Bioluminescence Imaging System Research Articles

Osteostaticytes: A novel osteoclast subset couples bone resorption and bone formation

BackgroundOsteomyelitis (OM) is an inflammatory condition of bone characterized by cortical bone devascularization and necrosis. Dysregulation of bone remodelling is triggered by OM. Bone remodelling is precisely coordinated by bone resorption and formation via a reversal phase. However, the cellular and molecular mechanisms underlying bone remodelling failure after osteomyelitis remain elusive. MethodsTo elucidate the cellular and molecular mechanism underlying bone healing after osteomyelitis, we employed single-cell RNA sequencing (scRNA-seq) to depict the atlas of human cortical bone in normal, infected and reconstructed states. Dimensionality reduction by t-stochastic neighbourhood embedding (t-SNE) and graph-based clustering were applied to analyse the detailed clusters of osteoclast lineages. After trajectory analysis of osteoclast lineages over pseudotime, real-time PCR and immunofluorescence (IF) staining were applied to identify marker gene expression of various osteoclast lineages in the osteoclast induction model and human bone sections, respectively. The potential function and communication of osteoclasts were analysed via gene set enrichment analysis (GSEA) and CellChat. The chemotactic ability of mesenchymal stem cells (MSCs) and osteoclast lineage cells in various differentiation states was determined by transwell assays and coculture assays. The effects of various osteoclast lineages on the osteogenic differentiation potential of MSCs were also determined by using this coculture system. A normal mouse tibia fracture model and an osteomyelitis-related tibia fracture model were generated via injection of luciferase-labelled Staphylococcus aureus to verify the relationships between a novel osteoclast lineage and MSCs. Then, the infection was detected by a bioluminescence imaging system. Finally, immunofluorescence staining was used to detect the expression of markers of MSCs and novel osteoclast lineages in different remodelling phases in normal and infected bone remodelling models. ResultsIn this study, we constructed a cell atlas encompassing normal, infected, and reconstructed cortical bone. Then, we identified a novel subset at the earlier stage of the osteoclast lineage that exhibited increased expression of IDO1, CCL3, and CCL4. These IDO1highCCL3highCCL4high cells, termed osteostaticytes (OSCs), were further regarded as the reservoir of osteoclasts in the reversal phase. Notably, OSCs exhibited the highest chemotactic activity, surpassing other lineage subsets. We also discovered that cells at the earlier stage of the osteoclast lineage play a significant role in recruiting mesenchymal stem cells (MSCs). Finally, the data revealed that OSCs might be positively related to the occurrence of bone MSCs and the contribution of bone remodelling. ConclusionCollectively, our findings revealed a novel stage (OSC) within the osteoclast lineage, potentially representing elusive bone reversal cells due to its increased chemotactic ability towards MSCs and potential contribution to bone remodelling. This study provides valuable insights into the intricate mechanisms of the reversal phase during bone remodelling and unveils potential therapeutic strategies for diseases associated with bone uncoupling. Translational potential of this articleThis study identified a new subset, referred to as IDO1(plus symbol) CCL3(plus symbol) CCL4(plus symbol) osteostaticytes which displayed the highest chemotactic activity among all osteoclast lineages and may serve as reversal cells in bone remodelling. These findings offer new insights and insights for understanding bone reversal-related diseases and may serve as novel therapeutic targets for conditions such as osteomyelitis and delayed bone healing.

Dynamics of Trypanosoma cruzi infection in hamsters and novel association with progressive motor dysfunction.

Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Clinical outcomes range from long-term asymptomatic carriage to cardiac, digestive, neurological and composite presentations that can be fatal in both acute and chronic stages of the disease. Studies of T. cruzi in animal models, principally mice, have informed our understanding of the biological basis of this variability and its relationship to infection and host response dynamics. Hamsters have higher translational value for many human infectious diseases, but they have not been well developed as models of Chagas disease. We transposed a real-time bioluminescence imaging system for T. cruzi infection from mice into female Syrian hamsters (Mesocricetus auratus). This enabled us to study chronic tissue pathology in the context of spatiotemporal infection dynamics. Acute infections were widely disseminated, whereas chronic infections were almost entirely restricted to the skin and subcutaneous adipose tissue. Neither cardiac nor digestive tract disease were reproducible features of the model. Skeletal muscle had only sporadic parasitism in the chronic phase, but nevertheless displayed significant inflammation and fibrosis, features also seen in mouse models. Whereas mice had normal locomotion, all chronically infected hamsters developed hindlimb muscle hypertonia and a gait dysfunction resembling spastic diplegia. With further development, this model may therefore prove valuable in studies of peripheral nervous system involvement in Chagas disease.

