Mouse Model Of Transplantation Research Articles

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.

Microneedle-Mediated Delivery of Immunomodulators Restores Immune Privilege in Hair Follicles and Reverses Immune-Mediated Alopecia.

Disorders in the regulatory arm of the adaptive immune system result in autoimmune-mediated diseases. While systemic immunosuppression is the prevailing approach to manage them, it fails to achieve long-lasting remission due to concomitant suppression of the regulatory arm and carries the risk of heightened susceptibility to infections and malignancies. Alopecia areata is a condition characterized by localized hair loss due to autoimmunity. The accessibility of the skin allowslocal rather than systemic intervention to avoid broad immunosuppression. It is hypothesized that the expansion of endogenous regulatory T cells (Tregs) at the site of antigen encounter can restore the immune balance and generate a long-lasting tolerogenic response. A hydrogel microneedle (MN) patch is therefore utilized for delivery of CCL22, a Treg-chemoattractant, and IL-2, a Tregsurvival factor to amplify them. In an immune-mediated murine model of alopecia, local bolstering of Tregnumbers is shown, leading to sustained hair regrowth and attenuation of inflammatory pathways. In a humanized skin transplant mouse model, expansion of Tregs within human skin is confirmed without engendering peripheral immunosuppression. The patch offers high-loading capacity and shelf-life stability for prospective clinical translation. By harmonizing immune responses locally, the aim is to reshape the landscape of autoimmune skin disease management.

DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer

ObjectiveHighly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments...

Pirfenidone treatment reduces cardiac allograft vasculopathy in a mouse model

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ISHLT research grant Objectives Chronic rejection in the form of cardiac allograft vasculopathy after heart transplantation remains without effective treatment. Main characteristics of cardiac allograft vasculopathy are immigration of leukocytes and proliferation of smooth muscle cells - collectively resulting in development of a neointima narrowing the vessel lumen and leading to graft ischemia. Pirfenidone is an anti-fibrotic drug with anti-inflammatory properties that is already approved for treatment of idiopathic pulmonary fibrosis. The aim of this study was to evaluate whether application of pirfenidone leads to reduced luminal occlusion corresponding with decreased chronic rejection in a mouse transplant model. Methods Murine thoracic aortas were transplanted into the abdominal aorta of CBA mice (H2k). For allogeneic transplantation, C57BL/6 mice (H2b) were used as donors (n = 8 / group). After transplantation, mice were fed a 0.5% pirfenidone diet versus conventional diet. Preliminary tests were performed determining the blood pirfenidone level by mass spectrometry throughout the study period on days 1, 3, 7, 14, 21 and 28, respectively, as well as after obtaining the aortic grafts on day 30 for histological analysis. Results Preliminary tests confirmed the successful uptake of pirfenidone by achieving constant plasma levels of pirfenidone and its metabolite 5-carboxy-pirfenidone over the whole examination period. Pirfenidone treated murine aortic grafts showed significantly reduced neointima formation compared to control allografts (luminal occlusion: 32.92 % ± 13.02 % vs. 42.44 % ± 19.81 %, p=0.046). As expected, syngeneic aortic grafts with and without pirfenidone treatment showed no luminal occlusion. Conclusion The results indicate that pirfenidone application after aortic transplantation reduces neointima formation and thus chronic rejection in a mouse aortic transplant model. Further analyses of underlying mechanisms are currently underway.

