Intravital Microscopy Research Articles

In Situ Reprogrammings of Splenic CD11b+ Cells by Nano‐Hypoxia to Promote Inflamed Damage Site‐Specific Angiogenesis

AbstractClinical translation of nanoparticles is limited because of their short circulation time, which hampers targeting to prolong therapeutic effects. Angiogenesis is required to regenerate damaged sites under inflammation, and CD11b+ cells turn vasculogenic under hypoxia. As a turning‐point strategy to increase the circulation time, this study explores liposomal targeting of splenic CD11b+ cells, which are gathered in the spleen and move to inflamed sites inherently. Moreover, nano‐hypoxia is strategized as a therapeutic method by loading liposomes with a hypoxic‐mimetic agent (CoCl2) to induce in situ reprogramming of splenic CD11b+ cells upon venous injection. Consequently, the vasculogenic potential of reprogrammed cells accelerates regeneration through inflammation‐responsive homing. Hydrophilic coating of liposomes improves the selectivity of splenic targeting in contrast to fast targeting without coating. Hypoxia chambers and surgical induction of splenic hypoxia are compared to validate the reprogramming effect. The strategy is validated in mouse models of inflamed skin, ischemic hindlimbs, and 70% hepatectomy compared with a conventional approach using bone marrow cells. Intravital multiphoton microscopy, 19F 2D/3D MRI, and microchannel hydrogel chips for 3D tissue culture are used as advanced tools. Overall, nanocarrier change to CD11b+ cells prolong targeting by inducing in situ reprogramming for inflammation‐responsive vasculogenic therapy.

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.

Real-Time Intravital Imaging of Acoustic Cluster Therapy-Induced Vascular Effects in the Murine Brain.

The blood-brain barrier (BBB) is an obstacle for cerebral drug delivery. Controlled permeabilization of the barrier by external stimuli can facilitate the delivery of drugs to the brain. Acoustic Cluster Therapy (ACT®) is a promising strategy for transiently and locally increasing the permeability of the BBB to macromolecules and nanoparticles. However, the mechanism underlying the induced permeability change and subsequent enhanced accumulation of co-injected molecules requires further elucidation. In this study, the behavior of ACT® bubbles in microcapillaries in the murine brain was observed using real-time intravital multiphoton microscopy. For this purpose, cranial windows aligned with a ring transducer centered around an objective were mounted to the skull of mice. Dextrans labeled with 2 MDa fluorescein isothiocyanate (FITC) were injected to delineate the blood vessels and to visualize extravasation. Activated ACT® bubbles were observed to alter the blood flow, inducing transient and local increases in the fluorescence intensity of 2 MDa FITC-dextran and subsequent extravasation in the form of vascular outpouchings. The observations indicate that ACT® induced a transient vascular leakage without causing substantial damage to the vessels in the brain. The study gave novel insights into the mechanism underlying ACT®-induced enhanced BBB permeability which will be important considering treatment optimization for a safe and efficient clinical translation of ACT®.

Imaging of lung resident memory CD8 +T cells reveals antigen-specific early protective responses during secondary infection

Abstract Tissue-resident memory T (T RM) cells are poised at mucosal sites to confer robust protection against secondary infection. In contrast to skin and intestine, lung T RMcells are short lived with a decline in number coinciding with a decline in protective immunity against subsequent heterosubtypic influenza infections. Despite this causal relationship, the localization, dynamics and function of lung Trm during secondary responses remains poorly defined. To address this, we used two-photon intravital microscopy of lungs to assess in real time the spatiotemporal dynamics of lung T RMcells during recall infection. At a memory time point, lung T RMcells (representing both CD103 +and CD103 −subsets) exhibit significantly lower motility and less displacement compared to an CD8 +effector T cells at an earlier time point post infection. Depletion of circulating CD8 +T cells using a low dose antibody administered systemically led to enrichment of lung T RMcells that exhibited slow to moderate speed scanning patterns. A fate mapping model for CD103 +T RMcells specifically revealed limited speed (<5 um/min) and patrolling behavior compared to CD103 −T RMcells. Within 24 hr of rechallenge, CD103 +T RMcells co-localized with infected cells and by 48 hr, lung-resident CD8 +T cells exhibit significantly lower motility with recruitment and clustering in infected regions in an antigen-specific manner. Protective immune responses by lung T RMcells is further accompanied by CXCR3-dependent migration of dendritic cells to draining lymph nodes as well as recruitment of inflammatory monocytes. Overall, these data unravel the spatiotemporal dynamics of lung T RMcells and interactions with recruited immune cells for early protective immunity. Supported by grants from NIH (GM134880, AI42767, AI114543, AI151183, AI167249, the Holden Cancer Center at the University of Iowa and its National Cancer Institute Award P30CA086862

