Mouse Model Of Transplantation Research Articles

Reduction in Renal Interstitial Fibrosis in Aged Male Mice by Intestinal Microbiota Rejuvenation.

Renal interstitial fibrosis is an important pathological basis for kidney ageing and the progression of ageing nephropathy. In the present research, we established an aged mouse model of faecal microbiota transplantation (FMT), identified the rejuvenation features of the kidney in aged male mice, and preliminarily analysed the possible mechanism by which the rejuvenation of the intestinal microbiota reduces renal interstitial fibrosis and delays senescence in aged male mice. We established an aged male mice model that was treated with FMT (FMT-Old) and a normal aged male mice control group (Old). Differentially expressed cytokines were identified using a cytokine array, and changes in protein expression related to signal transduction pathways in renal tissues were detected using a signalling pathway array. Senescence-associated β-galactosidase and Masson staining were performed to observe the degrees of renal senescence and tubule interstitial fibrosis. Immunohistochemistry was utilized to detect changes in the expression of the ageing markers p53 and p21 and the inflammation-related protein nuclear factor (NF-κB) subunit (RelA/p65). The pathological features of renal senescence in the FMT-Old group were significantly alleviated, and the levels of the ageing indicators p53 and p21 were decreased (p < 0.05). Ingenuity Pathway Analysis revealed that six differentially expressed cytokines, MIP-3β (CCL-19), E-selectin (SELE), Fas ligand (Fas L/FASLG), CXCL-11 (I-TAC), CXCL-1 and CCL-3 (MIP-1α) were related to a common upstream regulatory protein, RelA/p65, and the expression of this protein was significantly different between groups according to the signalling pathway array. Our findings suggest that the intestinal microbiota regulates the renal microenvironment by reducing immune inflammatory responses through the inhibition of the NF-κB signalling pathway, thereby delaying renal senescence in aged male mice.

Early graft-infiltrating lymphocytes are not associated with graft rejection in a mouse model of skin transplantation.

Graft-infiltrating lymphocytes (GILs) play an important role in promoting rejection after organ transplantation. We recently reported that GILs that accumulated up to 3 days post-transplantation did not promote rejection, whereas GILs present 3-5 days post-transplantation promoted rejection in a mouse heart transplantation model. However, the immunological behaviour of GILs in murine skin transplantation remains unclear. GILs were isolated on days 3, 5 or 7 post-transplantation from C57BL/6 (B6) allogeneic skin grafts transplanted onto BALB/c mice. BALB/c Rag2-/- γc-/- mice (BRGs) underwent B6 skin graft transplantation 10 weeks after adoptive transfer of day 3, 5, or 7 GILs. BRGs reconstituted with day 5 or 7 GILs completely rejected B6 grafts. However, when B6 grafts harvested from recipient BALB/c mice on day 5 or 7 were re-transplanted into BRGs, half of the re-transplanted day 5 grafts established long-term survival, although all re-transplanted day 7 grafts were rejected. BRGs reconstituted with day 3 GILs did not reject B6 grafts. Consistently, re-transplantation using day 3 skin grafts resulted in no rejection. Administration of anti-CD25 antibodies did not prevent the phenomenon observed for the day 3 skin grafts. Furthermore, BRGs reconstituted with splenocytes from naïve BALB/c mice immediately rejected the naïve B6 skin grafts and the re-transplanted day 3 B6 grafts, suggesting that day 3 GILs were unable to induce allograft rejection during the rejection process. In conclusion, the immunological role of GILs depends on the time since transplantation. Day 3 GILs had neither protective nor alloreactive effects in the skin transplant model.

HnRNPA1 Prevents Endothelial-to-mesenchymal Transition-induced VSMC Activation and Neointimal Hyperplasia in Vein Grafts.

