uninjured tissue in the same kidney and over time. Thus, we occluded only one branch of the left renal artery for 21 min, rendering half the kidney ischemic. We then tracked tubule necrosis and remodeling alongside myofibroblast dynamics for up to 3 weeks. Necrotic cell death was detected by in vivo propidium iodide staining. PDGFRβ-CreERT2-Salsa6F mice (N=5) identified main myofibroblast precursor cells, by tdTomato-expression. Myofibroblast-differentiation was confirmed by ex vivo αSMA staining. Proximal tubule (PT) function was assessed by in vivo uptake of fluorescent albumin.Necrotic cell death was solely detectable upon reperfusion. Border areas in between post-ischemic and non-ischemic tissue presented severely necrotic PTs alongside uninjured PTs. From 1 day after partial IRI, we observed formation of granular casts in the lumen of necrotic tubules, which 1-2 days later also appeared in initially non-necrotic PTs downstream. Tubule necrosis and granular cast accumulation preceded thinned epithelium that lacked albumin endocytosis (day 3-7) and recruitment of myofibroblasts (peaking D4-10). Of note, non-necrotic tubules, without granular cast accumulation, didn’t remodel nor recruit myofibroblasts.Among all remodeling tubule segments, 24% turned atrophic. Importantly, preceding necrotic cell death was higher in atrophic than in recovering tubules (55±13% vs. 39±18% necrotic cells/total segment cell number, (mean±SD), p<.0001). Preceding granular casts accounted for 38±15% vs. 12±11% (mean ±SD) of the luminal area in atrophic and recovering tubules, respectively (p<.0001). Ex vivo staining for pro-fibrotic VCAM-1 demonstrated selective positivity in atrophic tubules, which appeared scattered at D8 while at D21, entire atrophic tubules stained positive (n=3, each). Consistently, we detected persistent myofibroblast enclosure of atrophic tubule, while recruitment around recovered PTs resolved by day 14 (50±21% vs. 23±18% of coverage, p<.001).Tracking the same tubules in vivo and over time, we demonstrated early proximal tubule necrosis, which induced luminal granular cast formation and injury propagation. These events preceded myofibroblast accumulation and predicted tubule atrophy. We further documented de novo expression of pro-inflammatory VCAM-1 and persistent myofibroblast recruitment selectively in atrophic tubules, suggesting an active role of this tubule state in AKI-CKD transition. Novo Nordisk Foundation, Augustinus Foundation, Aarhus University Research Foundation (AUFF) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

Corneal tissue-resident memory T cells form a unique immune compartment at the ocular surface.

The eye is considered immune privileged such that immune responses are dampened to protect vision. As the most anterior compartment of the eye, the cornea is exposed to pathogens and can mount immune responses that recruit effector Tcells. However, presence of immune memory in the cornea is not defined. Here, we use intravital 2-photon microscopy to examine Tcell responses in the cornea in mice. We show that recruitment of CD8+ Tcells in response to ocular virus infection results in the formation of tissue-resident memory T (TRM) cells. Motile corneal TRM cells patrol the cornea and rapidly respond in situ to antigen rechallenge. CD103+ TRM cell generation requires antigen and transforming growth factor β. Invivo imaging in humans also reveals highly motile cells that patrol the healthy cornea. Our study finds that TRM cells form in the cornea where they can provide local protective immunity.

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations. In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Frömter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p < 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 ± 0.002 in untreated MWFs to 0.045 ± 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations. In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Frömter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p < 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 ± 0.002 in untreated MWFs to 0.045 ± 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Dynamic tracing using ultra-bright labeling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriers in vivo

The potential of poly(lactic-co-glycolic acid) (PLGA) to design nanoparticles (NPs) and target the central nervous system remains to be exploited. In the current study we designed fluorescent 70-nm PLGA NPs, loaded with bulky fluorophores, thereby making them significantly brighter than quantum dots in single-particle fluorescence measurements. The high brightness of NPs enabled their visualization by intravital real-time 2-photon microscopy. Subsequently, we found that PLGA NPs coated with pluronic F-68 circulated in the blood substantially longer than uncoated NPs and were taken up by cerebro-vascular endothelial cells. Additionally, confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 h after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer.