Regulatory T cells aid Leishmania majorimmune evasion by suppressing effector responses against the parasite in an antigen-specific manner

Abstract Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete and leaves a pool of chronically infected cells. Understanding of the persistence is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells at the site of infection has been proposed to prevent parasite clearance in vivo. Here, we used a novel TCR transgenic mouse model, where CD4 +T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4 +T cell responses and characterize mechanisms which restrain their effector function. We show that PEPCK-specific Tregs are able to suppress nearly 80% of IFNg produced by PEPCK-specific Th1 cells in vitro though the production of IL-10, while decreasing IL-12, TNF, and IL-2. Intravital microscopy studies characterizing PEPCK-specific CD4 +T cell migration dynamics in live mice show a significant recruitment of adoptively transferred cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition, yet augmented at the chronic stage, indicating a fundamentally altered environment. Injection of heat-killed L. major into mice which have already healed a L. major infection, distal to the original lesion, induced a rapid expansion of PEPCK-specific Tregs at the original site of infection, which suppressed endogenous anti-Leishmania response through IL-10 and led to parasite expansion and lesion reactivation. Our results show that antigen recognition by Tregs plays an important role in suppressing effector responses against L. major and thus maintaining concomitant immunity. Research funded by a grant from the GSK-CIHR Partnered Chair Program

Abstract A26: Age and Dose Related Changes to the Bone Marrow Microenvironment after Cytotoxic Conditioning with Busulfan

Abstract Hematopoietic Cell Transplantation (HCT) is a frequently used treatment for hematologic malignancies such as acute myeloid leukemia, multiple myeloma, lymphoma and non-malignant diseases. Preparative regimens before HCT damage the Bone Marrow (BM) niche, but it is not fully known how the cytotoxic preconditioning, whether High or Low intensity, impacts bone and BM remodeling, regeneration, and subsequent hematopoietic recovery over time. In addition, the effect of recipient age on these factors has not been completely described. In this study, we sought to longitudinally investigate bone and BM remodeling after Low and High intensity Busulfan (BU) conditioning with the aim of understanding the role that BM niche alterations play in the recovery of the hematopoietic system after transplantation. Using two-photon intravital microscopy, we visualized bone and BM changes in young and adult mice on days 2, 5, and 42 post-transplantation. Both Low and High intensity conditioning were administered with injections of 40 mg/kg and 80 mg/kg busulfan, respectively. Busulfan is a DNA alkylating drug that in combination with cyclophosphamide is being clinically used to treat leukemia. Mice were then transplanted with 1 × 106 whole BM cells from a C57BL/6-Tg(UBC-GFP) mouse. During live imaging, functionality of the vascular system and hematopoietic recovery were studied. For ex vivo imaging, vascular labeling fluorescent antibodies (Alexafluor 647 conjugated to anti-CD31, CD144, and Sca-1) and calcium binding dyes (dye1; Calcein, Dye2; Alizarin) were administered before intracardiac perfusion. Then long bones were harvested, frozen, and the cortical layer was shaved to enable visualization of the BM. Vascular analysis indicated increased leakage in both Low and High intensity conditioning even after 42 days suggesting delayed endothelial recovery following conditioning. Morphological evaluation of the vascular network revealed a decrease in the frequency and increase in the size and density of the vessels at early timepoints that was partially restored by day 42 in young mice only. Bone remodeling in young mice showed a reduction in the dye1/dye2 ratio at day 5 and a more significant decrease by day 42. In the adult mice, a low ratio was observed in BU-conditioned mice only at day 42 post treatment. To further investigate, we classified the metaphyseal and epiphyseal long bone cavities as deposition type, mixed type, and resorption type based on the ratio of the two dyes. The classification analysis indicates that the reduction in dye1/dye2 ratio in BU-conditioned mice over time is primarily due to an increase in resorption type bone cavities in BU-conditioned mice compared to the control. Although donor HSC engraftment is known to be age and dose dependent, our results indicate that long-term microenvironmental changes in the bone and bone marrow may further impact hematopoietic recovery. Overall, our results demonstrate new aspects of bone remodeling and bone marrow regeneration that may influence hematopoietic recovery and warrant further investigation. Citation Format: Joel A Spencer, Christian Burns, Ruth Verrinder, Farhad Ghazali, Nastaran Abbasizadeh. Age and Dose Related Changes to the Bone Marrow Microenvironment after Cytotoxic Conditioning with Busulfan [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A26.

