Syngeneic Transplantation Research Articles

Abstract A006: Development of a syngeneic transplant rat model for the identification of an mRNA signature associated with metastatic hepatocellular carcinoma

Abstract Approximately 70% of patients with hepatocellular carcinoma (HCC) die of metastasis, mainly in the lung, due in part to late diagnosis. Therefore, in vivo models that are representative of metastatic HCC biology in patients must include factors that are essential for metastasis development, such as an immunocompetent system and a tumor microenvironment with homotypic interactions. This will allow the identification of new prognostic biomarkers for patients with metastatic HCC that are efficient and accessible. We developed two hepatocellular carcinoma cell lines for syngeneic transplant models in immunocompetent rats: Samy (nonmetastatic) and Samy Met (metastatic). These cell lines allow the development of cancer and metastasis in a homotypic environment with a complete immune system; therefore, they are expected to be representative of what happens in patients with metastatic HCC. Both cell lines were biologically characterized, and the primary tumors they produced were used in a comparative transcriptomic study to identify a metastatic HCC molecular signature that may function as a prognostic biomarker. The differentially expressed genes (DEGs) were related to relevant biological processes, such as metastasis, cancer stem cells (CSCs), and tumor growth, and all of these processes are essential for cancer progression. Among the top 50 DEGs, an mRNA firm was assembled, including 3 upregulated genes (ITGB6, SEMA3F, and NDC80) and 7 downregulated genes (COL15A1, ATP13A4, C18ORF26, PRKG2, ITM2A, IGSF9B, and CNTN1). The firm was related to poor survival in patients with HCC and had good accuracy in predicting macrovascular invasion, which is a strong predictor of poor HCC outcome, both after surgical resection and after liver transplantation. Citation Format: Theresa G Azcárraga Acosta, Carolina Piña Vazquéz, Hury V Hernández Galdámez, Karla M Rubio Nava, Josué Guzmán Linares, Ivonne Ramírez Díaz, Guillermo Barreto, Mario A Aguilar Chaparro, Stefan Günther, Saul Villa Treviño, Mireya De la Garza, Teresita del niño Jesús Flores Téllez, Veronica R Vásquez Garzón. Development of a syngeneic transplant rat model for the identification of an mRNA signature associated with metastatic hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A006.

Allogeneic abdominal non-vascularized rectus fascia transplantation without immunosuppression equals syngeneic transplantation in a rabbit model at short-term follow-up

Complex abdominal wall repair remains a major surgical challenge. In transplant patients, non-vascularized rectus fascia (NVRF) is successfully used to bridge the defect. To extrapolate this to non-transplant patients, we developed a rabbit model of NVRF-transplantation without immunosuppression comparing syngeneic versus allogeneic transplants. Short-term outcome (4 weeks) was evaluated macroscopically (ingrowth, seroma/hematoma, herniation, infection), histologically at the graft interface and center (inflammation, neovascularization, collagen deposition) and by mechanical testing. In both groups a similar macroscopic ingrowth of the NVRF was observed. In the syn-group one seroma and one hematoma was seen. Two small herniations were detected at the suture line in the allo-group. No surgical site infections were observed. Histologically, graft neovascularization was observed in all animals. Infiltration of T-lymphocytes was seen at the graft interface in both groups, but more in the allo-group (p < 0.0001). Deposition of collagen was not different between groups. Macrophages were present in both groups around sutures and in the center more abundantly in the allo-group (p = 0.0001). Graft stiffness and strength were similar for both groups. With this model, we showed that allogeneic transplantation without immunosuppression results in favorable short-term inflammatory and mechanical outcomes. Long-term experiments are needed to further evaluate the effect on graft integration and hernia development.

Tumor microenvironment dynamics in oral cancer: unveiling the role of inflammatory cytokines in a syngeneic mouse model.