Mechanical load regulates the proliferation of multiple cell types in the heart

Abstract Funding Acknowledgements None. Introduction The adult mammalian heart has a poor regenerative and angiogenic potential. At the same time, both primary cardiac tumors and cardiac metastases are extremely rare. This suggests that shared mechanisms may halt the proliferation of cardiac (both cardiomyocyte and endothelial) and cancer cells. Among the mechanisms that have been claimed to be responsible for the loss of proliferative potential of cardiomyocytes early after birth is a sudden increase in mechanical load. Purpose Here, we investigate whether alterations in mechanical load affect the proliferation of multiple cell types in the heart. Methods Adult heart unloading in vivo was achieved using a mouse model of heterotopic heart transplantation, followed by EdU injection to label proliferating cells. Engineered heart tissues (EHTs) were generated using neonatal cardiomyocytes and fibroblasts, with a modified protocol allowing to finely tune mechanical load. Primary cardiac endothelial cells and multiple types of cancer cells (lung, melanoma and colon cancer) along with adult cardiac endothelial cells were included in EHTs. Results In line with our hypothesis, unloaded, heterotransplanted hearts showed a significant number of proliferating cardiomyocytes and endothelial cells, which were almost absent in native hearts. While cancer cells did not grow in native hearts, they massively proliferated in unloaded hearts. Consistent with in vivo results, mechanical unloading in EHTs significantly increased the percentage of EdU-positive cardiomyocytes, endothelial and cancer cells. In contrast, increasing afterload reduced cell proliferation in all cell types. Mechanistically, we identified Nesprin2 as an essential factor in sensing mechanical load and translating it into a cell proliferation arrest. Conclusions Overall, these data indicate that variations in mechanical load have a dramatic effect on the proliferation of multiple cell types within the heart tissue, including cardiomyocytes, endothelial and cancer cells and that Nesprin2 is required to mediate this effect.Unloading in the heartUnloading in EHT

Characterization of an orthotopic mouse transplant model reveals early changes in the tumor microenvironment of lung cancer

Characterization of an orthotopic mouse transplant model reveals early changes in the tumor microenvironment of lung cancer

Abstract A026: PIN1 isomerase promotes the initiation and progression of bladder cancer through SREBP2-mediated cholesterol biosynthesis pathway

Abstract Bladder cancer, one of the most commonly diagnosed cancers worldwide, is associated with high morbidity, mortality, and a poor prognosis. The PIN1 phospho-dependent prolyl isomerase is frequently overexpressed in many human cancer types and affects tumor initiation and progression through disrupting the balance of oncoprotein and tumor suppressor protein signaling. However, the functional pSer/Thr.Pro protein substrates of PIN1 and its effects on downstream signaling in bladder carcinogenesis remain largely unknown. Phenotypically, we discovered that ablation of Pin1 in human/mouse bladder cancer cells and organoids formed from mouse primary urothelial cells resulted in decreased cell proliferation, stemness maintenance, cell invasion, migration, urothelium clearance capacity, and reduced free/total cholesterol levels in vitro. Re-expressing Pin1 in Pin1 knockout cells reversed the defects caused by lack of Pin1. Moreover, in vivo subcutaneous xenograft and allograft transplantation mouse models combined with an orthotopic implantation model were utilized to confirm a positive role for Pin1 in controlling tumor growth and potential metastasis. Therapeutically, a combination of the sulfopin PIN1 inhibitor and the simvastatin HMGCR inhibitor was shown to suppress cell proliferation in vitro and tumor growth in vivo synergistically. Mechanistically, we observed a negative enrichment of SREBP2-driven cholesterol metabolism pathways in PIN1 knockout cells via RNA-sequencing and used a suite of diverse molecular and biochemical approaches to show that PIN1 directly binds to JNK-dependent phosphorylated SREBP2 (Ser455) and vice versa. These results indicate that active cholesterol biosynthesis pathway in bladder cancer cells is regulated by PIN1-mediated isomerization and JNK-mediated phosphorylation of its key transcriptional factor SREBP2. These findings emphasize that PIN1 can act as a regulator and potential therapeutic target in bladder cancer. Citation Format: Xue Wang, Tony Hunter. PIN1 isomerase promotes the initiation and progression of bladder cancer through SREBP2-mediated cholesterol biosynthesis pathway [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A026.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