Attenuated hyaluronan synthesis impairs glycocalyx integrity and leads to arterial dysfunction

The glycocalyx (GC) is a semi-solid layer lining the endothelium that plays a key role in maintaining barrier function and mechanosensing shear forces. With advanced age and pathological conditions, the GC deteriorates, and this is accompanied by arterial dysfunction characterized by impaired endothelial-dependent dilation (EDD) and large elastic artery stiffening. Alterations in hyaluronan (HA), a primary component of the GC, may contribute to the development of a dysfunctional artery phenotype. Here, we sought to determine the role of HA in GC integrity and arterial function. We have generated a tamoxifen-inducible, endothelial cell-specific, knockout mouse model of the hyaluronan producing enzyme, hyaluronan synthase 2 (HAS2, HAS2 f/f- Vecad-CreERT2). Endothelial HAS2 knockout (HAS2 f/f- Vecad-creERT2+: ecHAS2 KO, n=10) and wildtype (HAS2 +/+- Vecad-creERT2+: ecHAS2 WT, n=10) young mice were injected intraperitoneally with 4 mg of tamoxifen for 5 consecutive days. HAS2 mRNA expression was reduced by ~47% in carotid endothelial cells of ecHAS2 KO mice (P=0.004). GC barrier function (perfused boundary region [PBR]) was evaluated in vivo in the mesenteric microcirculation using an intravital microscope equipped with an automated capture and analysis system. PBR was 8.9% higher in ecHAS2 KO compared to ecHAS2 WT (P<0.0001). Subsequently, video recordings acquired were used to manually determine GC thickness from individual microvascular segments. GC thickness was significantly reduced in ecHAS2 KO vs ecHAS2 WT (7.9μm±0.02 vs 0.37μm±0.02, P<0.0001). Together, these results indicated that GC integrity was deteriorated in response to a reduction in endothelial HAS2. This GC deterioration impairs EDD, as evidenced by a 43.4% reduction in flow-induced dilation in isolated carotid arteries from ecHAS2 KO compared to ecHAS2 WT (P=0.013). To directly assess the role of GC plays in the differences in flow-induced EDD observed, the lumen of the arteries was infused with hyaluronidase, a HA degrading enzyme, and this abolished the differences in EDD between groups (P>0.05). Endothelial-independent dilation in response to sodium nitroprusside was not different between groups (P>0.05). Furthermore, aortic pulse wave velocity, a measure of large elastic artery stiffness, as well as systolic, diastolic, and mean arterial blood pressure were all greater in ecHAS2 KO compared to ecHAS2 WT (all P≤0.033). These results demonstrate that endothelial cell-specific impairment in HA synthesis compromises GC integrity and leads to arterial dysfunction and elevated blood pressure. Funded in part by awards from National Institutes of Health Awards R01 AG060395, R01 AG048366, T32 HL007576, T32 HL139451 and Veteran's Affairs Merit Review Award I01 BX004492. 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.

Feeding enhances fibronectin adherence of quiescent lymphocytes through non-canonical insulin signalling.

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of β-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin-integrin interaction.

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.

Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.

