Noninvasive Bioluminescence Imaging Research Articles

In Vivo Imaging of Immune Rejection of MIN6 Cells Transplanted in C3H Mice.

Recently, we successfully utilized noninvasive magnetic resonance and bioluminescence imaging to track MIN6 cells subcutaneously transplanted in immunocompromised nude mice for up to 64 days. In this study, we further used bioluminescence imaging to investigate the immune rejection of MIN6 cells in immunocompetent C3H mice. A total of 5 × 106 luciferase-transfected MIN6 cells were implanted into the subcutaneous space of each nude or C3H mouse. After transplantation, hypoglycemia and persistent bioluminescence signals were observed in eight of eight (100%) nude mice and five of nine (56%) C3H mice (p < 0.05). We then presensitized a group of C3H mice with C57BL/6 spleen cells just prior to transplantation (n = 14). Interestingly, none of them had hypoglycemia or persistent bioluminescence signals (p < 0.01 vs. C3H mice without presensitization). Histological examination of the grafts revealed a lack or minimal presence of insulin-positive cells in recipients without hypoglycemia and persistent bioluminescence signals. In contrast, recipients with hypoglycemia and persistent bioluminescence signals showed a significant presence of insulin-positive cells in their grafts. Our results indicate that rejection of MIN6 cells occurred in C3H mice and could be enhanced by presensitization with C57BL/6 spleen cells and that bioluminescence imaging is a useful noninvasive tool for detecting rejection of subcutaneously transplanted MIN6 cells.

EDI3 knockdown in ER-HER2+ breast cancer cells reduces tumor burden and improves survival in two mouse models of experimental metastasis

BackgroundDespite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis.MethodsTo inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively.ResultsInducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo.ConclusionsReduced metastasis upon silencing supports EDI3’s potential as a treatment target in metastasizing ER-HER2+ breast cancer.

Suppression of metastatic organ colonization and antiangiogenic activity of the orally bioavailable lipid raft-targeted alkylphospholipid edelfosine

Metastasis is the leading cause of cancer mortality. Metastatic cancer is notoriously difficult to treat, and it accounts for the majority of cancer-related deaths. The ether lipid edelfosine is the prototype of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs, and its antitumor activity involves lipid raft reorganization. In this study, we examined the effect of edelfosine on metastatic colonization and angiogenesis. Using non-invasive bioluminescence imaging and histological examination, we found that oral administration of edelfosine in nude mice significantly inhibited the lung and brain colonization of luciferase-expressing 435-Lung-eGFP-CMV/Luc metastatic cells, resulting in prolonged survival. In metastatic 435-Lung and MDA-MB-231 breast cancer cells, we found that edelfosine also inhibited cell adhesion to collagen-I and laminin-I substrates, cell migration in chemotaxis and wound-healing assays, as well as cancer cell invasion. In 435-Lung and other MDA-MB-435-derived sublines with different organotropism, edelfosine induced G2/M cell cycle accumulation and apoptosis in a concentration- and time-dependent manner. Edelfosine also inhibited in vitro angiogenesis in human and mouse endothelial cell tube formation assays. The antimetastatic properties were specific to cancer cells, as edelfosine had no effects on viability in non-cancerous cells. Edelfosine accumulated in membrane rafts and endoplasmic reticulum of cancer cells, and membrane raft-located CD44 was downregulated upon drug treatment. Taken together, this study highlights the potential of edelfosine as an attractive drug to prevent metastatic growth and organ colonization in cancer therapy. The raft-targeted drug edelfosine displays a potent activity against metastatic organ colonization and angiogenesis, two major hallmarks of tumor malignancy.

Abstract B010: Revealing the Influence of the Infantile Immune System on the Developmental Origins of Childhood Ependymoma: An Integrative Multi-Omic Investigation of Supratentorial Ependymoma Tumorigenesis Driven by ZFTA-RELA Fusion