The process of cervical lymph node metastasis is dependent on the phenotype of the tumor cells and their interaction with the host microenvironment and immune system; conventional research methods that focus exclusively on tumor cells are limited in their ability to elucidate the metastatic mechanism. In cancer tissues, a specialized environment called the tumor microenvironment (TME) is established around tumor cells, and inflammation in the TME has been reported to be closely associated with the development and progression of many types of cancer and with the response to anticancer therapy. In this study, to elucidate the mechanism of metastasis establishment, including the TME, in the cervical lymph node metastasis of oral cancer, we established a mouse-derived oral squamous cell carcinoma cervical lymph node highly metastatic cell line and generated a syngeneic orthotopic transplantation mouse model. In the established highly metastatic cells, epithelial-mesenchymal transition (EMT) induction was enhanced compared to that in parental cells. In the syngeneic mouse model, lymph node metastasis was observed more frequently in tumors of highly metastatic cells than in parental cells, and Cyclooxygenase-2 (COX-2) expression and lymphatic vessels in primary tumor tissues were increased, suggesting that this model is highly useful. Moreover, in the established highly metastatic cells, EMT induction was enhanced compared to that in the parent cell line, and CCL5 and IL-6 secreted during inflammation further enhanced EMT induction in cancer cells. This suggests the possibility of a synergistic effect between EMT induction and inflammation. This model, which allows for the use of two types of cells with different metastatic and tumor growth potentials, is very useful for oral cancer research involving the interaction between cancer cells and the TME in tumor tissues and for further searching for new therapeutic agents.

Development and Characterization of Syngeneic Orthotopic Transplant Models of Obesity-Responsive Triple-Negative Breast Cancer in C57BL/6J Mice.

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC), but preclinical studies to delineate the mechanisms underlying the obesity-TNBC link as well as strategies to break that link are constrained by the lack of tumor models syngeneic to obesity-prone mouse strains. C3(1)/SV40 T-antigen (C3-TAg) transgenic mice on an FVB genetic background develop tumors with molecular and pathologic features that closely resemble human TNBC, but FVB mice are resistant to diet-induced obesity (DIO). Herein, we sought to develop transplantable C3-TAg cell lines syngeneic to C57BL/6 mice, an inbred mouse strain that is sensitive to DIO. We backcrossed FVB-Tg(C3-1-TAg)cJeg/JegJ to C57BL/6 mice for ten generations, and spontaneous tumors from those mice were excised and used to generate four clonal cell lines (B6TAg1.02, B6TAg2.03, B6TAg2.10, and B6TAg2.51). We characterized the growth of the four cell lines in both lean and DIO C57BL/6J female mice and performed transcriptomic profiling. Each cell line was readily tumorigenic and had transcriptional profiles that clustered as claudin-low, yet markedly differed from each other in their rate of tumor progression and transcriptomic signatures for key metabolic, immune, and oncogenic signaling pathways. DIO accelerated tumor growth of orthotopically transplanted B6TAg1.02, B6TAg2.03, and B6TAg2.51 cells. Thus, the B6TAg cell lines described herein offer promising and diverse new models to augment the study of DIO-associated TNBC.

Non-cell-autonomous suppression of tumor growth by RECK in immunocompetent mice.

RECK is a candidate tumor suppressor gene isolated as a gene that induces flat reversion in a cell line transformed by the KRAS oncogene. Since RECK knockout mice die in utero, they are not suitable for studying the effects of RECK on tumor formation. In this study, we found an increased incidence of spontaneous pulmonary adenomas in mice with reduced RECK expression (RECK-Hypo mice). To evaluate the effects of RECK expressed by either tumor cells or host cells on tumor growth, we established a tumorigenic cell line (MKER) from the kidney of a C57BL/6 mouse and performed syngeneic transplantation experiments. Our results indicate that when RECK expression is low in host cells, transplanted MKER cells grow faster and kill the animal more rapidly. Since RECK is required for the formation of proper fibrillin fibers that serve as a tissue reservoir for precursors of TGFβ-family cytokines, we assessed the levels of TGFβ1 in the peripheral blood. We found a significant increase in TGFβ1 in RECK-Hypo mice compared to wild-type mice. We also found that the proportion of FOXP3-positive regulatory T (Treg) cells among splenocytes was higher in RECK-Hypo mice compared to the control mice. Furthermore, the number of FOXP3-positive cells in spontaneous hematopoietic neoplasms in the lungs as well as tumors that formed after MKER transplantation was significantly higher in RECK-Hypo mice compared to the control mice. These findings indicate that RECK-mediated tumor suppression involves a non-cell-autonomous mechanism and that possible roles of TGFβ1 and Treg cells in such a mechanism warrant further study.

Optimizing DCD Liver Grafts With Prolonged Warm Ischemic Time Using Stabilized Plasmin in a Static Cold Storage Orthotopic Rat Liver Transplant Model.