In situ expansion and reprogramming of Kupffer cells elicit potent tumoricidal immunity against liver metastasis.

Liver metastasis represents one of the most frequent malignant diseases with no effective treatment. Functional reprogramming of Kupffer cells (KCs), the largest population of hepatic macrophages, holds promise for treating liver cancer, but remains seldom exploited. Taking advantage of the superior capacity of KCs to capture circulating bacteria, we report that a single administration of attenuated Escherichia coli producing clustered regularly interspersed short palindromic repeats CasΦ (CRISPR/CasΦ) machinery enables efficient editing of genes of interest in KCs. Using intravital microscopy, we observed a failure of tumor control by KCs at the late stage of liver metastasis due to KC loss preferentially in the tumor core and periphery, resulting in inaccessibility of these highly phagocytic macrophages to cancer cells. Simultaneous disruption of MafB and c-Maf expression using the aforementioned engineered bacteria could overcome KC dysfunction and elicit remarkable curative effects against several types of metastatic liver cancer in mice. Mechanistically, bacterial treatment induced massive proliferation and functional reprogramming of KCs. These cells infiltrated into the tumor, dismantled macrometastases by nibbling cancer cells, and skewed toward proinflammatory macrophages to unleash antitumor T cell responses. These findings provide an immunotherapy strategy that could be applicable for treating liver metastasis and highlight the therapeutic potential of targeting tissue-resident macrophages in cancer.

Human intravital microscopy in the study of sarcomas: an early trial of feasibility.

ClinicalTrials.gov, identifier NCT03517852; ClinicalTrials.gov, identifier NCT03823144.

A Novel Preclinical Murine Model to Monitor Inflammatory Breast Cancer Tumor Growth and Lymphovascular Invasion

Inflammatory breast cancer (IBC), an understudied and lethal breast cancer, is often misdiagnosed due to its unique presentation of diffuse tumor cell clusters in the skin and dermal lymphatics. Here, we describe a window chamber technique in combination with a novel transgenic mouse model that has red fluorescent lymphatics (ProxTom RFP Nu/Nu) to simulate IBC clinicopathological hallmarks. Various breast cancer cells stably transfected to express green or red fluorescent reporters were transplanted into mice bearing dorsal skinfold window chambers. Intravital fluorescence microscopy and the in vivo imaging system (IVIS) were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140 h. This short-term, longitudinal imaging time frame in studying transient or dynamic events of diffuse and collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility, and vessel characteristics can be expanded to investigate other cancer cell types exhibiting lymphovascular invasion, a key step in metastatic dissemination. It was found that these models were able to effectively track tumor cluster migration and dissemination, which is a hallmark of IBC clinically, and was recapitulated in these mouse models.

Mechanosensing via a GpIIb/Src/14-3-3ζ axis critically regulates platelet migration in vascular inflammation

AbstractPlatelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ–dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

Abstract 2397: An advanced approach technique for tracking point between immune check point inhibitor and cancer cell mechanism in immune response motility by infiltration a breast cancer cell

Abstract In Breast Cancer, Tumor-infiltrating lymphocyte (TIL) motility has been reported that it is reflecting tumor microenvironment response by adoptive cell therapy (ACT). ACT is well-known to be best applied for suppressing cancer cell growth with immune response and it also was well-known the immunotherapy. However, ACT response tracking by general analysis method included immunohistochemistry and flow cytometry was provided limited information for cancer therapy in recent years. Therefore, by assessing relationship immunotherapy marker, PD-L1, known as immune check point inhibitor and TIL using intravital microscopy in breast cancer model, we also imaged whether TIL response was related to immunotherapy following dependent on time point in a various breast cancer cell implanted mouse model such as 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple negative breast cancer cell) and MCF7 (human breast cancer cell). These images result in PD-L1 expression was decreased in cancer cell implanted until 10days, whereas TIL expression increased in a various breast cancer cell when it is measuring displacement length, track mean velocity and confinement ratio called tumor microenvironment. These results indicated that we can imaged immune response related to TIL response in breast cancer cell line by intravital microscopy instead of general microscopy. It means that intravital microscope was an advanced approach technique to understand cancer mechanism accompanied by the immune response and established the method of high-resolution intravital cellular visualization compared to a general histopathological method for evaluating immune response in cancer tissue Citation Format: Hyunseok Kim, Soyeon Ahn, Hyungjin Kwon, Hsuan-Ju Chen, Inwon Park, Pilhan Kim, Akyildiz Kubra. An advanced approach technique for tracking point between immune check point inhibitor and cancer cell mechanism in immune response motility by infiltration a breast cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2397.

