Orthotopic Transplantation Model Research Articles

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer [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 287.

Bulk and single-cell RNA sequencing analysis with 101 machine learning combinations reveal neutrophil extracellular trap involvement in hepatic ischemia-reperfusion injury and early allograft dysfunction

BackgroundHepatic ischaemia–reperfusion injury (HIRI) is a major clinical concern during the perioperative period and is closely associated with early allograft dysfunction (EAD), acute rejection (AR) and long-term graft survival. Neutrophil extracellular traps (NETs) are extracellular structures formed by the release of decondensed chromatin and granular proteins following neutrophil stimulation. There is growing evidence that NETs are involved in the progression of various liver transplantation complications, including ischaemia–reperfusion injury (IRI). This study aimed to comprehensively analyse the expression patterns of NET-related genes (NRGs) in HIRI, identify HIRI subtypes with distinct characteristics, and develop a reliable EAD prediction model. MethodsMicroarray, bulk RNA-seq, and single-cell sequencing datasets were obtained from the GEO database. Initially, differentially expressed NRGs (DE-NRGs) were identified using differential gene expression analyses. We then utilised a non-negative matrix factorisation (NMF) algorithm to classify HIRI samples. Subsequently, we employed machine learning algorithms to screen the hub NRGs related to EAD and developed an EAD prediction model based on these hub NRGs. Concurrently, we assessed the expression patterns of hub NRGs at the single-cell level using the HIRI. Additionally, we validated C5AR1 expression and its effect on HIRI and NETs formation in a rat orthotopic liver transplantation (OLT) model. ResultsIn this study, we identified 11 DE-NRGs in the HIRI context. Based on these 11 DE-NRGs, HIRI samples were classified into two distinct clusters. Cluster1 exhibited a low expression of DE-NRGs, minimal neutrophil infiltration, mild inflammation, and a low incidence of EAD. Conversely, Cluster2 displayed the opposite phenotype, with an activated inflammatory subtype and a higher incidence of EAD. Furthermore, an EAD prediction model was developed using the four hub NRGs associated with EAD. Based on risk scores, HIRI samples were classified into high- and low-risk groups. The OLT model confirmed substantial upregulation of C5AR1 expression in the liver tissue, accompanied by increased formation of NETs. Treatment with a C5AR1 antagonist improved liver function, reduced tissue inflammation, and decreased NETs formation. ConclusionsThis study distinguished two apparent HIRI subtypes, established a predictive model for EAD, and validated the effect of C5AR1 on HIRI. These findings provide novel perspectives for the development of advanced clinical strategies to enhance the outcomes of liver transplant recipients.

Abstract B020: A clinically relevant orthotopic endometrial carcinosarcoma mouse model using human patient-derived organoids

Abstract Uterine carcinosarcoma (CS) is a rare but aggressive endometrial malignancy. The incidence of CS has been increasing and is projected to continue to rise. African American women in particular have an increased incidence of CS and overall increased mortality related to endometrial cancer, underscoring known disparities in cancer care across racial and ethnic groups. Compared to other histologic subtypes, CS remains rare and understudied on a molecular and cellular basis, and under-represented in clinical trials. Therefore, current treatment therapies available for CS have been extrapolated from treatments used in other histologies of endometrial cancer, highlighting a significant need for effective treatment targeted at this subtype. Due to this paucity of data surrounding targeted therapies for CS, patients who are diagnosed with this aggressive and lethal cancer tend to have worse outcomes. We have developed a clinically relevant CS murine model by injecting human CS patient-derived organoids (PDO) from diverse ancestries into the endometrium of immunocompromised mice. Tumor progression in the mice was monitored with physical examination, PET/CT scan, contrast enhanced CT scan, ultrasound, and bioluciferase imaging. The tumor was established after three months, and the mice were sacrificed following the experiment. The diagnosis of invasive CS was confirmed by macroscopic and histologic analyses in the orthotopic model. Our orthotopic CS PDO transplantation model has many potential applications for studying tumor biology of CS in the pre-clinical setting. Future directions include trialing the efficacy of standard of care chemotherapy in the orthotopic model. Using this model, we aim to identify targeted therapy for CS and potentially advance to Phase I human clinical trials. Citation Format: Megan Gorman, Divya Gowthaman, Arielle Katcher, Charlie Chung, Brian Yueh, Mali Barbi, Libia Garcia, Marina Frimer, Gary L. Goldberg, Semir Beyaz. A clinically relevant orthotopic endometrial carcinosarcoma mouse model using human patient-derived organoids [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B020.