Abstract PO3-24-08: Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells

Abstract Purpose Cancer stem cells are considered to cause recurrence and metastasis due to their resistance to drug and radiation therapy. Breast cancer stem cells are therefore the true target for the treatment of breast cancer. In this study, we established a method to selectively culture breast cancer stem cells from patient-derived breast cancer tissue, and we analyzed their characteristics using immunostaining to identify gene expression. Method Breast cancer tissues from ER+, HER2- and drug-naive patients were fragmented and cultured using low-adherent plates (spheroid culture). We investigated the stemness of the breast cancer cells obtained by the spheroid culture by immunostaining the breast cancer stem cell markers and examining their proliferation and differentiation in a mouse transplantation model. The diversity of the cells was further examined by means of the expression of the EMT markers and genetic analysis. Results In 48 cases, the proportion of CD44+/CD24- breast cancer cells increased from 13.8% to 61.6% in the spheroid culture. In addition, even small numbers of cells obtained by the spheroid culture were transplanted into mice, and the transplanted cells subsequently differentiated in the mice, demonstrating their stemness. ER expression was negatively changed in 52.1% of cases, and the expression of EMT markers, Twist, Snail, and Vimentin, was increased from 43.8% to 75.0%, 12.9% to 58.1%, and 7.71% to 37.7%, respectively. A DNA microarray with 14 cases showed that breast cancer stem cells obtained from ER+ and HER2- breast cancer tissue were classified into two groups. Conclusion We demonstrated that breast cancer stem cells were selectively cultured from patient-derived breast cancer tissue in a spheroid culture. Furthermore, breast cancer stem cells have different characteristics from breast cancer cells in primary tumors and are diverse at an EMT level, and in genetic analyses. Table. Expression of primary tumors and spheroids Citation Format: Satoshi Sueoka, Takayuki Kadoya, Kai Azusa, Shinsuke Sasada, Akiko Emi, Morihito Okada, Yukino Kobayashi, Koh Nakayama. Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells [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 PO3-24-08.

Targeted depletion of PD-1-expressing cells induces immune tolerance through peripheral clonal deletion.

Thymic negative selection of the T cell receptor (TCR) repertoire is essential for establishing self-tolerance and acquired allograft tolerance following organ transplantation. However, it is unclear whether and how peripheral clonal deletion of alloreactive T cells induces transplantation tolerance. Here, we establish that programmed cell death protein 1 (PD-1) is a hallmark of alloreactive T cells and is associated with clonal expansion after alloantigen encounter. Moreover, we found that diphtheria toxin receptor (DTR)-mediated ablation of PD-1+ cells reshaped the TCR repertoire through peripheral clonal deletion of alloreactive T cells and promoted tolerance in mouse transplantation models. In addition, by using PD-1-specific depleting antibodies, we found that antibody-mediated depletion of PD-1+ cells prevented heart transplant rejection and the development of experimental autoimmune encephalomyelitis (EAE) in humanized PD-1 mice. Thus, these data suggest that PD-1 is an attractive target for peripheral clonal deletion and induction of immune tolerance.

Bazedoxifene attenuates dextran sodium sulfate-induced colitis in mice through gut microbiota modulation and inhibition of STAT3 and NF-κB pathways

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract for which treatment options remain limited. In this study, we used a dual-luciferase-based screening of an FDA-approved drug library, identifying Bazedoxifene (BZA) as an inhibitor of the NF-κB pathway. We further investigated its therapeutic effects in a dextran sodium sulfate (DSS)-induced colitis model and explored its impact on gut microbiota regulation and the underlying molecular mechanisms. Our results showed that BZA significantly reduced DSS-induced colitis symptoms in mice, evidenced by decreased colon length shortening, lower histological scores, and increased expression of intestinal mucosal barrier-associated proteins, such as Claudin 1, Occludin, Zo-1, Mucin 2 (Muc2), and E-cadherin. Used independently, BZA showed therapeutic effects comparable to those of infliximab (IFX). In addition, BZA modulated the abundance of gut microbiota especially Bifidobacterium pseudolongum, and influenced microbial metabolite production. Crucially, BZA's alleviation of DSS-induced colitis in mice was linked to change in gut microbiota composition, as evidenced by in vivo gut microbiota depletion and fecal microbiota transplantation (FMT) mice model. Molecularly, BZA inhibited STAT3 and NF-κB activation in DSS-induced colitis in mice. In general, BZA significantly reduced DSS-induced colitis in mice through modulating the gut microbiota and inhibiting STAT3 and NF-κB activation, and its independent use demonstrated a therapeutic potential comparable to IFX. This study highlights gut microbiota's role in IBD drug development, offering insights for BZA's future development and its clinical applications.