Abstract Despite advances in treatment, the recurrence rate of childhood supratentorial ependymomas remains distressingly high, with approximately 60% of patients experiencing relapse. Moreover, the rigorous treatment regimen poses significant challenges to the quality of life of affected children. Among supratentorial ependymomas, those driven by the ZFTA-RELA fusion represent over 70% of cases. Here, we present the pioneering development of a spontaneous genetically engineered mouse model (GEMM) for ZFTA-RELA fusion-driven ependymoma. By targeting the Nestin-Flx-STOP-Flx-ZFTA-RELA open reading frame with luciferase to the Rosa-26 locus of C57B/L6 mice, and subsequently crossing them with the Nestin-CreERT2 line, we successfully induced recombination in neural progenitor cells. Consequently, resulting in the development of forebrain tumors that could be monitored through non-invasive bioluminescence imaging from postnatal day 20. Recognizing the neonatal origins of ZFTA-RELA ependymoma, our study employed an integrated multi-omic approach to investigate the complete developmental trajectory of this tumor, spanning from embryonic stages to postnatal tumor formation. Notably, we uncovered a reservoir of immature immune populations in the developing brain, which we implicate in the central tolerance of ependymoma tumorigenesis. The coordinated interaction between these immature immune populations in the developing brain and immune cell progenitors in the skull bone marrow niche establishes a unique environment that fosters tumor formation. In light of these findings, we propose that immunotherapeutic strategies aimed at augmenting these immature immune populations hold promise as potential therapeutic interventions. By exploiting the intricate interplay between these populations, we may unlock new avenues for targeted immunotherapies in ZFTA-RELA ependymoma. Our research sheds vital light on the critical role played by the pediatric immune system in the developmental origins of this devastating malignancy, opening up exciting possibilities for improved treatments and outcomes in the future. Citation Format: Elizabeth Cooper, Sigourney Bonner, Amir Jassim, Gunjan Katyal, Alisha Kardian, Stephen C. Mack, Katherine Masih, Jessica T. Taylor, Claire King, Katherine Wickham Rahrmann, Linda Hu, Richard J. Gilbertson. Revealing the Influence of the Infantile Immune System on the Developmental Origins of Childhood Ependymoma: An Integrative Multi-Omic Investigation of Supratentorial Ependymoma Tumorigenesis Driven by ZFTA-RELA Fusion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B010.

Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free &amp;gt;180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Type of anesthesia for cancer resection surgery: No differential impact on cancer recurrence in mouse models of breast cancer.

Surgery is essential for curative treatment of solid tumors. Evidence from recent retrospective clinical analyses suggests that use of propofol-based total intravenous anesthesia during cancer resection surgery is associated with improved overall survival compared to inhaled volatile anesthesia. Evaluating these findings in prospective clinical studies is required to inform definitive clinical guidelines but will take many years and requires biomarkers to monitor treatment effect. Therefore, we examined the effect of different anesthetic agents on cancer recurrence in mouse models of breast cancer with the overarching goal of evaluating plausible mechanisms that could be used as biomarkers of treatment response. To test the hypothesis that volatile anesthesia accelerates breast cancer recurrence after surgical resection of the primary tumor, we used three mouse models of breast cancer. We compared volatile sevoflurane anesthesia with intravenous propofol anesthesia and used serial non-invasive bioluminescent imaging to track primary tumor recurrence and metastatic recurrence. To determine short-term perioperative effects, we evaluated the effect of anesthesia on vascular integrity and immune cell changes after surgery in animal models. Survival analyses found that the kinetics of cancer recurrence and impact on survival were similar regardless of the anesthetic agent used during cancer surgery. Vascular permeability, immune cell infiltration and cytokine profiles showed no statistical difference after resection with inhaled sevoflurane or intravenous propofol anesthesia. These preclinical studies found no evidence that choice of anesthetic agent used during cancer resection surgery affected either short-term perioperative events or long-term cancer outcomes in mouse models of breast cancer. These findings raise the possibility that mouse models do not recapitulate perioperative events in cancer patients. Nonetheless, the findings suggest that future evaluation of effects of anesthesia on cancer outcomes should focus on cancer types other than breast cancer.

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.

Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase-Luciferin Pair.

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.

Powerful and Real-Time Quantification of Antifungal Efficacy against Triazole-Resistant and -Susceptible Aspergillus fumigatus Infections in Galleria mellonella by Longitudinal Bioluminescence Imaging.

Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies.

The Image-Histology Correlation of Subcutaneous mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cell Grafts in Nude Mice.