Improving the predictive accuracy of efficacy evaluation using tumor orthotopic transplant and resection model.

Preclinical efficacy evaluation and tumor drug sensitivity analysis are two main applications of efficacy evaluation. Preclinical efficacy evaluation is to predict whether candidate drugs or therapies may improve patient outcomes in clinical trials. Tumor drug sensitivity analysis is an approach for the personalized evaluation and optimization of approved anti-cancer drugs and treatment regimens. Overall survival (OS) is the gold standard to evaluate the outcome of drugs or therapies in both clinical trials and clinical treatment. Many efficacy evaluation models, such as cell model, tumor cell-line transplant model, patient-derived tumor xenograft model, tumor organoid model, have been developed to assess the inhibitory effect of tested drugs or therapies on tumor growth. In fact, many treatments may also lead to malignant progression of tumors, such as chemotherapy, which can lead to metastasis. Therefore, tumor growth inhibition does not necessarily predict OS benefit. Whether it can prevent or inhibit tumor recurrence and metastasis is the key to whether drugs and therapies can improve patient outcomes. In this perspective, we summarize the current understanding of the pathological progression of tumor recurrence and metastasis, point out the shortcomings of existing tumor transplant models for simulating the clinical scenario of malignant progression of tumors, and propose five improved indicators for comprehensive efficacy evaluation to predict OS benefit using tumor orthotopic transplant and resection model. Improvement in the accuracy of efficacy evaluation will accelerate the development process of anti-cancer drugs or therapies, optimize treatment regimens to improve OS benefit, and reduce drug development and cancer treatment costs.

Evaluating the Effects of Kidney Preservation at 10 °C with Hemopure and Sodium Thiosulfate in a Rat Model of Syngeneic Orthotopic Kidney Transplantation.

Kidney transplantation is preferred for end-stage renal disease. The current gold standard for kidney preservation is static cold storage (SCS) at 4 °C. However, SCS contributes to renal graft damage through ischemia-reperfusion injury (IRI). We previously reported renal graft protection after SCS with a hydrogen sulfide donor, sodium thiosulfate (STS), at 4 °C. Therefore, this study aims to investigate whether SCS at 10 °C with STS and Hemopure (blood substitute), will provide similar protection. Using in vitro model of IRI, we subjected rat renal proximal tubular epithelial cells to hypoxia-reoxygenation for 24 h at 10 °C with or without STS and measured cell viability. In vivo, we preserved 36 donor kidneys of Lewis rats for 24 h in a preservation solution at 10 °C supplemented with STS, Hemopure, or both followed by transplantation. Tissue damage and recipient graft function parameters, including serum creatinine, blood urea nitrogen, urine osmolality, and glomerular filtration rate (GFR), were evaluated. STS-treated proximal tubular epithelial cells exhibited enhanced viability at 10 °C compared with untreated control cells (p < 0.05). Also, STS and Hemopure improved renal graft function compared with control grafts (p < 0.05) in the early time period after the transplant, but long-term function did not reach significance. Overall, renal graft preservation at 10 °C with STS and Hemopure supplementation has the potential to enhance graft function and reduce kidney damage, suggesting a novel approach to reducing IRI and post-transplant complications.

Safe Procedure for Efficient Hydrodynamic Gene Transfer to Isolated Porcine Liver in Transplantation.

Although calcineurin inhibitors are very effective as immunosuppressants in organ transplantation, complete graft acceptance remains as a challenge. Transfer of genes with immunosuppressant functions could contribute to improving the clinical evolution of transplantation. In this sense, hydrodynamic injection has proven very efficacious for liver gene transfer. In the present work, the hIL-10 gene was hydrofected 'ex vivo' to pig livers during the bench surgery stage, to circumvent the cardiovascular limitations of the procedure, in a model of porcine orthotopic transplantation with a 10-day follow-up. We used IL-10 because human and porcine proteins can be differentially quantified and for its immunomodulatory pleiotropic functions. Safety (biochemical parameters and histology), expression efficacy (RNA transcription and blood protein expression), and acute inflammatory response (cytokines panel) of the procedure were evaluated. The procedure proved safe as no change in biochemical parameters was observed in treated animals, and human IL-10 was efficaciously expressed, with stationary plasma protein levels over 20 pg/mL during the follow-up. Most studied cytokines showed increments (interferon-α, IFN-α; interleukin-1β, IL-1β; tumor necrosis factor α, TNFα; interleukin-6, IL-6; interleukin-8, IL-8; interleukin-4, IL-4; and transforming growth factor-β, TGF-β) in treated animals, without deleterious effects on tissue. Collectively, the results support the potential clinical interest in this gene therapy model that would require further longer-term dose-response studies to be confirmed.