The clinical success of liver transplantation has led to increased demand, requiring further expansion of the donor pool. Therapeutic interventions to optimize organs from donation after circulatory death (DCD) have significant potential to mitigate the organ shortage. Dysfunction in DCD liver grafts is mediated by microvascular thrombosis during the warm ischemic period, and strategies that reduce this thrombotic burden may improve graft function. We hypothesized that the administration of the fibrinolytic enzyme plasmin to the donor organ during the cold storage period would reduce the thrombotic burden and improve DCD liver graft function. In 2 separate cohorts, 32 syngeneic orthotopic rat liver transplants were performed in Lewis rats. Livers were procured from donors with 45 min of warm ischemic injury. Liver grafts were flushed with histidine-tryptophan-ketoglutarate preservation solution mixed with either plasmin (experimental group) or albumin (control group). All investigators were blinded to treatment group. After preparing the liver for implant using a modified cuff technique, the liver was stored for 1 h by static cold storage at 4 °C. Immediately before implantation, the liver graft was flushed, and this effluent was analyzed for fibrin degradation products to determine graft clot burden. Twenty-four hours following transplantation, animals were euthanized, and samples were collected. Recipient survival was significantly higher for DCD liver grafts treated with plasmin compared with control. Moreover, histology of liver graft tissue immediately before implant reflected significantly reduced congestion in plasmin-treated livers (score, mean ± SD: 0.73 ± 0.59 versus 1.12 ± 0.48; P = 0.0456). The concentration of fibrin degradation products in the final flush before implantation was significantly reduced in plasmin-treated livers (743 ± 136 versus 10 919 ± 4642 pg/mL; P = 0.0001), reflecting decreased clot burden in the graft. The present study demonstrates that plasmin improves survival and may reduce thrombotic burden in DCD liver grafts with prolonged warm ischemic injury, meriting further study.

Collagen modification remodels the sarcoma tumor microenvironment and promotes resistance to immune checkpoint inhibition.

Molecular mechanisms underlying immune checkpoint inhibitor (ICI) response heterogeneity in solid tumors, including soft tissue sarcomas (STS), remain poorly understood. Herein, we demonstrate that the collagen-modifying enzyme, procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (Plod2), which is over-expressed in many tumors relative to normal tissues, promotes immune evasion in undifferentiated pleomorphic sarcoma (UPS), a relatively common and aggressive STS subtype. This finding is consistent with our earlier observation that Plod2 promotes tumor metastasis in UPS, and its enzymatic target, collagen type VI (ColVI), enhances CD8+ T cell dysfunction. We determined that genetic and pharmacologic inhibition of Plod2 with the pan-Plod transcriptional inhibitor minoxidil, reduces UPS growth in an immune competent syngeneic transplant system and enhances the efficacy of anti-Pd1 therapy. These findings suggest that PLOD2 is an actionable cancer target and its modulation could augment immunotherapy responses in patients with UPS, and potentially other sarcomas and carcinomas.

Optimizing drug combinations for T-PLL: restoring DNA damage and P53-mediated apoptotic responses

AbstractT-prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm associated with marked chemotherapy resistance and continued poor clinical outcomes. Current treatments, that is, the CD52-antibody alemtuzumab, offer transient responses, with relapses being almost inevitable without consolidating allogeneic transplantation. Recent more detailed concepts of T-PLL’s pathobiology fostered the identification of actionable vulnerabilities: (1) altered epigenetics, (2) defective DNA damage responses, (3) aberrant cell-cycle regulation, and (4) deregulated prosurvival pathways, including T-cell receptor and JAK/STAT signaling. To further develop related preclinical therapeutic concepts, we studied inhibitors of histone deacetylases ([H]DACs), B-cell lymphoma 2 (BCL2), cyclin-dependent kinase (CDK), mouse double minute 2 (MDM2), and classical cytostatics, using (1) single-agent and combinatorial compound testing in 20 well-characterized and molecularly profiled primary T-PLL (validated by additional 42 cases) and (2) 2 independent murine models (syngeneic transplants and patient-derived xenografts). Overall, the most efficient/selective single agents and combinations (in vitro and in mice) included cladribine, romidepsin ([H]DAC), venetoclax (BCL2), and/or idasanutlin (MDM2). Cladribine sensitivity correlated with expression of its target RRM2. T-PLL cells revealed low overall apoptotic priming with heterogeneous dependencies on BCL2 proteins. In additional 38 T-cell leukemia/lymphoma lines, TP53 mutations were associated with resistance toward MDM2 inhibitors. P53 of T-PLL cells, predominantly in wild-type configuration, was amenable to MDM2 inhibition, which increased its MDM2-unbound fraction. This facilitated P53 activation and downstream signals (including enhanced accessibility of target-gene chromatin regions), in particular synergy with insults by cladribine. Our data emphasize the therapeutic potential of pharmacologic strategies to reinstate P53-mediated apoptotic responses. The identified efficacies and their synergies provide an informative background on compound and patient selection for trial designs in T-PLL.