Discovery of Novel Dual Inhibitors Targeting Mutant IDH1 and NAMPT for the Treatment of Glioma with IDH1Mutation.

The targeting of cancer cell intrinsic metabolism has emerged as a promising strategy for antitumor intervention. In the study, we identified the first-in-class small molecules that effectively inhibit both mutant isocitrate dehydrogenase 1 (mIDH1) and nicotinamide phosphoribosyltransferase (NAMPT), two crucial targets in cancer metabolism, through structure-based drug design. Notably, compound 23h exhibits excellent and balanced inhibitory activities against both mIDH1 (IC50 = 14.93 nM) and NAMPT (IC50 = 12.56 nM), leading to significant suppression of IDH1-mutated glioma cell (U87 MG-IDH1R132H) proliferation. Significantly, compound 23h has the ability to cross the blood-brain barrier (B/P ratio, 0.76) and demonstrates remarkable in vivo antitumor efficacy (20 mg/kg) in the U87 MG-IDH1R132H orthotopic transplantation mouse models without any notable toxicity. This proof-of-concept investigation substantiates the viability of discovering small molecules that concurrently target mIDH1 and NAMPT, providing valuable leads for the treatment of glioma and an efficient approach for the discovery of multitarget antitumor drugs.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism

Long noncoding RNAs HAND2-AS1 ultrasound microbubbles suppress hepatocellular carcinoma progression by regulating the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis.

Increasing data indicated that long noncoding RNAs (lncRNAs) were directly or indirectly involved in the occurrence and development of tumors, including hepatocellular carcinoma (HCC). Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues, but its role in HCC progression is unclear. Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes. To study the role of ultrasound microbubbles (UTMBs) mediated HAND2-AS1 in the progression of HCC, in order to provide a new reference for the treatment of HCC. In vitro, we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs, and detected cell proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) by cell counting kit-8 assay, flow cytometry, Transwell invasion assay and Western blotting, respectively. In addition, we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior. Next, the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2 (TIMP2) overexpression vector, and we detected cell proliferation, apoptosis, invasion and EMT. In vivo, we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability. We found that UTMBs carrying HAND2-AS1 restricted cell proliferation, invasion, and EMT, encouraged apoptosis, and HAND2-AS1 silencing eliminated the effect of UTMBs. Additionally, miR-873-5p targets the gene HAND2-AS1, which also targets the 3'UTR of TIMP2. And miR-873-5p mimic counteracted the impact of HAND2-AS1. Further, miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs. We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase (MMP) 2/MMP9. In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice. LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.

Oxaliplatin-Loaded Mil-100(Fe) for Chemotherapy-Ferroptosis Combined Therapy for Gastric Cancer.

Oxaliplatin (Oxa) is a commonly used chemotherapy drug in the treatment of gastric cancer, but its toxic side effects and drug resistance after long-term use have seriously limited its efficacy. Loading chemotherapy drugs with nanomaterials and delivering them to the tumor site are common ways to overcome the above problems. However, nanomaterials as carriers do not have therapeutic functions on their own, and the effect of single chemotherapy is relatively limited, so there is still room for progress in related research. Herein, we construct Oxa@Mil-100(Fe) nanocomposites by loading Oxa with a metal-organic framework (MOF) Mil-100(Fe) with high biocompatibility and a large specific surface area. The pore structure of Mil-100(Fe) is conducive to a large amount of Oxa loading with a drug-loading rate of up to 27.2%. Oxa@Mil-100(Fe) is responsive to the tumor microenvironment (TME) and can release Oxa and Fe3+ under external stimulation. On the one hand, Oxa can inhibit the synthesis of DNA and induce the apoptosis of gastric cancer cells. On the other hand, Fe3+ can clear overexpressed glutathione (GSH) in TME and be reduced to Fe2+, inhibiting the activity of glutathione peroxidase 4 (GPX4), leading to the accumulation of intracellular lipid peroxides (LPO), and at the same time releasing a large number of reactive oxygen species (ROS) through the Fenton reaction, inducing ferroptosis in gastric cancer cells. With the combination of apoptosis and ferroptosis, Oxa@Mil-100(Fe) shows a good therapeutic effect, and the killing effect on gastric cancer cells is obvious. In a nude mouse model of subcutaneous tumor transplantation, Oxa@Mil-100(Fe) shows a significant inhibitory effect on tumor growth, with an inhibition rate of nearly 60%. In addition to its excellent antitumor activity, Oxa@Mil-100(Fe) has no obvious toxic or side effects. This study provides a new idea and method for the combined treatment of gastric cancer.

Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM

BackgroundTumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood.MethodsWe depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn−/−) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays.ResultsWe found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden.ConclusionThese findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

Lipocalin-2: a novel potential therapy for GVHD

Czech et al. used mouse models of allogeneic hematopoietic stem cell transplantation (allo-HCT) to investigate the role of lipocalin-2 (LCN2) as a newfound regulator of intestinal graft-versus-host disease (GVHD). Administration of recombinant LCN2 protein after disease onset prevented GVHD progression, suggesting that it may play a role in reversing tissue damage that has already begun.

Abstract 2367: STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma

Abstract Background: Immunotherapy with CLDN18.2/CD3 BiTEs (bispecific T-cell engagers) has shown clinical activity in pancreatic adenocarcinoma. However, only few patients benefit from the treatment, and most responders develop acquired resistance. STING agonists may promote the development of stem-like central memory CD8+ T cells and enhance antitumor responses. This study aimed to investigate the synergistic effect and related mechanism of STING agonist plus CLDN18.2/CD3 BiTEs regimen. Methods: Single-cell RNA sequencing and flow cytometry were used to examine the tumor immune landscape in humanized PDX mouse models. By qPCR, western blot and flow cytometry, an orthotopic transplantation mouse model of pancreatic cancer and an in vitro tumor-PBMC coculture system were established to reveal the potential molecular mechanism of STING agonist ADU-S100 in optimizing the effects of CLDN18.2/CD3 BiTEs (IBI389). Results: In PDAC organoids, IBI389 induced potent, specific cytotoxicity, the CLDN18.2 BiTEs significantly engaged human CD3+ T cells with tumor cells in an organoid-PBMC coculture system in vitro, resulting in the formation of the immunological synapse (IS). IBI389 monotherapy significantly suppressed tumor growth and improved survival in vivo in hCD3e C57BL/6 and humanized hCD34+ PDX mouse models. Further, single-cell RNA sequencing and flow cytometry analysis revealed that the intertumoral TCF1+PD-1+ stem-like T cells and STING signaling activity of T cells were enriched in the early stage of tumor development and sharply decreased in the late stage during CLDN18.2 BiTEs administration. Furthermore, we discovered that the STING agonist ADU-S100 could significantly induce the enrichment of TCF1+PD-1+ stem-like CD8+ T cells in vitro and in vivo, increasing IBI389’s therapeutic effects. Furthermore, humanized PDX mice that were given the combination of IBI389 and ADU-S100 and survived after complete tumor rejection were rechallenged with PDX tumors. Humanized PDX tumors and hCD3e KPC orthotopic tumors from mice treated with the combination contained increased numbers of TCF1+PD-1+ stem-like CD8+ T cells, CD4+CD127+ memory T cells, CD8+CD127+ memory T cells, and fewer numbers of CD4+PD-1+Tim-3+ exhausted T cells, CD8+PD-1+Tim-3+ exhausted T cells and CD4+CD25+ Treg cells. Conclusions: Pancreatic tumors appear to evade immune response by inducing immune-suppressive tumor microenvironment development. In mice, the combination of STING agonist ADU-S100 and CLDN18.2 BiTEs increased the proportion of memory and stem-like CD4+ and CD8+ T cells while decreasing the proportion of regulatory and exhausted T cells in pancreatic tumors, eradicating all detectable tumors. This data may be utilized to formulate immune-based combination therapies for PDAC. Citation Format: Tianxing Zhou, Jingrui Yan, Bin Wang, Yongjie Xie, Chao Yang, Jun Yu, Jihui Hao. STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma [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 2367.