Endothelial-to-mesenchymal transition (EndoMT) is associated with neointimal hyperplasia and vein graft failure, and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has emerged as a major modulator of EMT. We aimed to investigate the functional consequence of EndoMT in neointimal hyperplasia and the precise role of hnRNPA1 in the regulation of EndoMT and neointimal hyperplasia. We investigated the spatial and temporal distribution characteristics of EndoMT cells in a mouse model of vein graft transplantation. In vitro, we studied the interaction between EndoMT cells and VSMCs, and the underlying mechanism was investigated by cytokine antibody assays. In cultured HUVECs, we studied the effect of hnRNPA1 on EndoMT and the cellular interactions by using siRNA-mediated knockdown and adenovirus-mediated overexpression. We further investigated the role of hnRNPA1 in EndoMT and neointimal hyperplasia in vivo with an AAV-mediated EC-specific hnRNPA1 overexpression murine model. We demonstrated the presence of EndoMT cells during the initial stage of neointimal formation, and that EndoMT cells promoted the proliferation and migration of VSMCs in vitro. Mechanistic studies revealed that EndoMT cells express and secrete a higher level of PDGF-B. Furthermore, we found a regulatory role for hnRNPA1 in EndoMT in vitro and in vivo. Similarly, we found that hnRNPA1 overexpression in ECs reduced the expression and secretion of PDGF-B during EndoMT, effectively inhibiting EndoMT cell-mediated activation of VSMCs in vitro and neointimal formation in vivo. Taken together, these findings indicate that EndoMT cells can activate VSMCs through a paracrine mechanism mediated by hnRNPA1 and lead to neointimal hyperplasia.

Transcriptional evidence for transient regulation of muscle regeneration by brown adipose transplant in the rotator cuff.

Chronic rotator cuff(RC) injuries can lead to a degenerative microenvironment that favors chronic inflammation, fibrosis, and fatty infiltration. Recovery of muscle structure and function will ultimately require a complex network of muscle resident cells, including satellite cells, fibro-adipogenic progenitors (FAPs), and immune cells. Recent work suggests that signaling from adipose tissue and progenitors could modulate regeneration and recovery of function, particularly promyogenic signaling from brown or beige adipose (BAT). In this study, we sought to identify cellular targets of BAT signaling during muscle regeneration using a RCBAT transplantation mouse model. Cardiotoxin injured supraspinatus muscle had improved mass at 7 days postsurgery (dps) when transplanted with exogeneous BAT. Transcriptional analysis revealed transplanted BAT modulates FAP signaling early in regeneration likely via crosstalk with immune cells. However, this conferred no long-term benefit as muscle mass and function were not improved at 28 dps. To eliminate the confounding effects of endogenous BAT, we transplanted BAT in the "BAT-free" uncoupling protein-1diphtheria toxin fragment A (UCP1-DTA) mouse and here found improved muscle contractile function, but not mass at 28 dps. Interestingly, the transplanted BAT increased fatty infiltration in all experimental groups, implying modulation of FAP adipogenesis during regeneration. Thus, we conclude that transplanted BAT modulates FAP signaling early in regeneration,but does not grant long-term benefits.

Pexidartinib and Immune Checkpoint Inhibitors Combine to Activate Tumor Immunity in a Murine Colorectal Cancer Model by Depleting M2 Macrophages Differentiated by Cancer-Associated Fibroblasts.

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are known to play supportive roles in tumor development and progression, but their interactions in colorectal cancer (CRC) remain unclear. Here, we investigated the effects of colon-cancer-derived CAFs on TAM differentiation, migration, and tumor immunity, both in vitro and in vivo. When co-cultured with monocytes, CAFs attracted monocytes and induced their differentiation into M2 macrophages. Immunohistology of surgically resected human CRC specimens and orthotopically transplanted mouse tumors revealed a correlation between numbers of CAFs and numbers of M2 macrophages. In a mouse model of CRC orthotopic transplantation, treatment with an inhibitor of the colony-stimulating factor-1 receptor (PLX3397) depleted M2 macrophages and increased CD8-positive T cells infiltrating the tumor nest. While this treatment had a minor effect on tumor growth, combining PLX3397 with anti-PD-1 antibody significantly reduced tumor growth. RNA-seq following combination therapy showed activation of tumor immunity. In summary, CAFs are involved in the induction and mobilization of M2 macrophage differentiation in the CRC tumor immune microenvironment, and the combination of cancer immunotherapy and PLX3397 may represent a novel therapeutic option for CRC.