Study on the Extrapolability of Current Tumorgenicity Test With Mice by Comparing the Syngeneic or Allogeneic Mouse Transplantation Model.

The extrapolability of the current tumorigenicity test performed by transplanting human cell product into immunodeficient (NOG) mice was investigated. For this purpose, the susceptibility to form teratomas of NOG mice was assessed by transplanting undifferentiated human-induced pluripotent stem cells (hiPSCs) as positive control cells via the liver, striatum, or tail vein and evaluating the TPD50 value (dose required to form teratomas in half of the transplanted mice). This was then compared to the TPD50 of syngeneic or allogeneic mouse models. The TPD50 of C57/BL/6(B6)-iPSC or 129/Ola(129)-embryonic stem cell (ESC) transplanted into the liver of syngeneic mice was 4.08 × 105 and 4.64 × 104 cells, respectively, while the TPD50 of hiPSC administered into the liver of NOG mice was 4.64 × 104 cells. The TPD50 of B6-miPSC-synergic, 129-mESC-synergic, or 129-cell/B6 allogeneic transplantation into the striatum was 5.09 × 102, 1.0 × 104, and 3.73 × 104 cells, respectively, while that of hiPSC/NOG mice was 1.0 × 103 cells. The TPD50 for B6-miPSC or 129-mESC syngeneic tail vein infusion was 3.16 × 106 or 5.62 × 106 cells, respectively, while no incidence was observed from 1 × 107 B6-miPSCs in 129 mice or hiPSCs in NOG mice infusion study. Although the number of data sets was limited, these data indicate that the teratoma formation from transplanted undifferentiated hiPSCs via the liver or striatum in NOG mice is comparable to that in syngeneic or allogeneic mouse transplantation model, suggesting that the result of the current tumorigenicity test in NOG mice would provide useful information to infer the incidence of teratoma from residual undifferentiated hPSCs in hPSC-derived products after transplantation.

Differential response of injured and healthy retinas to syngeneic and allogeneic transplantation of a clonal cell line of immortalized olfactory ensheathing glia: a double-edged sword.

JOURNAL/nrgr/04.03/01300535-202508000-00029/figure1/v/2024-09-30T120553Z/r/image-tiff Olfactory ensheathing glia promote axonal regeneration in the mammalian central nervous system, including retinal ganglion cell axonal growth through the injured optic nerve. Still, it is unknown whether olfactory ensheathing glia also have neuroprotective properties. Olfactory ensheathing glia express brain-derived neurotrophic factor, one of the best neuroprotectants for axotomized retinal ganglion cells. Therefore, we aimed to investigate the neuroprotective capacity of olfactory ensheating glia after optic nerve crush. Olfactory ensheathing glia cells from an established rat immortalized clonal cell line, TEG3, were intravitreally injected in intact and axotomized retinas in syngeneic and allogeneic mode with or without microglial inhibition or immunosuppressive treatments. Anatomical and gene expression analyses were performed. Olfactory bulb-derived primary olfactory ensheathing glia and TEG3 express major histocompatibility complex class II molecules. Allogeneically and syngenically transplanted TEG3 cells survived in the vitreous for up to 21 days, forming an epimembrane. In axotomized retinas, only the allogeneic TEG3 transplant rescued retinal ganglion cells at 7 days but not at 21 days. In these retinas, microglial anatomical activation was higher than after optic nerve crush alone. In intact retinas, both transplants activated microglial cells and caused retinal ganglion cell death at 21 days, a loss that was higher after allotransplantation, triggered by pyroptosis and partially rescued by microglial inhibition or immunosuppression. However, neuroprotection of axotomized retinal ganglion cells did not improve with these treatments. The different neuroprotective properties, different toxic effects, and different responses to microglial inhibitory treatments of olfactory ensheathing glia in the retina depending on the type of transplant highlight the importance of thorough preclinical studies to explore these variables.