Animal models of neglected parasitic diseases: In vivo multimodal imaging of experimental trypanosomatid infections.

African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (μCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.

Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost N-acetylglucosamine residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous knockout (Fut8−/−) or heterozygous knockout (Fut8+/−) mice, compared with the wide-type (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome (BAC) transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this BAC transgenic monitoring system. Then we examined the effects of L-fucose on neuroinflammation in the Fut8+/− mice. The lectin blot and mass spectrometry analysis showed that L-fucose pre-administration increased the core fucosylation levels in the Fut8+/− mice. Notably, exogenous L-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues induced by LPS, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by L-fucose pre-administration. L-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2, Akt, and STAT3. L-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/− mice, which was further confirmed in BV-2 cells. These results indicate that L-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/− mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that L-fucose is a potential prophylactic compound against neuroinflammation.

Co-overexpression of OPN, IGF-1 and CNTF augment the therapeutic effect of DPSC on spinal cord injury

Background aimsSpinal cord injury (SCI) is one of the most complex and destructive diseases of the nervous system, which can lead to permanent loss of tactile perception. But existing treatment methods have limited effects. To establish a novel method that may be therapeutic in repairing the injured spinal cord, gene-modified dental pulp stem cells (DPSCs) were injected in situ. MethodsAdenovirus carrying osteopontin (OPN), Insulin-like growth factor 1 (IGF-1) and cailiary-derived neurotrophic factor (CNTF) (Ad-OIC) was constructed. After modified with Ad-OIC, supernatant of DPSC were co-cultured with HT-22 cells and the effect of DPSC-OIC on the HT-22 cells were evaluated via Cell Counting Kit-8 (CCK-8) assay, Real-Time polymerase chain reaction (PCR) analysis, laser confocal microscopy and fluorescence activating cell sorter (FACS). DPSC-OIC were injected in the lesion area of injured spinal cord and the survival time of transplanted cells were measured by bioluminescence imaging system. The recovery of the injured spinal cord was evaluated by behavioral score, radiological evaluation and immunopathological analysis. ResultsDPSC-OIC could enhance the proliferation and axon growth of HT-22 cells, and protect HT-22 cells from H2O2 induced apoptosis. The transplanted DPSC-Null or DPSC-OIC could survive for more than two weeks in local injection site. DPSC-OIC treatment could increase Basso-Mouse Scale (BMS) scores, improve Magnetic Resonance Imaging (MRI) manifestation and promote bladder function recovery. Less apoptotic neurons and more proliferative cells were found in the lesion area of DPSC-OIC treated spinal cord. Nestin+ cells and neural stem cell marker (Sox2) were both up-regulated after DPSC-OIC treatment. Additionally, inhibitory extracellular matrix proteoglycan Neural/Glial Antigen 2 (NG2) was down-regulated and axon growth promotive factor fibronectin was up-regulated after both DPSC-Null (DPSCs infected with Ad-Null) and DPSC-OIC treatments. ConclusionsDPSC-OIC could be a novel effective method for treating SCI.