Abstract 2190: Transcriptomic signatures of obesity-driven colon cancer: A multi-species transdisciplinary approach

Abstract Obesity is a highly prevalent metabolic disease and an established risk factor for colon cancer. However, critical biological pathways underpinning this relationship remain undetermined. Herein, we integrate human and murine colon tumor transcriptomic data to identify obesity-responsive pathways conserved between species. Human colon cancer samples (n=155) were collected as part of the clinical ColoCare Study, a prospective cohort study of patients newly diagnosed with colorectal cancer. Enrolled patients were diagnosed with stage I-III colon cancer and treatment naïve at time of tissue collection. Patients were classified as having a BMI &amp;lt;30 kg/m2 (i.e., non-obese) or BMI ≥30 kg/m2 (i.e., obese) at baseline. Age, sex, tumor stage, and collection site were adjusted for in all analyses. Patients were 63.3 ± 13.7 years, 51% male, and 34% had a BMI ≥30. C57BL/6J adult female mice were fed a low-fat control diet (Research Diets D12450J, n= 8) or a high-fat diet-induced obesity regimen (D12492, n=7) for ~23 weeks, at which time 700 Apc-null;KrasG12D/+;Trp53-null;Smad4-null;tdTomato (i.e., AKPS) organoids were orthotopically transplanted into the colonic submucosa via colonoscopy-guided injection. Obese mice weighed more than controls (controls 26.4 ± 3.7 g vs. DIO 45.9 ± 9.7 g) and had more mesenteric fat surrounding the colon (controls 201 ± 143 mg vs. 1176 ± 589 mg). Tumors were excised after 4 weeks of growth. RNA was extracted from bulk human tumor samples and from the epithelial-enriched EpCAM+ cell population in murine tumor samples for RNA sequencing. Genes differentially expressed between the reference and obese groups were identified using DESeq2. For gene set enrichment analysis, genes were preranked by log2 fold change for both human and mouse datasets. There were 76 and 132 differentially expressed genes (DEGs; FDRq&amp;lt;0.10) identified in the human and mouse datasets, respectively. None of these 208 DEGs were common to both datasets. Thirty-five Hallmark gene sets in the human dataset and 25 Hallmark gene sets in the mouse dataset were significantly enriched in the obese group; 18 of these gene sets were common to both mouse and human datasets. These overlapping pathways were largely related to inflammation (e.g., inflammatory response, IL6 JAK STAT3 signaling, TNFɑ signaling) and metabolism (e.g., cholesterol homeostasis, glycolysis, and fatty acid metabolism). Additional analysis revealed 204 genes common between the leading edges of both transcriptomic datasets. These transdisciplinary data suggest that obesity induces a set of metabolic and inflammatory processes and/or pathways in colon tumors which is conserved between mice and humans. Future studies will mechanistically evaluate the role of candidate genes and pathways in obesity-associated colorectal carcinogenesis in our AKPS orthotopic organoid transplant mouse model. Citation Format: Elaine M. Glenny, Tengda Lin, Victoria M. Bandera, Biljana Gigic, Aik Choon Tan, Babak Mirminachi, Saratchandra S. Khumukcham, Alessandro Carpanese, Christy A. Warby, Olena Aksonova, Caroline Himbert, Jennifer Ose, Chris Stubben, David Nix, Kenneth Boucher, Peter Schirmacher, Ildiko Strehli, Jolanta Jedrzkiewicz, Alexander Brobeil, Martin A. Schneider, Christoph Kahlert, Erin M. Siegel, Adetunji T. Toriola, David Shibata, Christopher I. Li, Jane C. Figueiredo, Jatin Roper, Cornelia M. Ulrich, Stephen D. Hursting. Transcriptomic signatures of obesity-driven colon cancer: A multi-species transdisciplinary approach [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 2190.