Previously, we have successfully used noninvasive magnetic resonance (MR) and bioluminescence imaging to detect and monitor mPEG-poly(Ala) hydrogel-embedded MIN6 cells at the subcutaneous space for up to 64 days. In this study, we further explored the histological evolution of MIN6 cell grafts and correlated it with image findings. MIN6 cells were incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) and then 5 × 106 cells in the 100 μL hydrogel solution were injected subcutaneously into each nude mouse. Grafts were removed and examined the vascularization, cell growth and proliferation with anti-CD31, SMA, insulin and ki67 antibodies, respectively, at 8, 14, 21, 29 and 36 days after transplantation. All grafts were well-vascularized with prominent CD31 and SMA staining at all time points. Interestingly, insulin-positive cells and iron-positive cells were scattered in the graft at 8 and 14 days; while clusters of insulin-positive cells without iron-positive cells appeared in the grafts at 21 days and persisted thereafter, indicating neogrowth of MIN6 cells. Moreover, proliferating MIN6 cells with strong ki67 staining was observed in 21-, 29- and 36-day grafts. Our results indicate that the originally transplanted MIN6 cells proliferated from 21 days that presented distinctive bioluminescence and MR images.

Preclinical evaluation of targeting PRAME with TCR mimic CAR T cells in AML.

2553 Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, but has had limited success against AML in part due to overlap of cell surface antigens expressed in AML and normal hematopoietic cells. To identify AML-restricted targets, we interrogated the transcriptome from over 3000 AML patients and found PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein, to be a highly expressed AML-restricted target. Using a novel approach to target intracellular antigens, we developed a PRAME CAR T cell using a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface. To conduct final IND-enabling studies, we generated an in vivo patient derived xenograft (PDX) model to treat with PRAME mTCRCAR T cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against a PDX AML model. Methods: We used the VL and VH sequences from the PRAME TCR mimic antibody (Pr20) to construct the single chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME+/HLA-A2+ pediatric patient with AML. PDX cells were transduced with ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. PDX leukemia-bearing mice were treated with unmodified T-cells or PRAME mTCRCAR T cells at 5x106 cells (1:1 CD4:CD8) per mouse 1 week after leukemia injection. Leukemia burden was measured by IVIS imaging and peripheral blood analysis. Results: Treatment with PRAME mTCRCAR T cells led to eradication of leukemia with all mice alive &gt;100 days post-treatment, while control mice (unmodified T cells) had disease progression at Day 50 (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75 and 429.6 fold increase in radiance by IVIS at 6 and 11 weeks post-treatment. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. Human T cells (CD45+/CD3+) were detectable in the peripheral blood at Day 7 and 14 post-treatment in both the unmodified and CAR T cell treated groups. Conclusions: We demonstrate the therapeutic potential of targeting PRAME with mTCRCAR T cells in AML. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival. These results provide a novel approach to target PRAME with CAR T cells and provide compelling data to evaluate PRAME mTCRCAR T cells for use in clinical trials against AML. [Table: see text]

Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.

Direct monitoring of meropenem therapeutic efficacy against Klebsiella pneumoniae respiratory infection by bioluminescence imaging.

Introduction. Klebsiella pneumoniae is a major threat to public health worldwide. It is the causative agent for multiple disease presentations including urinary tract infection, septicemia, liver abscess, wound infection and respiratory tract infection. K. pneumoniae causes community- and hospital-acquired pneumonia, which is a devastating disease associated with high mortality rates.Hypothesis. There is a growing concern about the emergence of multidrug-resistant K. pneumoniae strains complicating the treatment with the current available therapeutics; therefore, there is an urgent need for the development of new antimicrobial agents.Aim. K. pneumoniae causes an acute respiratory disease in mice and in the current work we investigated the capability to perform non-invasive monitoring of bioluminescent Klebsiella to monitor therapeutic efficacy.Methodology. We engineered a bioluminescence reporter strain of K. pneumoniae to monitor the impact of antibiotics in a murine respiratory disease model.Results. We demonstrate that bioluminescence correlates with bacterial numbers in host tissues allowing for a non-invasive enumeration of bacterial replication in vivo. Light production is directly linked to bacterial viability, and this novel bioluminescent K. pneumoniae strain enabled monitoring of the efficacy of meropenem therapy in arresting bacterial proliferation in the lung.Conclusion. The use of non-invasive bioluminescent imaging improves preclinical animal model testing to detect study outcome earlier and with higher sensitivity.