Bio-Orthogonally Redirected Bispecific Lentiviral Vectors for In Vivo CAR-T Cell Generation

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a remarkably efficacious treatment modality in recent years for refractory and relapsed hematopoietic malignancies. However, the exorbitant cost and intricate manufacturing procedure of ex vivo CAR-T cell generation have impeded its broader application. In vivo induced CAR-T therapy offers the potential to circumvent cumbersome manufacturing logistics, thereby providing novel insights into the field of CAR-T treatment. In this study, we have developed a bio-orthogonally redirected bispecific lentiviral vector platform for the reprogramming of circulating T lymphocytes into CAR-T cells and subsequent elimination of tumor cells in vivo. Methods: To engineer dual-targeting lentiviral vectors (D-LVs), the packing cell lines 293T was labelled with azide groups by glycometabolic bio-orthogonal chemistry (designated N 3-293T) firstly, and a CD3 promoter (CD3p) was fused to CAR transfer plasmid. Secondly, azide groups modified LVs were further surface engineered with anti-CD3 antibody (OKT3) via click chemistry. The conjunction of OKT3 and was analyzed using dot immunobinding assay, confocal microscopy and the in vitro transduction efficiency was evaluated using flow cytometry. To demonstrate the ability of targeted transduction and specific cytotoxicity in vivo, D-LVs were intravenously infused into humanized NOD-scid-IL2Rγnull (huNSG) mice engrafted with Nalm6-luc cells. Subsequently, the tumor burden was monitored using a noninvasive bioluminescence imaging system and the in-vivo CD19 -CAR-T cells existence were detected by flow cytometry. Results: By displaying OKT3 on the single lentiviral surface and fused CD3p into the key construct, we could achieve targeted delivery of CD19-CAR genes to T cells both in vitro and in vivo. Conclusion: These results demonstrate the great potential applications of this engineered lentiviral system as a new strategy for inducing CAR-T immunotherapy in vivo and a promising approach for leading personalized treatment.

Bioluminescent imaging systems boosting near-infrared signals in mammalian cells.

Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.

S-Series Coelenterazine-Driven Combinatorial Bioluminescence Imaging Systems for Mammalian Cells.

A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named "S-series". For this study, ten kinds of novel S-series CTZ analogues were synthesized and characterized concerning the BL intensities, spectra, colors, and specificity of various marine luciferases. The characterization revealed that the S-series CTZ analogues luminesce with blue-to-orange-colored BL spectra with marine luciferases, where the most red-shifted BL spectrum peaked at 583 nm. The colors completed a visible light color palette with those of our precedent C-series CTZ analogues. The synthesized substrates S1, S5, S6, and S7 were found to have a unique specificity with marine luciferases, such as R86SG, NanoLuc (shortly, NLuc), and ALuc16. They collectively showed unique BL intensity patterns to identify the marine luciferases together with colors. The marine luciferases, R86SG, NLuc, and ALuc16, were multiplexed into multi-reporter systems, the signals of which were quantitatively unmixed with the specific substrates. When the utility was applied to a single-chain molecular strain probe, the imaging system simultaneously reported three different optical indexes for a ligand, i.e., unique BL intensity and color patterns for identifying the reporters, together with the ligand-specific fold intensities in mammalian cells. This study directs a new combinatorial BL imaging system to specific image molecular events in mammalian cells with multiple optical indexes.

Quantifying bacterial concentration in water and sand media during flow-through experiments using a non-invasive, real-time, and efficient method.

Monitoring the dynamics of bacteria in porous media is of great significance to understand the bacterial transport and the interplay between bacteria and environmental factors. In this study, we reported a non-invasive, real-time, and efficient method to quantify bioluminescent bacterial concentration in water and sand media during flow-through experiments. First, 27 column experiments were conducted, and the bacterial transport was monitored using a real-time bioluminescent imaging system. Next, we quantified the bacterial concentration in water and sand media using two methods-viable count and bioluminescent count. The principle of the bioluminescent count in sand media was, for a given bioluminescence image, the total number of bacteria was proportionally allocated to each segment according to its bioluminescence intensity. We then compared the bacterial concentration for the two methods and found a good linear correlation between the bioluminescent count and viable count. Finally, the effects of porous media surface coating, pore water velocity, and ionic strength on the bioluminescent count in sand media were investigated, and the results showed that the bioluminescence counting accuracy was most affected by surface coating, followed by ionic strength, and was hardly affected by pore water velocity. Overall, the study proved that the bioluminescent count was a reliable method to quantify bacterial concentration in water (106 to 2 × 108 cell mL-1) or sand media (5 × 106-5 × 108 cell cm-3). This approach also offers a new way of thinking for in situ bacterial enumeration in two-dimensional devices such as 2D flow cells, microfluidic devices, and rhizoboxes.