Abstract 2736: Locoregional modified vaccinia ankara (MVA) encoding a single-chain IL-12 achieves efficacy antitumor immune response in peritoneal carcinomatosis

Abstract Peritoneal carcinomatosis is an advanced stage of cancer developed in the peritoneal cavity without effective treatment. In order to restore antitumor immunity subverted by tumor cells, we evaluated in this study intraperitoneal administration of vaccinia virus Ankara (MVA) genetically modified to express the proinflammatory cytokine single-chain interleukin 12. MVA encoding scIL-12 was evaluated in peritoneal carcinomatosis models based on intraperitoneal administration of tumor cells. The immune response was evaluated by ELISpot, flow cytometry, intravital microscopy, and depleting monoclonal antibodies. Safety was assessed by measuring body weight and a hemogram test. Tissue tropism after intravenous or intraperitoneal administration was determined by bioluminescence analysis after the administration of MVA encoding luciferase. Intraperitoneal or locoregional administration, but not other routes of administration, elicited a potent immune response characterized by increased levels of tumor-specific T CD8 lymphocytes. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result, a single intraperitoneal administration of MVA encoding scIL-12 completely eradicates MC38 cells implanted in the peritoneum, protecting cured mice from a subcutaneous rechallenge with MC38 cells. The safety profile of intraperitoneal administration was also superior to that of intravenous administration since there was no weight loss or hematological toxicity when the vector was infused into the peritoneal cavity. In conclusion, intraperitoneal administration of MVA vectors encoding scIL-12 induces a potent tumor-specific immune response that leads to the eradication of peritoneal metastasis. Citation Format: Ángela Bella, Leire Arrizabalaga, Claudia Augusta Di Trani, José González-Gomariz, Assunta Cirella, Irene Olivera, Maite Alvarez, Álvaro Teijeira, Cigdem Atay Langbein, José Medina-Echeverz, Maria Hinterberger, Hubertus Hochrein, Ignacio Melero, Pedro Berraondo, Fernando Aranda. Locoregional modified vaccinia ankara (MVA) encoding a single-chain IL-12 achieves efficacy antitumor immune response in peritoneal carcinomatosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2736.

Abstract 3589: The use of a novel orthotopic xenograft model of fusion negative rhabdomyosarcoma to study the effect of ASAP1 on tumor progression and metastasis

Abstract ASAP1 is a multidomain ADP-ribosylation factor (Arf) GTPase-activating protein (GAP) involved in the regulation of the actin cytoskeleton, focal adhesions dynamics and receptor tyrosine kinases trafficking. ASAP1 expression levels correlate with progression of solid tumors (e.g. breast, colorectal, pancreatic, ovarian, gastric, and prostate cancer). The first evidence for a role of ASAP1 in cancer progression was the discovery that the amplification of ASAP1 gene on chromosome 8 correlates with poor prognosis in uveal melanoma. A similar amplification of chromosome 8 has been reported in ~72% of fusion negative rhabdomyosarcoma (FN-RMS) tumors, suggesting a potential role for ASAP1 signaling in the progression of FN-RMS. To determine the function of ASAP1 in tumor growth, invasion and metastasis, we used a tongue orthotopic xenograft of FN-RMS which recapitulates physiologically relevant tumor progression and metastasis to lymph nodes and lungs, two of the common metastatic sites in patients. Using intravital microscopy, we observed that injected FN-RMS cells formed elongated structures and exhibited dynamic branching, which might represent local invasion. H&E stained of fixed tissue revealed invasion into the tongue and local metastasis. Metastases to the regional lymph nodes, lower mandible and lungs were also observed. Using this system, we are now able to determine effect of knocking down and knocking out ASAP1 on the progression of the xenografts, including invasion in real time and metastasis. Citation Format: Sarah M. Hammoudeh, Yeap Ng, Mukesh P. Yadav, Roberto Weigert, Paul A. Randazzo. The use of a novel orthotopic xenograft model of fusion negative rhabdomyosarcoma to study the effect of ASAP1 on tumor progression and metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3589.