Abstract 4654: Differential immunomodulation following checkpoint blockade in the orthotopic ID8-luc ovarian model

Abstract Syngeneic tumor models for ovarian cancer are limited in preclinical research. One of these models, ID8-luc, is established intraperitoneally with disease progression resulting in the accumulation of ascites fluid. Treatment efficacy can be determined by non-invasive bioluminescence imaging (BLI), but solid ID8-luc tumors do not form in the peritoneal cavity, making assessment of immunomodulation challenging. To this end, we have developed the ID8-luc model as a tool to investigate immuno-modulatory drug candidates by utilizing tissues in the peritoneal cavity of diseased animals for ex vivo analyses. Specifically, flow cytometry of diseased ovaries was employed to examine the effects of checkpoint inhibition on T cell phenotypes and NK cell infiltration. To identify tumor deposits in organs from mice bearing ID8-luc disease for analysis tumor infiltrate, the model was established in untreated C57BL/6 albino mice, and ovaries, liver, peritoneal wall, pancreas, and spleen were evaluated pathologically. Progressive establishment of tumor deposits were seen in the ovaries, pancreas, and peritoneal wall of untreated mice, but not in naïve mice. Further, immune infiltration in ovaries were markedly higher compared to naïve mice. To assess immunomodulation, disease was established in a second cohort of mice, and mice were treated with anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4. Disease progression was monitored by BLI, and following treatment, ovaries containing tumor deposits were collected and lymphoid infiltrate was analyzed by flow cytometry. Checkpoint blockade did not result in efficacy under the conditions tested. However, differences in T cell infiltration were notable with treatment of anti-PD-1 and anti-mPD-L1. Particularly, a marked increase of five to ten fold CD8+ and CD4+ T cells by absolute counts was seen with anti-mPD-1 and anti-mPD-L1 treatment compared to control. Further, the percentage of CD8+ T cells expressing the activation markers CD69 and ICOS as well as PD-1, TIM-3, and LAG-3 checkpoint proteins increased with anti-mPD-1 treatment. Less prominent yet similar trends in this biomarker expression were seen with anti-mPD-L1 treatment. NK and NKT infiltration also increased five to eight fold in ovaries following anti-mPD-1 treatment. Interestingly, anti-mCTLA-4 treatment did not result in any remarkable changes in infiltration or activation marker dynamics. Taken together, these findings are suggestive of a mounting immune response following PD-1 and PD-L1 blockade, but not CTLA-4 blockade, characterized by changes in the lymphocyte compartment in the ovaries. This response is seen in the absence of efficacy, suggesting the potential that this immunomodulation can be exploited in rational combination strategies with checkpoint blockade for immune-oncology drug candidates using the ID8-luc ovarian tumor model. Citation Format: Sheri Barnes, Anita Zaitouna, Lauren Kucharczyk, Scott Wise. Differential immunomodulation following checkpoint blockade in the orthotopic ID8-luc ovarian model. [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 4654.

In Vivo Imaging of Bone Marrow Long-Chain Fatty Acid Uptake.

In vivo imaging enables the detection and visualization of many different processes occurring within the body. Fatty acid uptake is a fundamental cellular process which is essential for the use of free fatty acids (FFAs) as a fuel source for metabolism. Detection and visualization of in vivo FFA uptake in the bone marrow has been relatively unknown. Here, we describe the process of non-invasive bioluminescent imaging of in vivo FFA uptake within the bone marrow.