Abstract B070: Histone deacetylation inhibition promotes bi-directional subtype switch in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with 5-year survival of just 11%. Recent large-scale transcriptomic studies have identified two prognostically relevant neoplastic tumor subtypes, i.e. the well-differentiated classical (CLA) subtype with better prognosis, characterized by epithelial gene expression, as well as the dedifferentiated, invasive basal-like (BL) subtype, which displays mesenchymal stemness-related gene signatures. These subtypes have shown to be highly plastic and responsive to extrinsic signals, e.g. from the microenvironment or treatment-induced. Here, we investigate master transcription factor networks governing lineage determination in the different subtypes and the role of histone deacetylation (HDAC) co-regulators in their maintenance. We utilized established, subtype-defined PDAC cell lines in vitro and in orthotopic transplantation models to determine subtype plasticity in response to HDAC inhibition by the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). By integration of RNA-seq, ATAC-seq as well as HiChIP for the activating mark histone 3 lysine 27 acetylation (H3K27ac), we show that the subtype-dependent chromatin organization and regulatory networks can be influenced by pharmacological inhibition of chromatin remodelers, in turn promoting changes in transcription of lineage-determining factors. Targeting these transcription factor networks by HDAC inhibition can lead both to a favorable switch from BL to CLA identity, as well as a detrimental CLA to BL switch. Functional assay such as trans-well invasion further supported the changes in subtype identity following SAHA treatment. This bi-directional subtype switch was additionally validated in patient-derived xenograft cell lines. Together, our findings highlight the importance of understanding the underlying transcriptional and epigenetic mechanisms of the pancreatic cancer subtypes, as they may exhibit opposing responses to targeted therapies. Citation Format: Lukas Klein, Mengyu Tu, Eva K. Schmitt, Frederike Penz, Athanasia Mizi, Stefan Küffer, Elisabeth Hessmann, Argyris Papantonis, Volker Ellenrieder, Shiv K. Singh. Histone deacetylation inhibition promotes bi-directional subtype switch in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B070.

Antitumor activity of three new azoloazine derivatives in orthotopic transplantation model of human breast cancer cells into mice

Breast cancer (BC) is one of the most common types of malignant tumors, which makes scientific research in this area extremely relevant. The difficulties of breast cancer chemotherapy stimulate the search for new drugs to treat this nosology. Derivatives of imidazotriazine and imidazotetrazine, which are analogues of the antitumor drug temozolamide, can be ones of the promising drugs in this regard.The aim of the work was to evaluate the antitumor activity of three new azoloazine derivatives in a xenogeneic breast cancer model in mice in vivo.Materials and methods. A study was conducted on a xenogeneic model of BC. After the immunosuppression with azathioprine, 48 white BALB/c mice were injected with MCF-7 cells, test derivatives, and the reference drug epirubicin at doses of 1/2 IC50 and 1/10 IC50, into the base of the mammary gland once. The body weight of the mice was monitored; on the 15th day, at the end of the experiment, the relative volume was assessed.Results and discussion. Among the three compounds studied, imidazotetrazine 1 showed the most encouraging results: stopping the loss of body weight in the mice caused by the administration of tumor cells, and reducing the tumor volume on the 15th day of the experiment to 50.6% of that in the control when using a dose of 1/10 IC50, up to 39.2% – when using a dose of 1/2 IC50, which significantly exceeded the values of the reference drug epirubicin and the values in the control group. In the histological examination, the signs of spread and preservation of tumor cells viability of the MCF-7 line after its administration were minimal, the value of the histological malignancy index decreased by 9.3% of the control value.Conclusion. Among the tested azoloazine derivatives, 3-cyclohexyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazine-8-N-piperidinylcarboxamide is the undisputed leader, causing inhibition of the tumor growth in a xenogeneic model in vivo.

Establishment of Mouse Orthotopic Urinary Bladder Tumor Model and Its Analysis by Light and Electron Microscopy.