Activated CMPs Act in Concert with the Spleen and Accelerate Hematopoietic Inflation after Transplantation

Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After transplantation, injected HSCs rapidly expand to reconstitute the entire blood system. This early post-transplant "hematopoietic inflation" is a crucial phase in sustaining life. However, the time course of hematopoietic inflation and the spatial transition within the whole body have remained a mystery. The discovery of colonies forming in the spleen early after HSC transplantation in the 1960s has been a strong advocate of the existence of HSCs, but biological role of transient nature of spleen colonies has not been understood. In this study, we elucidated the spatiotemporal dynamics of hematopoietic inflation in the same individual in a murine HSC transplantation model. Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize hematopoietic inflation in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and each progenitor cell has a different hematopoietic inflation pattern. We found that activated common myeloid progenitors (aCMPs) use the spleen as a site for hematopoietic inflation, producing much more cells early after transplantation than normal CMPs. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. In the allogeneic transplantation setting, universal exCMPs could sustain life for a long time by a serial transplantation. Human exCMPs could be easily expanded as well, and the clinical use of human exCMPs is highly anticipated as a supportive therapy after HSCT. Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that aCMPs and the spleen cooperated to contribute to hematopoietic inflation post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

The Evaluation of Novel Targets for CAR T Therapy in Glioblastoma

Abstract AIMS The basis of this research revolves around the issue of limited expression of targetable tumour-specific antigens, heterogeneity, and mutation, which impede the development and success of CAR T therapy for glioblastoma. The research will identify an off-tumour cell target involved in tumour angiogenesis. The principal aim is to generate an off-tumour targeted CAR T and test their efficacy and safety in cell culture and animal models. METHOD Analyse the prevalence and distribution of the target molecules via immunohistochemistry (IHC). Confirm IHC results via mRNA extraction. Analyse the target molecule’s serum concentrations via the Luminex instrument. Generate the off-tumour targeted CAR T using lentivirus. Assess tumour burden and progression via a bioluminescent imaging system. Assess toxicity in vitro against human pericytes and in vivo by monitoring mouse weight and submitting histologic tissue for sectioning. RESULTS Utilise a scoring system to analyse the staining and correspond this to the serum concentrations to identify a blood-borne treatment response biomarker. If murine toxicity is observed, the CAR T construct will be affinity matured to selectively target molecules expressed at a cancerous level. A tandem bi-specific CAR T cells may also be created to mitigate immune escape and enhance anti-tumour efficacy. CONCLUSION The search for an off-tumour cell target for CAR T therapy in glioblastoma has not yet been completed. The discovery and validation of the novel molecule as a biomarker of treatment response would be a unique finding.

Eupalinolide O Induces Apoptosis in Human Triple-Negative Breast Cancer Cells via Modulating ROS Generation and Akt/p38 MAPK Signaling Pathway.

Background Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited therapeutic options. Eupalinolide O (EO) was reported to inhibit tumor growth. This study is aimed at exploring the role of EO on TNBC both in vivo and in vitro. Methods. In in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assay were conducted to measure the impact of EO on TNBC cell growth at different concentrations and time points. Flow cytometry was conducted to evaluate cell apoptosis. Mitochondrial membrane potential (MMP) loss, caspase-3 activity, and reactive oxygen species (ROS) generation were assessed. The expressions of apoptosis-related mRNAs and Akt/p38 MAPK signaling pathway-related proteins were measured. In in vivo experiments, by injecting TNBC cells into the nude mice to induce xenograft tumor, mice were treated with EO for 20 days. Then, in vivo bioluminescence imaging system was utilized to monitor the growth and distribution of TNBC cells. Tumor volume and weight were also recorded. Hematoxylin-eosin (HE) staining and ELISA assay were applied to observe tumor tissue morphology and ROS levels. Furthermore, western blotting was conducted to observe the expression of apoptosis-related proteins and Akt/p38 MAPK signaling pathway-associated proteins. Results EO inhibited the cell viability and proliferation of TNBC cells but not normal epithelial cells. Furthermore, EO induced apoptosis, decreased MMP, and elevated caspase-3 activity and ROS content in TNBC cells. Meanwhile, the expression of apoptosis-related mRNAs and Akt/p38 MAPK pathway-related proteins was regulated by EO treatment. Besides, in vivo experiments demonstrated EO not only suppressed tumor growth, Ki67 expression, ROS generation, and Akt phosphorylation but also upregulated caspase-3 expression and p-38 phosphorylation. Conclusion EO may induce cell apoptosis in TNBC via regulating ROS generation and Akt/p38 MAPK pathway, indicating EO may be a candidate drug for TNBC.