Exogenous Metabolic Modulators Improve Response to Carboplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks targeted therapies, leaving cytotoxic chemotherapy as the current standard treatment. However, chemotherapy resistance remains a major clinical challenge. Increased insulin-like growth factor 1 signaling can potently blunt chemotherapy response, and lysosomal processes including the nutrient scavenging pathway autophagy can enable cancer cells to evade chemotherapy-mediated cell death. Thus, we tested whether inhibition of insulin receptor/insulin-like growth factor 1 receptor with the drug BMS-754807 and/or lysosomal disruption with hydroxychloroquine (HCQ) could sensitize TNBC cells to the chemotherapy drug carboplatin. Using in vitro studies in multiple TNBC cell lines, in concert with in vivo studies employing a murine syngeneic orthotopic transplant model of TNBC, we show that BMS-754807 and HCQ each sensitized TNBC cells and tumors to carboplatin and reveal that exogenous metabolic modulators may work synergistically with carboplatin as indicated by Bliss analysis. Additionally, we demonstrate the lack of overt in vivo toxicity with our combination regimens and, therefore, propose that metabolic targeting of TNBC may be a safe and effective strategy to increase sensitivity to chemotherapy. Thus, we conclude that the use of exogenous metabolic modulators, such as BMS-754807 or HCQ, in combination with chemotherapy warrants additional study as a strategy to improve therapeutic responses in women with TNBC.

Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1β, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Abstract PO1-24-05: EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.

A new bicornuate model of rat uterus transplantation.

Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child. We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and, therefore, comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Leukotriene B4: A potential mediator and biomarker for cardiac allograft vasculopathy

BackgroundCardiac allograft vasculopathy (CAV) remains the leading cause of long-term graft failure and mortality after heart transplantation. Effective preventive and treatment options are not available to-date, largely because underlying mechanisms remain poorly understood. We studied the potential role of leukotriene B4 (LTB4), an inflammatory lipid mediator, in development of CAV. MethodsWe used an established pre-clinical rat CAV model to study the role of LTB4 in CAV. We performed syngeneic and allogeneic orthotopic aortic transplantation, after which neointimal proliferation was quantified. Animals were then treated with Bestatin, an inhibitor of LTB4 synthesis, or vehicle control for 30 days post-transplant, and evidence of graft CAV was determined by histology. We also measured serial LTB4 levels in a cohort of 28 human heart transplant recipients with CAV, 17 matched transplant controls without CAV, and 20 healthy non-transplant controls. ResultsWe showed that infiltration of the arterial wall with macrophages leads to neointimal thickening and a rise in serum LTB4 levels in our rat model of CAV. Inhibition of LTB4 production with the drug Bestatin prevents development of neointimal hyperplasia, suggesting that Bestatin may be effective therapy for CAV prevention. In a parallel study of heart transplant recipients, we found non-significantly elevated plasma LTB4 levels in patients with CAV, compared to patients without CAV and healthy, non-transplant controls. ConclusionsThis study provides key evidence supporting the role of the inflammatory cytokine LTB4 as an important mediator of CAV development, and provides preliminary data suggesting the clinical benefit of Bestatin for CAV prevention.

Loss of NLRP6 expression increases the severity of intestinal injury after syngeneic hematopoietic stemcell transplantation.

NLRP6 plays a crucial role in maintaining intestinal homeostasis by regulating the interaction between the intestinal mucosa and the microbiota. However, the impact of NLRP6 deficiency on intestinal damage following hematopoietic stem cell transplantation (HSCT) remains poorly understood. In this study, we established a syngeneic HSCT mouse model using C57BL/6 mice as donors and NLRP6-/- or C57BL/6 mice as recipients. Our findings revealed that NLRP6 deficiency had minimal influence on peripheral blood cell counts and splenic immune cell proportions in transplanted mice. However, it exacerbated pathological changes in the small intestine on day 14 post-transplantation, accompanied by increased proportions of macrophages, dendritic cells, and neutrophils. Furthermore, the NLRP6 deficiency resulted in elevated expression of MPO and CD11b, while reducing the levels mature caspase-1 and mature IL-1β in the intestine. Moreover, the NLRP6 deficiency disturbed the expression of apoptosis-related molecules and decreased the tight junction protein occludin. Notably, recipient mice with NLRP6 deficiency exhibited lower mRNA expression levels of antimicrobial genes, such as Reg3γ and Pla2g2a. The short-term increase in inflammatory cell infiltration caused by NLRP6 deficiency was associated with intestinal damage, increased apoptosis, reduced expression of antimicrobial peptides, and impaired intestinal repair. Taken together, our findings demonstrate that the loss of NLRP6 exacerbates post-transplantation intestinal damage in recipient mice.