Mouse tumor models are an important tool in cancer research, and the orthotopic cancer cell transplantation model is the most widely used among them. Methods for establishing tumor models may differ in many ways, including the selection of cancer cell lines and the type of urinary bladder pretreatment. Here, we describe our mouse orthotopic bladder tumor model using a labeled MB49 urothelial cancer cell line and chemical pretreatment with the cationic polypeptide poly-L-lysine to traumatize the bladder epithelium. Double labeling of MB49 cancer cells by their transduction with GFP and internalization of metal nanoparticles allows the study of their implantation process from the first hours to several days after intravesical injection, as well as the analysis of developed tumors after 3weeks. Thus, our model provides a comprehensive analysis of the early and late stages of tumor development in the bladder at the light and electron microscopic level.

Abstract PR02: Lyn kinase regulates myeloid-cell responses to mammary tumors

Abstract Myeloid-cell enrichment in tumors is correlated with increased metastasis, reduced survival times, and poor treatment outcomes in multiple cancers, including breast cancer. Many signals (CSF, Il-6, chemokine) to myeloid cells in the tumor microenvironment are transduced through the Src-family kinase Lyn. Expression of both isoforms of Lyn (LynA and LynB) is regulated by cytokine signaling and cell type, and total Lyn expression is negatively correlated with breast cancer patient survival. Lyn expression in macrophages contributes to leukemogenesis, but the functional contributions of LynA and LynB in solid tumors have not been studied. We are examining the contributions of Lyn in trafficking of myeloid cells to mammary tumors, pro-tumor polarization of tumor-associated macrophages, and growth and metastasis of mammary tumors. In an E0771 orthotopic transplant model in hosts expressing or lacking LynA or LynB expression, we have discovered that Lyn expression in the mammary tumor microenvironment promotes the early recruitment of monocytes, macrophages, neutrophils, and dendritic cells, as determined by flow cytometry analysis of tumors early in development. Furthermore, Lyn promotes pro-tumor polarization of tumor-associated macrophages. Tumors from Lyn knockout animals contained populations of inflammatory, anti-tumor macrophages that were not significantly different in size from WT, but the CD206+, anti-inflammatory population was reduced. Finally, we have observed that expression of either Lyn isoform in the tumor microenvironment can promote tumor growth and decrease survival in orthotopically transplanted mice. In contrast, our studies suggest that only LynB promotes metastasis to the lungs of transplanted mice, as determined by in vivo imaging of a hyper-metastatic, luciferase-expressing variant of E0771. Together, our findings suggest that Lyn kinase plays a role in the signaling response of myeloid cells, which are major factors in disease course and treatment response, to tumor-associated stimuli. Modulation of Lyn activity, specifically the LynB isoform, or the activity of its downstream signaling partners, is thus a potential strategy for influencing the trafficking and polarization of myeloid cells within breast tumors to benefit patients. Citation Format: Joseph T Greene, Ben F Brian, Aditi Bapat, Tanya S Freedman. Lyn kinase regulates myeloid-cell responses to mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR02.

A modified nucleoside O6-methyl-2′-deoxyguanosine-5′-triphosphate exhibits anti-glioblastoma activity in a caspase-independent manner

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2′-deoxyguanosine-5′-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Tumor cell-induced macrophage senescence plays a pivotal role in tumor initiation followed by stable growth in immunocompetent condition

BackgroundThe cancer stem cell theory proposes that tumor formation in vivo is driven only by specific tumor-initiating cells having stemness; however, clinical trials conducted to test drugs that target the...

Anti-CD20 Antibody and Calcineurin Inhibitor Combination Therapy Effectively Suppresses Antibody-Mediated Rejection in Murine Orthotopic Lung Transplantation.

Antibody-mediated rejection (AMR) is a risk factor for chronic lung allograft dysfunction, which impedes long-term survival after lung transplantation. There are no reports evaluating the efficacy of the single use of anti-CD20 antibodies (aCD20s) in addition to calcineurin inhibitors in preventing AMR. Thus, this study aimed to evaluate the efficacy of aCD20 treatment in a murine orthotopic lung transplantation model. Murine left lung transplantation was performed using a major alloantigen strain mismatch model (BALBc (H-2d) → C57BL/6 (BL/6) (H-2b)). There were four groups: isograft (BL/6→BL/6) (Iso control), no-medication (Allo control), cyclosporine A (CyA) treated, and CyA plus murine aCD20 (CyA+aCD20) treated groups. Severe neutrophil capillaritis, arteritis, and positive lung C4d staining were observed in the allograft model and CyA-only-treated groups. These findings were significantly improved in the CyA+aCD20 group compared with those in the Allo control and CyA groups. The B cell population in the spleen, lymph node, and graft lung as well as the levels of serum donor-specific IgM and interferon γ were significantly lower in the CyA+aCD20 group than in the CyA group. Calcineurin inhibitor-mediated immunosuppression combined with aCD20 therapy effectively suppressed AMR in lung transplantation by reducing donor-specific antibodies and complement activation.