P1418: EXPANDED COMMON MYELOID PROGENITORS ACT IN CONCERT WITH THE SPLEEN AND SUPPORT EARLY HEMATOPOIETIC RECOVERY

Background: Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After intravenous injection, HSCs are thought to home directly toward the bone marrow niche, and their homing mechanisms and interactions with the microenvironment have been intensively studied. However, the time course of the transplanted HSCs to reconstitute the entire blood system and the spatial transition within the whole body have remained a mystery. Aims: We aim to elucidate the spatiotemporal dynamics of hematopoiesis in the same individual in a murine HSC transplantation model. Methods: Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize HSC migration in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Results: Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and we found that common myeloid progenitors (CMPs) use the spleen as a site for hematopoiesis, producing many cells early after transplantation. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. Image:Summary/Conclusion: Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that CMPs and the spleen cooperated to contribute to early hematopoiesis post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery.

Abstract 695: Patient derived organoids model lung adenocarcinoma metastases

Abstract Introduction: While lung cancer patient-derived xenografts (PDX) models predict response to therapy, PDX tumors rarely metastasize thus offering little personalized assessment of the biology of metastases. We hypothesize that the development of an in vivo patient-derived organoid (PDO) model will improve our understanding of the underlying genomic and transcriptomic alterations that help drive the biology of metastases for specific lung adenocarcinoma (LUAD) histologic subtypes. Methods: Fresh LUAD specimens were processed and cultured using optimized organoid culture conditions. Established PDOs were characterized by histologic analyses, RNA-sequencing and whole genomic sequencing (WGS). Response to therapy of PDOs was evaluated by treatment with combined chemotherapies, targeted therapies, or co-culturing with patient-matched peripheral blood mononuclear cells (PMBC) in vitro and/or in vivo. The metastatic capabilities of PDOs were assessed in vivo using intracardiac injection and scoring for tumor outgrowth in distant organs using in vivo bioluminescence imaging system. Metastatic tumors from specific organs were isolated, PDOs reestablished, and those tumors underwent additional genomic and transcriptomic characterization. Phylogenetic analyses of tumor metastatic progression and putative metastasis markers were investigated by WGS and RNA-sequencing. Results: We successfully generated 14 PDOs from early-stage (I-II) primary LUAD specimens and 12 from human LUAD metastatic sites (brain, bone and pleural effusion). All PDOs recapitulate genetic alterations and transcriptomic signatures of their original tumors. Moreover, we found that individual PDOs exhibit distinct responses to different chemotherapy regimens and targeted therapies. Pre-treatment of PDOs with low doses of platinum-based doublet chemotherapy regimens activated patient-matched tumor specific T cells and enhanced PD-1 checkpoint inhibitor Nivolumab induced CD8+ T cell-mediated tumor killing. Importantly, utilizing our in vivo PDO metastasis model we successfully developed a platform to evaluate: 1) metastatic potential, 2) response to targeted therapies, 3) tumor evolution with respect of genomic instability, tumor phylogeny, and colonization, and 4) molecular signatures promoting tumor metastases. Conclusion: Our PDO system reliably models human LUAD in vitro and in vivo, by recapitulating histologic architectures, genomic alterations and transcriptomic signatures of the parent LUAD. Using LUAD PDOs we have successfully created multiple platforms to assess response to chemo- and targeted- therapies, develop an in vivo PDO metastasis model to assess tumor evolution, and use of patient matched PMBCs and PDOs to assess immune-priming strategies for individual patient tumors. Citation Format: Yuan Liu, David Jones. Patient derived organoids model lung adenocarcinoma metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 695.