"Next top" mouse models advancing CTCL research.

This review systematically describes the application of in vivo mouse models in studying cutaneous T-cell lymphoma (CTCL), a complex hematological neoplasm. It highlights the diverse research approaches essential for understanding CTCL's intricate pathogenesis and evaluating potential treatments. The review categorizes various mouse models, including xenograft, syngeneic transplantation, and genetically engineered mouse models (GEMMs), emphasizing their contributions to understanding tumor-host interactions, gene functions, and studies on drug efficacy in CTCL. It acknowledges the limitations of these models, particularly in fully replicating human immune responses and early stages of CTCL. The review also highlights novel developments focusing on the potential of skin-targeted GEMMs in studying natural skin lymphoma progression and interactions with the immune system from onset. In conclusion, a balanced understanding of these models' strengths and weaknesses are essential for accelerating the deciphering of CTCL pathogenesis and developing treatment methods. The GEMMs engineered to target specifically skin-homing CD4+ T cells can be the next top mouse models that pave the way for exploring the effects of CTCL-related genes.

Liraglutide Protects Pancreatic Islet From Ischemic Injury by Reducing Oxidative Stress and Activating Akt Signaling During Cold Preservation to Improve Islet Transplantation Outcomes.

Islet transplantation is a promising therapy for patients with type 1 diabetes. However, ischemic injury to the donor islets during cold preservation leads to reduced islet quality and compromises transplant outcome. Several studies imply that liraglutide, a glucagon-like peptide-1 receptor agonist, has a positive effect on promoting islet survival, but its impact on islet cold-ischemic injury remains unexplored. Therefore, the aim of this study was to investigate whether liraglutide can improve islet transplantation efficacy by inhibiting cold-ischemic injury and to explore the underlying mechanisms. Liraglutide was applied in a mouse pancreas preservation model and a human islets cold-preservation model, and islet viability, function, oxidative stress levels were evaluated. Furthermore, islet transplantation was performed in a syngeneic mouse model and a human-to-nude mouse islet xenotransplantation model. The supplementation of liraglutide in preservation solution improved islet viability, function, and reduced cell apoptosis. Liraglutide inhibited the oxidative stress of cold-preserved pancreas or islets through upregulating the antioxidant enzyme glutathione levels, inhibiting reactive oxygen species accumulation, and maintaining the mitochondrial membrane integrity, which is associated with the activation of Akt signaling. Furthermore, the addition of liraglutide during cold preservation of donor pancreas or donor islets significantly improved the subsequent transplant outcomes in both syngeneic mouse islet transplantation model and human-to-nude mouse islet xenotransplantation model. Liraglutide protects islets from cold ischemia-related oxidative stress during preservation and hence improved islet transplantation outcomes, and this protective effect of liraglutide in islets is associated with the activation of Akt signaling.