Brain metastases frequently occur in lung cancer and dramatically limit prognosis of affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized. We established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intravital 2-photon laser scanning microscopy. This allowed in vivo tracking of fluorescence-expressing tumor cells and TAM/M on a single-cell level over weeks. Intracarotid injection of red tdTomato-fluorescent Lewis lung carcinoma cell was performed in transgenic mice either proficient or deficient for CX3CR1. After intracarotid cell injection, intravascular tumor cells extravasated into the brain parenchyma and formed micro- and mature macrometastases. We observed potential phagocytosis of extravasated tumor cells by TAM/M. However, during later steps of metastasis formation, these anti-tumor effects diminished and were paralleled by TAM/M accumulation and activation. Although CX3CR1 deficiency resulted in a lower number of extravasated tumor cells, progression of these extravasated cells into micro metastases was more efficient. Overall, this resulted in a comparable number of mature macrometastases in CX3CR1-deficient and -proficient mice. Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic option to prevent dissemination of lung cancer cells to the brain. Given the close interaction between TAM/M and tumor cells during metastasis formation, other therapeutic approaches targeting TAM/M function may warrant further evaluation. The herein established orthotopic mouse model may be a useful tool to evaluate such concepts in vivo.

Ly6G deficiency alters the dynamics of neutrophil recruitment and pathogen capture during Leishmania major skin infection

Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils’ ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system.

Developmental vascular malformations in EPAS1 gain-of-function syndrome.

Mutations in EPAS1, encoding hypoxia-inducible factor-2α (HIF-2α), were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to the NIH for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1 gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital 2-photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retinas from corresponding developmental time points (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental time points. These findings add vascular malformation as a new clinical feature of EPAS1 gain-of-function syndrome.

Intravital Imaging Allows Organ-Specific Insights Into Immune Functions.

Leukocytes are among the most mobile and versatile cells that have many essential functions in homeostasis and survival. Especially cells from the innate immune system, i.e., neutrophils and macrophages, play an important role as rapid first responders against invading microorganisms. With the advent of novel imaging techniques, new ways of visualizing innate immune cells have become available in recent years, thereby enabling more and more detailed discoveries about their nature, function and interaction partners. Besides intravital spinning-disc and 2-photon microscopy, clearing and 3D-imaging techniques provide new insights into the mechanism of innate immune cell behavior in their natural environment. This mini review focuses on the contributions of novel-imaging techniques to provide insight into the functions of neutrophils and macrophages under homeostasis and in infections. Imaging setups for different organs like the liver, kidney, heart, lung, and the peritoneal cavity are discussed as well as the current limitations of these imaging techniques.

Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p &amp;lt; 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p &amp;lt; 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p &amp;lt; 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Dual CXCR4 and E-Selectin Inhibition (GMI-1359) Rapidly Increases AML Cellular Motility Prior to Intravasation and Vascular Niche Depletion Observed By Intravital Bone Marrow Two-Photon Microscopy