Modified mRNA delivery to the heart using Lipid Nanoparticles

Funding AcknowledgementsType of funding sources: Foundation. Main funding source(s): Dutch heart foundation Dekker Senior Scientist grantMyocardial infarction is a global health burden for which there is no treatment available that aims to recover the damaged tissue after the ischemic event. Lipid nanoparticles (LNPs) represent a well characterized class of mRNA delivery systems, which were recently approved for clinical usage in their application for mRNA-based covid-19 vaccines.After myocardial infarction, endogenous mechanisms that enable repair of the functional damaged tissue can be triggered by modified mRNA (modRNA) delivery, locally in the infarcted area. As a first step, in order to optimize the LNP formulation for effective myocardial delivery and study cellular tropism of the LNPs in the heart, different LNPs formulations will be evaluated as delivery systems in a murine healthy heart model.Different LNP formulations varying in type and amount of helper lipid were used as delivery systems for modRNA encoding the reporter genes luciferase or eGFP. In vitro, LNPs were evaluated for modRNA delivery in a human endothelial cell line (HMEC-1), induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and induced pluripotent stem cell -derived fibroblasts (iPS-FBs). In vivo, modRNA delivery was evaluated in C57BL-6 mice, undergoing open chest heart surgery under general anaesthesia in order to infuse LNPs into the left ventricular wall. For determination of luciferase expression levels, animals were infused with luciferin substrate intraperitoneally 24 hrs after injection. Heart, liver, lungs, spleen and kidneys were extracted for imaging in a bioluminescence imaging system. The organs were then stored in liquid nitrogen for further ex-vivo modRNA delivery analysis. For determining cellular tropism, histology was performed on mice treated with eGFP modRNA.Both bioluminescence imaging and luminescence analysis in tissue lysates showed that mRNA transfection is achieved in the myocardium 24 hours after LNP intramyocardial administration. However, all LNP formulations also resulted in high expression levels in other organs, including liver and spleen. Changes in type or amount of helper lipid in LNPs strongly affected transfection levels. Histology of the treated hearts revealed a distinct transfection pattern. The targeted, interstitial cells were negative for CD31 (marker for endothelial cells and monocytes) and Troponin I3 (marker for cardiomyocytes) (Figure 1).We show that, using an optimized LNP formulation, a significant degree of modRNA local transfection of the heart can be achieved. However, despite the local route of administration (into the left ventricular wall), the highest LNP transfection is shown in remote organs such as liver and spleen. More improvements of the LNP formulations must be done to increase their tropism towards the heart tissue for their optimization as cardiac delivery systems. Determining which cell types are being targeted is also important in order to establish a therapeutic target when applying the LNPs for cardiac therapy. Immunofluorescence of heart sections

Validation of a combined ultrasound and bioluminescence imaging system with magnetic resonance imaging in orthotopic pancreatic murine tumors

Preclinical mouse solid tumor models are widely used to evaluate efficacy of novel cancer therapeutics. Recent reports have highlighted the need for utilizing orthotopic implantation to represent clinical disease more accurately, however the deep tissue location of these tumors makes longitudinal assessment challenging without the use of imaging techniques. The purpose of this study was to evaluate the performance of a new multi-modality high-throughput in vivo imaging system that combines bioluminescence imaging (BLI) with robotic, hands-free ultrasound (US) for evaluating orthotopic mouse models. Long utilized in cancer research as independent modalities, we hypothesized that the combination of BLI and US would offer complementary advantages of detection sensitivity and quantification accuracy, while mitigating individual technological weaknesses. Bioluminescent pancreatic tumor cells were injected into the pancreas tail of C57BL/6 mice and imaged weekly with the combination system and magnetic resonance imaging (MRI) to serve as a gold standard. BLI photon flux was quantified to assess tumor activity and distribution, and US and MRI datasets were manually segmented for gross tumor volume. Robotic US and MRI demonstrated a strong agreement (R2 = 0.94) for tumor volume measurement. BLI showed a weak overall agreement with MRI (R2 = 0.21), however, it offered the greatest sensitivity to detecting the presence of tumors. We conclude that combining BLI with robotic US offers an efficient screening tool for orthotopic tumor models.