Immuno-protective vesicle-crosslinked hydrogel for allogenic transplantation

The longevity of grafts remains a major challenge in allogeneic transplantation due to immune rejection. Systemic immunosuppression can impair graft function and can also cause severe adverse effects. Here, we report a local immuno-protective strategy to enhance post-transplant persistence of allografts using a mesenchymal stem cell membrane-derived vesicle (MMV)-crosslinked hydrogel (MMV-Gel). MMVs are engineered to upregulate expression of Fas ligand (FasL) and programmed death ligand 1 (PD-L1). The MMVs are retained within the hydrogel by crosslinking. The immuno-protective microenvironment of the hydrogel protects allografts by presenting FasL and PD-L1. The binding of these ligands to T effector cells, the dominant contributors to graft destruction and rejection, results in apoptosis of T effector cells and generation of regulatory T cells. We demonstrate that implantation with MMV-Gel prolongs the survival and function of grafts in mouse models of allogeneic pancreatic islet cells and skin transplantation.

Rapamycin Liposomes Targeted to Lymph Nodes Inhibit Dendritic Cell Maturation for the Treatment of Transplant Rejection

AbstractThe activation of naive T cells by mature dendritic cells (DCs) presenting allograft antigens marks a pivotal stage in triggering transplant rejection. A critical intervention in this process involves the administration of rapamycin, which disrupts the mTOR signaling pathway, thereby impeding DC maturation. Nevertheless, systemic administration of rapamycin faces challenges due to its limited bioavailability, non‐specific targeting, and notable side effects. To address these limitations, LNP@rapa (liposome‐encapsulated rapamycin) is developed, administered via subcutaneous injection. This formulation selectively targets lymph nodes, inhibiting DC maturation within these nodes and mitigating transplant rejection. This study validates the in vivo efficacy of LNP@rapa, demonstrating its ability to hinder DC maturation, reduce inflammatory cytokine secretion, and significantly prolong graft survival in two distinct mouse transplantation models. This study introduces an innovative strategy targeting lymph nodes to impede DC maturation, offering a promising approach to address transplant rejection.

GuiErBai: a potent inhibitor, exhibiting broadly antitumor effect against cervical cancer in vitro and in vivo.

Introduction: Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai (GEB), a compound preparation developed by our research team, is derived from the ancient Chinese medicine of the Miao nationality and is comprised of podophyllotoxin (PTOX), imperatorin, isoimperatorin, and A. dahurica alkaloids. These individual components have demonstrated notable efficacy in tumor treatment. However, the specific anti-tumor effect of the compound Chinese medicine GEB in the context of CC has yet to be validated. Methods: HeLa and SiHa cell lines were utilized for in vitro experiments and treated with 5mg/mL and 10mg/mL GEB concentrations, respectively. The cell cycle changes after GEB treatment were assessed using flow cytometry. Transmission electron microscopy was employed to observe autophagic bodies and apoptotic bodies, while MDC staining evaluated the occurrence of autophagy. CCK-8 was used to observe the effect of GEB on cell proliferation, and Transwell assays assessed cell migration and invasion. Western blotting detected cell cycle and apoptosis-related protein expression, along with the expression level of autophagy-related protein LC3I/II. Changes in ROS and mitochondrial membrane potential in cervical cancer cells following GEB treatment were determined using ROS detection and mitochondrial membrane potential detection kits. For the in vivo experiment, a nude mouse model of cervical cancer transplantation based on HeLa cells was established. Experimental animals were divided into negative control, positive control, high-dose GEB (10mg/mL), and low-dose GEB (5mg/mL) groups. Results: In HeLa and SiHa cell lines, the G0/G1 phase of tumor cells significantly decreased (p < 0.001), while the G2/M phase increased notably (p < 0.001) following various GEB treatments. Electron microscopy showed GEB promoted apoptotic body and autophagosome formation in both cell lines. Compared to untreated HeLa and SiHa cells, GEB-treated cells exhibited significantly reduced caspase3 protein expression, and substantially increased autophagy-related protein LC3I/II expression. GEB treatment significantly reduced migration and invasion capabilities in both cell lines (p < 0.001), while ROS content and mitochondrial membrane potential were significantly elevated (p < 0.001). GEB effectively inhibited cervical cancer cell proliferation, with the optimal concentration being 10mg/mL. A successful nude mouse model of cervical cancer transplantation was established using HeLa cells. Post-GEB treatment, the tumor volume and weight in nude mice significantly decreased (p < 0.001), with diminished expression of CD34, VEGF, and caspase3 proteins in tumor tissues. Discussion: GEB exhibits a robust antitumor effect against cervical cancer, both in vitro and in vivo, in a concentration-dependent manner, by regulating autophagy and apoptosis of tumor cells.

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.