BACKGROUND: Acute myelogenous leukemia (AML) cells that reside in bone marrow (BM) receive a great deal of protection from the cytotoxic effects of therapeutic agents. In contrast, the circulating leukemia cells are typically more chemosensitive compared to those embedded in BM niches. The BM homing of AML cells is mediated by multiple adhesive and chemokinetic interactions including, respectively, by sialylated glycoproteins on the cancer cells binding to E-selectin on the endothelium and by CXCR4-mediated sensing of SDF-1/CXCL12 gradients. Consequently, both E-selectin and CXCR4 inhibitors have been pursued as targets for add-on therapeutic strategies aimed to "mobilize" AML cells out of the BM. A next generation "mobilizing" agent GMI-1359 combines, in one molecular structure, the E-selectin and CXCR4 inhibitory moieties. In a previous study, GMI-1359 markedly reduced leukemia cell adhesion to endothelial cells, leukemia cellularity in BM (Zhang et al., 2016) and it enhanced the survival of cytarabine/daunorubicin therapy in a FLT3-mutated AML model (Zhang et al., 2015). APPROACH: Here, we used intravital 2-photon microscopy of calvarial BM to study the behavioral response of AML cells to the dual CXCR4 and E-selectin inhibition (GMI-1359) in vivo. We used a mTurquoise2 fluorescence-tagged transplantable mouse AML model (characterized by MLL, ENL-FLT3, ITD, p53-/-). To delineate the bone and vascular niches, the syngeneic immune-competent recipient mice harbored cell lineage fluorescence reporter genes such as Col2.3-GFP, hCD2-DsRed and CD11c-EYFP with the bone collagen and blood highlighted by, respectively, second harmonic generation (SHG) and fluorescent dextran. Intravenously infused AML cells (5x10E4) homed to BM where they gradually displaced most endogenous cells. In this experimental system, GMI-1359 or vehicle were infused into the tail vein while recording AML cell motilities in calvarial BM stroma in 3-D over 4 hours. RESULTS: In untreated or control vehicle treated mice, AML cells were slowly motile, migrating with the average velocity of ~2 µm/min. Cellular trajectories were random within the average 400 µm confinement radius corresponding to the size of BM cavities. Most AML cells localized in the vascular niche, defined as within a 50 µm distance to the nearest blood vessel. A minority of AML cells were also present in the proximity to osteoblasts and the bone (i.e. in the bone niche). In contrast, GMI-1359 infusion resulted in a 66% increase of the average speed of cellular motility of AML cells within 20 min and up to 100% increase within 3.5 hours. The motility pattern remained largely random within the bone cavity confinement volume and was concentrated in the vicinity of vasculature. Thereafter, we observed multiple instances of AML cell intravasation into the capillary lumens followed by several minute-long vessel wall attachment and sudden outflow. This cellular dynamics resulted in a substantial, yet structurally biased decrease of BM AML cellularity whereby the vascular niche was emptied preferentially whilst the bone niche remained populated by AML cells. In no case we observed a complete BM depletion of AML cells. Interestingly, as we reported before, a single, CXCR4-only inhibitor caused a protracted (24-48 h) depletion of AML cells in BM without significant cellular motility enhancement. CONCLUSIONS: Our observations reveal an unexpected mechanism of action by a dual E-selectin/CXCR4 inhibitor involving cellular migratory motility enhancement in BM stroma prior to AML cell intravasation. We envisage that, besides the designed blocking of E-selectin and CXCR4 molecules from binding their corresponding ligands, the dual moiety GMI-1359 agent has a capacity to generate motility-enhancing signals in AML cells that single inhibitors do not seem to trigger. Among several mechanisms possible, this action could involve E-selectin and CXCR4 crosslinking by a dual moiety molecular structure or binding avidity enhancement, requiring further investigation. In addition, our results highlight a likely mechanism of AML resistance to therapeutic "mobilization", one that involves cancer cell persistence in the bone/osteoblastic niches. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Optimization of a Rhabdomyolysis Model in Mice With Exertional Heat Stroke Mouse Model of EHS-Rhabdomyolysis.

Exertional heat stroke (EHS) is a life-threatening disease characterized by high mortality and incidence of rhabdomyolysis (RM). It would therefore be valuable to establish a stable EHS-induced RM model that accurately reflects the clinical characteristics of EHS patients and provides an objective animal model for further study of the pathogenesis of RM. In the current study, 8∼9-week-old, male, wild-type C57BL/6J mice, at the stage of sexual maturity, were randomly divided into four groups: the EHS group, the classical heat stroke (CHS) group, the sham heat exercise group, and sham heat rest group. The survival rate of mice was determined under relatively high levels of temperature and humidity (37.5°C, 65% relative humidity (RH); 37.5°C, 70% RH; 39.5°C, 65% RH; and 39.5°C, 70% RH) as well as a high core temperature (Tc; 42, 42.5, and 43°C). Results showed that the environmental condition of 39.5°C and 65% RH was most suitable for EHS modeling. The end point of EHS evaluation was exhaustion or an individual’s core temperature reaching 43°C. The survival rate of mice in the EHS group within 24 h under these conditions was 37.34%, which is consistent with the high mortality characteristics noted in EHS patients. Severe RM was observed in the EHS group by H&E staining and transmission electron microscopy. Creatine kinase levels in the EHS group mostly exceeded 10,000 U/L, which was approximately 10 times higher than that in the sham heat rest group. Renal tubules of the EHS group exhibited severe necrosis, and calcium overload in the skeletal muscles of this group was also observed using intravital 2-photon microscopy. In conclusion, we made improvements to a stable EHS-induced RM animal model to truly reflect the clinical characteristics of EHS patients. This new model should be helpful in the further study of RM pathogenesis.