Abstract LBA051: Importance of drug osmotic pressure and viscosity for efficient drug delivery using lymphatic drug delivery system

Abstract Metastasis is known to be the prime cause of cancer associated fatality. It is therefore imperative to treat and prevent metastasis in order to curb cancer-associated morbidity and mortality. Metastatic lymph nodes (MLNs) are known to be crucial diagnostic and prognostic indicators. Furthermore, as per the lymph node mediated hematogenous theory, due to extant communication between the lymphatic network and the venous system, the lymphatic network facilitates the passage and subsequent colonization of tumor cells to distant sites, thus, serving as a starting point for distant metastasis even during the initial stages. Treatment of MLNs, therefore, is imperative. Proposed in 2015, the Lymphatic Drug Delivery System (LDDS) is a novel and promising drug delivery system for the treatment of MLNs that overcomes the limitations of conventional chemotherapy. Unlike systemic chemotherapy, LDDS entails a direct intranodal drug administration to sentinel lymph node, post its identification using radioisotopes and fluorescent molecules, in order to treat it and its downstream lymph nodes. Previous studies have documented the superior efficacy of LDDS as a MLN management strategy as compared to conventional therapy. Higher drug delivery to target sites and prolonged retention with lower systemic toxicity, and thereby stronger anti-tumor effect have been reported to have been achieved upon drug administration via LDDS. In the present study, MXH10/Mo/lpr mice and FM3A-Luc, mouse mammary carcinoma cells expressing the firefly luciferase gene, have been utilized for the establishment of a mouse model of Lymph node metastasis (LNM) to investigate the effect of drug osmotic pressure and viscosity on therapeutic efficacy of the LDDS. Two lymph nodes, exhibiting systemic lymphadenopathy, in the subiliac and axillary region, the subiliac lymph node (SiLN) and proper axillary lymph node (PALN) have been used to model a network of interconnected human lymph nodes. Drug solution of varying osmotic pressure and viscosity were administered via LDDS to the murine model of LNM and subsequently therapeutic efficacy was monitored using the high frequency ultrasound system, VEVO and bioluminescent imaging system, IVIS, and confirmed through histology. The study revealed an optimized window of drug osmotic pressure and viscosity (π = 1000 - 2000 kPa; µ = 0.9 - 11.5 mPa·s.) where a significantly stronger anti-tumor response was observed. Venturing below or beyond the optimized range results in decreased therapeutic efficacy. The study therefore concluded that drug osmotic pressure and viscosity are important fluid parameters that require careful consideration prior to preparation of drug cocktails as they can severely impact the prognosis. The findings of this study have the potential for translation to clinics and we anticipate that these results can be replicated in clinical trials, positively altering the fate of many cancer patients. Citation Format: Radhika Mishra, Ariunbuyan Sukhbaatar, Sora Shouta, Maya Sakamoto, Shiro Mori, Tetsuya Kodama. Importance of drug osmotic pressure and viscosity for efficient drug delivery using lymphatic drug delivery system [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA051.

Abstract LBA049: Possibility of total body irradiations for lymph node metastasis and lung metastasis