The preventive effects of inulin, cellulose, and their mixture on colorectal cancer liver metastasis in mice by regulating gut microbiota.

Many studies have found that dietary fiber can protect against colorectal cancer (CRC). Survival in CRC patients is significantly reduced due to metastasis. However, little is known regarding the impact of dietary fiber on the CRC metastasis. In this study, we analyzed the effects of inulin, cellulose, and their mixture on CRC metastasis in a murine orthotopic transplantation model. BALB/C male mice were divided into the normal control (NC) (AIN-93M diet), MOD (AIN-93M diet), INU (10% w/w inulin), CEL (10% w/w cellulose), and MIX (5% w/w inulin+5% w/w cellulose) groups. Dietary fiber intake inhibited the weights of the orthotopic tumors, liver weights, and liver metastasis area (p<0.05) and improved the survival rate of tumor-bearing mice. Compared to the NC, the expression of β-catenin and the epithelial marker E-cadherin were lower, and that of mesenchymal markers, such as N-cadherin, MMP-9, and VEGF, were higher in the MOD group. All inulin, cellulose, and their mixture restored the gut microbiota diversity, and they, respectively, increased the relative abundance of Bifidobacteriales, Lactobacillus, and Lachnospiraceae. Inulin restored the levels of acetic acid, propionic acid, isobutyric acid, and butyric acid. Spearman correlation analysis results showed that there was a positive correlation between five genera and six short-chain fatty acids (SCFAs) (adjusted p<0.05). In conclusion, all inulin, cellulose, and their mixture have inhibitory effects on CRC metastasis, which may be achieved by the regulation of gut microbiota, the production of SCFAs, and the inhibition of the epithelial-to-mesenchymal transition process. Among the three dietary fiber intervention groups, the inhibitory effect of inulin is more significant.

Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity

BackgroundPancreatitis is known to be an important risk factor for pancreatic ductal adenocarcinoma (PDAC). However, the exact molecular mechanisms of how inflammation promotes PDAC are still not fully understood. Regnase-1, an endoribonuclease, regulates immune responses by degrading mRNAs of inflammation-related genes. Herein, we investigated the role of Regnase-1 in PDAC.MethodsClinical significance of intratumor Regnase-1 expression was evaluated by immunohistochemistry in 39 surgically-resected PDAC patients. The functional role of Regnase-1 was investigated by pancreas-specific Regnase-1 knockout mice and Kras-mutant Regnase-1 knockout mice. The mechanistic studies with gene silencing, RNA immunoprecipitation sequencing (RIP-seq) and immune cell reconstitution were performed in human/mouse PDAC cell lines and a syngeneic orthotopic tumor transplantation model of KrasG12D-mutant and Trp53-deficient PDAC cells.ResultsRegnase-1 expression was negatively correlated with the clinical outcomes and an independent predictor of poor relapse-free and overall survival in PDAC patients. Pancreas-specific Regnase-1 deletion in mice promoteed pancreatic cancer with PMN-MDSC infiltration and shortened their survival. A syngeneic orthotopic PDAC model exhibited that Regnase-1 downregulation accelerated tumor progression via recruitment of intratumor CD11b+ MDSCs. Mechanistically, Regnase-1 directly negatively regulated a variety of chemokines/cytokines important for MDSC recruitment and activation, including CXCL1, CXCL2, CSF2, and TGFβ, in pancreatic cancer cells. We subsequently showed that IL-1β-mediated Regnase-1 downregulation recruited MDSCs to tumor sites and promoted pancreatic cancer progression via mitigation of cytotoxic T lympohocytes-mediated antitumor immunity.ConclusionsIL-1b-mediated Regnase-1 downregulation induces MDSCs and promotes pancreatic cancer through the evasion of anticancer immunity.

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

YY1 regulates the proliferation and invasion of triple-negative breast cancer via activating PLAUR.