Tertiary lymphoid organs in renal chronic allograft rejection

Abstract Chronic allograft rejection remains a major obstacle to long-term allograft survival. The immunologic role of tertiary lymphoid organs (TLO) in allograft rejection is unclear. Here, we employed a chronic renal allograft rejection model in mice and intravital 2-photon microscopy to investigate the function of TLO in transplant rejection. CB6F1 (F1) RIP-LTα (preformed TLO) or F1 (no TLO) kidney grafts were transplanted to WT B6 recipients and survival monitored. To investigate immunologic function of TLO, we adoptively transferred B6-RIPLTα CD11c-YFP mice with 10m naïve dsRed OT-I T cells or 10m CTR-labeled NP-specific B cells + 10m CFP+ OT-II cells and immunized with NP-OVA + alum. Intravital 2P imaging of renal TLO was performed at time points 0, 3, 6, 24 or 72 hours after immunization. 4D image analysis was performed and mean speed, displacement, arrest coefficient (AC) and contact times (CT) with DC were calculated for OT-I, OT-II and NP-B cells. F1 RIP-LTα grafts rejected significantly faster (MST= 54) than F1 grafts (MST= 225), demonstrating that TLO contribute to allograft rejection. F1 RIP-LTα grafts contained similar numbers of, but larger TLO than F1 grafts as demonstrated by histology. Mean speed and displacement of OT-I and OT-II cells significantly decreased over time after immunization while AC and mean CT significantly increased. B cell mean speed, displacement and AC increased after immunization. These data are consistent with B cell activation and productive T cell-DC interactions and mirror previously reported data in secondary lymphoid organs. We provide first evidence that TLO provide a local structure for T and B cell activation that propagates anti-graft immune responses in the setting of chronic rejection.

Resident memory CD8 T cells patrol the lung and mediate early protective immunity

Abstract In contrast to the long-lived nature of resident-memory CD8 T cells (Trms) residing in the skin or intestine, Trms located in the lung parenchyma wane and their loss coincides with the decline of protective immunity against subsequent heterosubtypic Influenza infections. We utilized intravital 2-photon microscopy of live mice to dissect the relative contribution and examine the real-time spatiotemporal dynamics of secondary CD8 T cell responses containing or lacking lung Trms. Early post-Influenza infection, the lung contained a mixture of phenotypically defined circulating and CD69+/CD103+ resident-memory transgenic P14 cells which exhibited heterogeneous motility patterns, comprised of both highly motile cells and those exhibiting a slow to moderate scanning pattern. The incorporation of IV exclusion during intravital imaging predominantly labeled highly dynamic cells, and largely spared slower CD103+ Trms in the lung parenchyma and surrounding airways. Conversely, the selective loss of this slow to moderate population was observed with either CD103 antibody-mediated depletion of early memory or at 6 months post infection, suggesting that CD103+ CD8 Trms exhibit slow to moderate motility dynamics in the lung at homeostasis. Importantly, upon secondary infection, hosts containing Trms exhibited a higher proportion of CD8 T cells with altered dynamics, more pronounced clustering around infected cells in the lung in an antigen-specific manner and provided superior early heterosubtypic protection compared to hosts containing only circulating memory. Overall, these data indicate that lung Trms exhibit slow to moderate motility patterns, rapidly respond to and mediate early immunity to subsequent infections in the lung.