Abstract Metastatic lymph nodes (MLNs) are one of the most common causes of distant metastasis, and their presence signals a serious deterioration in the life prognosis of an affected patient. The treatment of MLNs at an early stage is necessary to inhibit metastases to distant LNs and organs. Radiation therapy is an effective therapeutic modality for local tumor control in many cancers, and its effect is enhanced by fractionated radiation therapy or in combination with other therapeutic regimens. In addition, recent studies have reported that total body irradiation (TBI) increases the activation of immune cells such as NK cells and infiltration of CD4+ T cells to tumor tissue. However, the effects of TBI on LN metastasis remains unclear. This study assessed the effect of TBI by changing the radiation dose and number of irradiations in an MLN mouse model. To induce metastasis into the proper axillary lymph node (PALN) and lung, mouse breast cancer FM3A-Luc cells were inoculated into the subiliac lymph node (SiLN) of MXH10/Mo/lpr mice. Mice were assigned to 4 groups thus: received no TBI; received a single dose of 0.2 Gy as low-dose TBI (single L-TBI); received a single dose of 1.0 Gy as middle-dose TBI (single M-TBI); or received two fractions of 2.0 Gy (fractionated M-TBI). The irradiation was carried out on day 4 post-inoculation in the single L-TBI and M-TBI groups. In the fractionated M-TBI group the irradiation was carried out on days 4 and 7 post-inoculation. Tumor growth was evaluated using an in vivo bioluminescence imaging system. In the non-irradiated group, tumor activity in the SiLN and PALN increased over time and lung metastasis was confirmed on day 28 post-inoculation. In addition, the single L-TBI dose did not delay tumor growth in the SiLN and PALN or inhibit lung metastasis. In other words, the tumor activity was not significantly changed compared to the non-irradiated group. Single and fractionated M-TBI doses significantly delayed tumor growth in the SiLN and PALN and inhibited lung metastasis. Furthermore, the fractionated M-TBI group exhibited the greatest delay in tumor growth in the PALN compared to the other three groups. These results suggest that the TBI at a dose > 1.0 Gy can delay tumor growth of MLNs in the early stage and distant metastasis. Moreover, the radiation dose and number of irradiations may be essential factors for enhancing the anti-tumor effects of TBI. We anticipate that TBI will contribute to the prevention and treatment of cancer metastasis via LNs. Citation Format: Shouta Sora, Radhika Mishra, Ariunbuyan Sukhbaatar, Maya Sakamoto, Shiro Mori, Tetsuya Kodama. Possibility of total body irradiations for lymph node metastasis and lung metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA049.

Aloin induced apoptosis by enhancing autophagic flux through the PI3K/AKT axis in osteosarcoma

BackgroundOsteosarcoma is a malignant tumor of bone and soft tissue in adolescents. Due to its tumor biological behavior pattern, osteosarcoma usually generates poor prognosis. Autophagy is an important self-defense mechanism in osteosarcoma.MethodsCell viability in IC50 testing and reverse assays was examined by the MTT assay. Cell apoptosis conditions were examined by flow cytometry, Hoechst 33,342 staining and apoptosis-related protein immunoblotting. Autophagy conditions were tested by autophagy-related protein immunoblotting, transmission electron microscopic observation and dual fluorescence autophagy flux detection. The possible targets of aloin were screened out by network pharmacology and bioinformatic methods. Osteosarcoma xenografts in nude BALB/c mice were the model for in vivo research on tumor suppression, autophagy induction, pathway signaling and toxicity tests. In vivo bioluminescence imaging systems, immunohistochemical assays, and gross tumor volume comparisons were applied as the main research methods in vivo.ResultsAloin induced osteosarcoma apoptosis in a dose-dependent manner. Its possible effects on the PI3K/AKT pathway were screened out by network pharmacology methods. Aloin increased autophagic flux in osteosarcoma by downregulating the PI3K/AKT pathway. Aloin promoted autophagic flux in the osteosarcoma cell lines HOS and MG63 in a dose-dependent manner by promoting autophagosome formation. Chloroquine reversed the apoptosis-promoting and autophagy-enhancing effects of aloin. Autophagy induced by starvation and rapamycin significantly enhanced the autophagic flux and apoptosis induced by aloin, which verified the role of the PI3K/AKT axis in the pharmacological action of aloin. Therapeutic effects, autophagy enhancement and regulatory effects on the PI3K/AKT/mTOR pathway were demonstrated in a nude mouse xenogeneic osteosarcoma transplantation model.ConclusionsAloin inhibited the proliferation of osteosarcoma by inhibiting the PI3K/AKT/mTOR pathway, increasing autophagic flux and promoting the apoptosis of osteosarcoma cells.