128 NMDA-Receptor Antagonism for the Prevention of Neurological Dysfunction in Traumatic Brain Injury: Results of a Randomized Pre-Clinical Trial

INTRODUCTION: Cortical spreading depolarization (CSD) represents a pathomechanistic target for neuroprotective therapy given the association with lesion development and poor outcomes following traumatic brain injury (TBI). METHODS: Experiments were conducted using 9-week-old Sprague Dawley rats. Daily neurobehavioral scores were recorded by trained, blinded observers. Using an established weight-drop model, animals received either single moderate (modTBI; n = 23) or four daily mild (rmTBI; n = 30) head impacts. Sham animals received brief anesthetic without TBI (n = 40). Animals underwent cranial window surgery and CSDs were electrically triggered by cortical stimulation; electrophysiological, imaging and blood-flow were monitored using intravital microscopy. Ketamine (100uM topical and 25 mg/kg IP, respectively) and memantine (10 mg/kg IP) were tested in vivo. Subsequently, a RCT was conducted (N=31) using memantine (10 mg/kg) or saline (2.5 cc/kg). Primary outcome: proportion of animals resilient to rmTBI (neurobehavioral scores ≥ 6 out of 12). RESULTS: In modTBI, and rmTBI animal models, intraperitoneal ketamine and memantine reduced the proportion of electrically triggered CSDs by 79-88%, and 42-73%, respectively. The average relative amplitude of change in ECoG were reduced by 70-75%, and 51-60%, respectively. Memantine significantly reduced spreading depression and cortical hypoperfusion following CSD. RCT animals in the memantine group were more likely resilient to injury (93% vs. 56%; p = 0.023) and had higher mean neurological scores (9.27 (SD 3.08) vs. 5.56 (SD 3.05)), p < 0.001) compared to saline. CONCLUSIONS: The NMDA-receptor antagonist memantine supresses CSDs and reduces pathological cerebral hypoperfusion in vivo following CSDs. In a rmTBI RCT, memantine prevented neurological decline.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4+ anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive Tcell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance Tcell infiltration and the intratumoral performance of T lymphocytes.

LP342, a novel histone deacetylase inhibitor, decreases nitro-oxidative stress, mitochondrial dysfunction and hepatic ischaemia/reperfusion injury in mice.

Some histone deacetylase (HDAC) isoforms contribute to ischaemia/reperfusion (IR) injury (IRI). Here, we examined whether LP342, the lead candidate of a new generation of hydrazide-based HDAC inhibitors (HDACi), decreases hepatic IRI. IR was induced by clamping blood vessels to ~70% of the livers of mice for 1 h. At 6 h after reperfusion, ALT markedly increased, and wide-spread necrosis, leukocyte infiltration, and apoptosis occurred. LP342 treatment (1 mg/kg, ip) at 20 h or 1 h before ischaemia markedly decreased IRI whereas LP342 treatment upon reperfusion was marginally protective. Nitro-oxidative stress, c-Jun-N-terminal kinase (JNK) activation, and mitochondrial dysfunction contribute to IRI. 4-Hydroxynonenal, 3-nitrotyrosine, inducible nitric oxide synthase (iNOS), JNK activation and Sab binding increased markedly after IR, which LP342 blunted. LP342 also induced thioredoxin-1 expression before and after IR. LP342 also decreased mitochondrial depolarisation as detected by intravital microscopy at 2 h after IR. Lastly, LP342 increased acetylation of both histone-3 (class I HDAC substrate) and NFκB p65 but not tubulin (class II HDAC substrate) before and after IR. This novel HDACi protects against IRI most likely by epigenetic upregulation of antioxidant proteins and post-translational modifications of NFκB thus inhibiting iNOS expression and inflammatory responses.