Inhibiting Matrix Metalloproteinases Hinders Acute Myeloid Leukaemia and Prevents Healthy Stem Cell Loss

Haematopoietic stem cells (HSCs), despite being very rare (&lt;0.015% of bone marrow haematopoietic cells), maintain the turnover of all blood cells through a balance of quiescence, self-renewal and differentiation. Disruption of HSCs function and of the bone marrow (BM) microenvironment are key aspects of Acute Myeloid Leukaemia (AML). AML develops in adults and symptoms arise due to the loss of healthy haematopoietic cells. It is unknown exactly what factors contribute to this although it is clear that there is a progressive loss of BM HSCs in this disease. One hypothesis is that HSCs are pushed out of the BM niche by competing leukaemic blasts. To explore this, we used intravital 2-photon confocal microscopy in a live mouse model of leukaemia which allows us to visualise the dynamics of healthy haematopoietc cells at various stages of the disease. We monitored the development of extramedullary haematopoiesis (EMH) during AML growth and tested the function of HSCs found in these alternative sites to determine whether EMH acts as an alternative mechanism of blood cell maintenance in response to AML. Furthermore, we investigated the influence of an extracellular matrix metalloproteinase inhibitor, Prinomastat, on the loss of HSCs in this model. Prinomastat has been studied extensively in solid organ cancers as it has been shown capable of inhibiting cancer metastasis. In this study, we examined whether it might have a function in preventing the loss of HSCs from the bone during leukaemia infiltration. C57BL/6 mice were injected with 100k of YFP-AML blasts and peripheral blood (PB) checked every four days for AML progression. Cellular dynamics were assessed by intravital microscopy (IVM) of the mouse calvarium and spleen at early (10%), medium (25%) and late (&gt;25%) PB infiltration. To calculate the number of circulating HSCs and progenitors (HSPCs) blood was taken by cardiac puncture and analysed by flow cytometry for HSCPs absolute number. The same was done for BM, spleen and liver HSPCs. HSC functionality was determined by transplanting sorted Lin- c-Kit+ Sca-1+ CD48- CD150+ (LKS Slam) cells from CD45.1 BM, spleen and liver of AML-burdened mice into lethally irradiated C57BL/6 mice. BM reconstitution was then analysed every four weeks. To analyse the role of extracellular matrix remodelling, C57BL/6 mice were transplanted with 100k AML cells tagged with yellow-fluorescent-protein (YFP) and then administered intravenous prinomastat daily. These mice were imaged and had bone marrow analysed using flow cytometry together with a control group at early, medium and late AML based on PB infiltration. AML progression leads to a dramatic and progressive loss HSCPs in the BM. Intravital imaging showed an enhanced egress of healthy cells from the BM into the circulation. Conversely, we found a clear association between the extent of infiltration of the marrow and the number of HSCs found in the spleen and liver. Our transplantation experiments show that the extramedullary HSCs are functional and able to reconstitute the BM of lethally irradiated mice irrespective of the organ from which they were sorted. Treatment with prinomastat significantly reduced the number of HSCs and progenitors leaving the bone marrow (P=0.0001). In the treated mice, the number of BM HSCs was consistently higher at every infiltration time point when compared to untreated mice. In addition, prinomastat caused a reduction in the extent of extramedullary haematopoesis in both the spleen and liver. This study provides a unique insight into the effect of AML on the dynamics of HSCs as disease progresses. Contrary to expectations, HSCs are not lost, but rather a majority appear to migrate from the bone marrow to sites of extramedullary haematopoiesis. These cells remain functional and are capable of regenerating haematopoiesis when transplanted into a recipient mouse. Further to this, we have demonstrated that by inhibiting the function of metalloproteinases using Prinomastat it is possible to prevent this loss of HSCs thus retain these cells within the bone marrow. These findings highlight the importance of the extracellular matrix in acute myeloid leukaemia and suggest that metalloproteinase inhibitors could potentially have a significant role in resisting the perturbations caused by AML on haematopoiesis. Disclosures No relevant conflicts of interest to declare.