Abstract 5318: SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells

Abstract Background: T cells require both signal 1 TCR engagement and signal 2 co-stimulatory signal to achieve complete activation and tumor recognition. CD28 is a signal 2 co-stimulatory receptor and activating CD28 increases T cell sensitivity, cytokine production and T cell survival. PD-1/PD-L1 signaling attenuates the T cells functional activity by inhibiting CD28 co-stimulation in the presence of TCR stimulation. Therefore, we developed SM2275, a tetraspecific VHH antibody against EGFR, PD-L1, CD28 and HSA, for the treatment of EGFR+/PD-L1+ solid tumors. Preclinical study demonstrated promising efficacy and favorable safety profile via a unique mechanism of action. Methods: SM2275 was developed using StarMab’s proprietary Quadbody multifunctional VHH antibody platform. It features two monovalent, low-affinity binding domains for EGFR and PD-L1, along with nanomolar low affinity CD28 binding domains arranged in a tandem format, followed by a half-life extending HSA binding domain. Differential binding affinities of SM2275 to EGFR/PD-L1 single-expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity-enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single-positive cells was analyzed via competitive binding assay and Jurkat PD1 NFAT Luciferase reporter assay. EGFR/PD-L1 dual-target-dependent CD28 activation was confirmed using a primary human T cell IL2 &amp; IFN-γ release assay in the presence of TCR stimulation. In vivo efficacy against EGFR+/PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of MC38-hEGFR/hPDL1 cell line into hPD-L1/hCD28 humanized mice. Results: SM2275 displays dramatically increased affinity for cancer cells co-expressing EGFR/PD-L1 compared to single-expressing cells. This leads to an avidity-enhanced PD-L1 blockade specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, enabling localized reactivation of tumor-infiltrating T cells without systemic immune toxicity. Simultaneously blocking the PD-L1 signaling unleashed the inhibition of CD28 co-stimulation for the full T cell activation. In the presence of SM2275, EGFR/PD-L1 double-positive cancer cells induce significantly enhanced CD28 activation and cytokine secretion compared to single-antigen-expressing cells. In a syngeneic tumor transplantation model, SM2275 effectively inhibits the growth of MC38-hEGFR/hPDL1 tumors in hPD-L1-hCD28 humanized mice, with no apparent cytokine release and immune toxicity. Conclusions: SM2275 is a novel dual-EGFR &amp; PD-L1-targeting, conditional CD28 agonistic antibody. It demonstrated promising efficacy and favorable safety profile in preclinical studies via a unique mechanism of action. Citation Format: Shihao Lu, Xiaodan Liu, Sheila Zhou, Wenjing Song, Huiqin Geng, Simin Yang, Hong Zhou, Siyao Xie, Yi Wei, Xing Zhang, Yanbin Liang. SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5318.

Abstract 5316: SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response

Abstract Background: Monoclonal antibodies targeting the co-stimulatory receptors CD137 (4-1BB) and CD40 have displayed encouraging activity against a wide range of solid tumors in preclinical animal models. However, their clinical development as monotherapy has been hindered by dose-related liver toxicity, leading to limited efficacy at safe doses. To address this challenge and bolster tumor-specific immunity by orchestrating the interaction between dendritic cells (DCs) and cytotoxic T cells, we introduce SM2256 - a bispecific conditional CD40 and 4-1BB dual agonist. SM2256 physically bridges DCs and T cells, enhancing T cell priming and reactivation. In preclinical animal studies, SM2256 exhibited both excellent tolerability and potent anti-tumor activity. Methods: SM2256 was engineered by fusing high-affinity, non-ligand-blocking VHH domains targeting 4-1BB and CD40 to a silenced IgG1 Fc backbone. This fusion resulted in a tetravalent bispecific antibody capable of conditionally activating CD40 and 4-1BB upon mutual receptor binding. We assessed the binding specificity and affinity of SM2256 to human CD40 and 4-1BB using ELISA and flow cytometry. The target-dependent activation of CD40 and 4-1BB was confirmed via reporter assays and co-culture systems using primary DCs and T cells. In vivo safety and anti-tumor efficacy were evaluated in a syngeneic transplantation model using the MC38 cancer cell line in B-h41BB/B-hCD40 double humanized mice. Results: SM2256 exhibited sub-nanomolar binding affinity to recombinant human and cynomolgus monkey CD40 and 4-1BB, activating 4-1BB-NFκB-luc and CD40-NFκB-luc reporter cell lines at picomolar concentrations in a strictly target-dependent manner. Furthermore, SM2256 physically facilitated the interaction between DCs and T cells, leading to increased expression of DC co-stimulatory ligands and elevated secretion of cytokines by T cells in mixed lymphocyte cultures. In a syngeneic tumor transplantation model, SM2256 significantly restrained the growth of MC38 tumors in B-h41BB/B-hCD40 double humanized mice. Conclusions: SM2256 is a novel conditional CD40 and 4-1BB dual agonist that demonstrates promising preclinical efficacy against solid tumors while boasting an improved safety profile when compared to existing monoclonal antibodies. Citation Format: Wenjing Song, Simin Yang, Yi Wei, Shihao Lu, Huiqin Geng, Xiaodan Liu, Hong Zhou, Siyao Xie, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5316.