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

MODL-01. TRACTABLE PATIENT-DERIVED MODELS FOR PRECLINICAL THERAPEUTIC STUDIES OF CNS METASTASES

Abstract INTRODUCTION Brain metastases are the most common brain tumors and occur in 10-30% of cancer patients, whereas leptomeningeal disease (LMD) occurs in approximately 5% of adults with systemic malignancies. Tractable preclinical disease models that faithfully represent metastasis to the brain and recapitulate LMD are needed to improve our understanding of the biological basis of CNS disease as well developing effective therapeutic strategies. Our goal in this study was to generate representative preclinical disease models using two methods. METHODS We isolated tumor cells from CSF of 16 patients with cytologically proven LMD (9 NSCLC, 1 melanoma, 1 ovarian cancer, 1 endometrial cancer, and 4 breast cancer) and implanted the cells subcutaneously into the flank of immunocompromised mice. Cell lines were also generated from PDX tissues. Models were characterized by next-generation sequencing (NGS). We also generated a model of CNS metastasis of kinase-driven sarcoma by intracardiac (IC) injection of human mesenchymal stem cells (HMSC) expressing a SPECC1L::RET fusion (CRISPR-Cas9 gene editing). The cells (HMSC-RET) were also labeled with a luciferase construct to allow non-invasive bioluminescence imaging. RESULTS We established three PDX models (2 lung, 1 ovarian) from CSF (19% success rate compared to approximately 33% for solid tumors) and matched cell lines from the resulting PDX tissues. Intracardiac injection of HMSC-RET cells resulted in tumors establishing in several peripheral organs and the brain. SUMMARY AND CONCLUSIONS We have established disease models of CNS metastasis and LMD. Translational studies where patients with clinical suspicion of LMD undergo CSF sampling, NGS/ctDNA analysis, and PDX modeling are crucial in improving our understanding of this metastatic compartment and investigating novel treatment paradigms. Future studies will be focused on examining the biochemical and genetic nature of these tumors as well as developing effective therapeutic strategies.

Monoclonal antibody-mediated immunosuppression enables long-term survival of transplanted human neural stem cells in mouse brain.

BackgroundAs the field of stem cell therapy advances, it is important to develop reliable methods to overcome host immune responses in animal models. This ensures survival of transplanted human stem cell grafts and enables predictive efficacy testing. Immunosuppressive drugs derived from clinical protocols are frequently used but are often inconsistent and associated with toxic side effects. Here, using a molecular imaging approach, we show that immunosuppression targeting costimulatory molecules CD4 and CD40L enables robust survival of human xenografts in mouse brain, as compared to conventional tacrolimus and mycophenolate mofetil.MethodsHuman neural stem cells were modified to express green fluorescent protein and firefly luciferase. Cells were implanted in the fimbria fornix of the hippocampus and viability assessed by non‐invasive bioluminescent imaging. Cell survival was assessed using traditional pharmacologic immunosuppression as compared to monoclonal antibodies directed against CD4 and CD40L. This paradigm was also implemented in a transgenic Alzheimer's disease mouse model.ResultsGraft rejection occurs within 7 days in non‐immunosuppressed mice and within 14 days in mice on a traditional regimen. The addition of dual monoclonal antibody immunosuppression extends graft survival past 7 weeks (p < .001) on initial studies. We confirm dual monoclonal antibody treatment is superior to either antibody alone (p < .001). Finally, we demonstrate robust xenograft survival at multiple cell doses up to 6 months in both C57BL/6J mice and a transgenic Alzheimer's disease model (p < .001). The dual monoclonal antibody protocol demonstrated no significant adverse effects, as determined by complete blood counts and toxicity screen.ConclusionsThis study demonstrates an effective immunosuppression protocol for preclinical testing of stem cell therapies. A transition towards antibody‐based strategies may be advantageous by enabling stem cell survival in preclinical studies that could inform future clinical trials.

Ionizing Radiation-Induced Tumor Cell-Derived Microparticles Prevent Lung Metastasis by Remodeling the Pulmonary Immune Microenvironment

The majority of cancer-related deaths are attributed to metastasis rather than localized primary tumor progression. However, the factors that regulate the premetastatic niche (PMN) and metastasis have not yet been clearly elucidated. We investigated the antimetastatic effects of irradiated tumor cell-derived microparticles (RT-MPs) and highlighted the role of innate immune cells in PMN formation. Mice were treated 3 times with isolated RT-MPs, followed by tumor cell injection via the tail vein. The hematoxylin and eosin staining was performed to assess the number of tumor nodules in the lungs, and in vivo luciferase-based noninvasive bioluminescence imaging was conducted to detected tumor burden. The mechanisms of RT-MPs mediated PMN formation was evaluated using flow cytometry, transwell assay, and reverse transcription-polymerase chain reaction. RT-MPs inhibited tumor cell colonization in the lungs. Neutrophils phagocytosed RT-MPs and secreted CCL3 and CCL4, which induced monocytes chemotaxis and maturation into macrophages. RT-MPs promoted the transition of neutrophils and macrophages into antitumor phenotypes, hence inhibiting cancer cell colonization and proliferation. RT-MPs inhibited PMN formation and lung metastasis in a neutrophil- and macrophage-dependent but T cell-independent manner.

Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.