It is well-established that breast cancer is a highly prevalent malignancy among women, emphasizing the need to investigate mechanisms underlying its pathogenesis and metastasis. In this study, the Gene Expression Omnibus (GEO) database was utilized to conduct differential expression analysis in breast cancer and adjacent tissues. Upregulated genes were selected for prognostic analysis of breast cancer. The expression of urokinase plasminogen activator receptor (uPAR), also known as PLAUR, was assessed using RT-qPCR and western blot. Immunofluorescence staining was employed to determine PLAUR localization. Various cellular processes were analyzed, including proliferation, migration, invasion, apoptosis, and cell cycle. Bioinformatics analysis was used to predict transcription factors of PLAUR, which were subsequently validated in a double luciferase reporter gene experiment. Rescue experiments confirmed the impact of PLAUR on the proliferation, apoptosis, and migration of MDA-MB-231 cells. Furthermore, the effects of PLAUR were evaluated in an orthotopic tumor transplantation and lung metastasis nude mouse model. Our findings substantiated the critical involvement of PLAUR in the progression of triple-negative breast cancer (TNBC) in vitro and among TNBC patients with a poor prognosis. Additionally, we demonstrated Yin Yang-1 (YY1) as a notable transcriptional regulator of PLAUR, whose activation could transcriptionally enhance the proliferation and invasion capabilities of TNBC cells. We also identified the downstream mechanism of PLAUR associated with PLAU, focal adhesion kinase (FAK), and AKT. Overall, these findings offer a novel perspective on PLAUR as a potential therapeutic target for TNBC.

Dual-regulation by Cx32 in hepatocyte to trigger and worsen liver graft injury

Liver transplantation is the ultimate treatment option for end-stage liver failure. However, liver graft injury remains a challenge. This study aimed to investigate the role of connexin32 (Cx32) in liver graft injury and elucidate its mechanism of action. Through detecting liver graft samples from 6 patients, we observed that changes in the Cx32 level coincided with liver graft injury. Therefore, we established autologous orthotopic liver transplantation (AOLT) models using Cx32-knockout and wild-type mice and hypoxia/reoxygenation (H/R) and lipopolysaccharide (LPS) pretreatment models using alpha mouse liver 12 (AML12) cells, to explore Cx32 mechanisms in liver graft injury. Following in vivo and in vitro Cx32 knockout, oxidative stress and inflammatory response were inhibited through the regulation of PKC-α/NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thereby reducing Bak/Bax-related apoptosis and ameliorating liver graft injury. When the Cx32-based gap junction (GJ) was blocked with 2-aminoethoxydiphenyl borate (2-APB), ROS transfer was attenuated between neighboring cells, exacerbated oxidative stress and inflammatory response were prevented, and aggravation of liver graft injury was mitigated. These results highlight the dual regulation mechanism of Cx32 in liver graft injury. Through interaction with PKC-α, Cx32 regulated the NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thus directly triggering oxidative stress and inflammatory response. Simultaneously, mass-produced ROS were transferred to neighboring cells through Cx32 channels, for which oxidative stress and the inflammatory response were aggravated indirectly. Finally, Bak/Bax-related apoptosis was activated, thereby worsening liver graft injury. Our findings propose Cx32 as a dual mechanistic factor for oxidative stress and inflammatory signaling pathways in regulating cell apoptosis on liver graft injury, which suggests a promising therapeutic targets for liver graft injury.

Nanotherapeutics for solid organ transplantation: new kid onthe block.

Nanotherapeutics for solid organ transplantation: new kid onthe block.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying a bispecific T cell engager against hepatocellular carcinoma

ABSTRACT We previously established a hepatocellular carcinoma (HCC) targeting system of conditionally replicative adenovirus (CRAd) delivered by human umbilical cord-derived mesenchymal stem cells (HUMSCs). However, this system needed to be developed further to enhance the antitumor effect and overcome the limitations caused by the alpha-fetoprotein (AFP) heterogeneity of HCC. In this study, a bispecific T cell engager (BiTE) targeting programmed death ligand 1 controlled by the human telomerase reverse transcriptase promoter was armed on the CRAd of the old system. It was demonstrated on orthotopic transplantation model mice that the new system had a better anti-tumor effect with no more damage to extrahepatic organs and less liver injury, and the infiltration and activation of T cells were significantly enhanced in the tumor tissues of the model mice treated with the new system. Importantly, we confirmed that the new system eliminated the AFP-negative cells on AFP heterogeneous tumor models efficiently. Conclusion: Compared with the old system, the new system provided a more effective and safer strategy against HCC.