Population In Mice Research Articles

RF21 | PSAT90 Lineage Tracing Sonic Hedgehog-Expressing Cells in Adrenal Glands in Post-Weaning Mice

Abstract The Sonic Hedgehog (Shh) gene expressed in the subcapsular cortical region of the adrenal gland has been found to play a role in adrenal gland development. The Shh(+) cell population at the fetal stages contributes to different cortical layers in the adrenal gland. However, the (1) capability of these cells after weaning and (2) how soon they can renew the adrenal cortex in the postnatal stages is not fully understood. Here, we conducted a lineage tracing experiment to track Shh(+) cells and cells descended from them in post-weaning mice to ultimately better understand the processes of adrenal cortex renewal and remodeling over time in young adult mice. This experiment used the NuTRAP; Shh-Cre-ERT2 mice as the Shh-reporter mouse model. This tamoxifen-inducible mouse model allows us to specifically target and label Shh-expressing cells and all descendant cells with green fluorescence. Tamoxifen was given at postnatal days (P) 22, P24, and P26 to enable the Cre recombinase activity driven by the Shh promoter. Adrenal glands were then analyzed after two and four months. This lineage tracing experiment found that Shh(+) cells and their descendant cells reached the margin of the Cyp2f2(+) cortical zone in 2 months and the cortical-medullary boundary in 4 months. This finding indicates that the Shh(+) cell population in post-weaning mice can proliferate, differentiate, and eventually renew the entire adrenal cortex over a four-month period of time. Understanding the adrenal cortex's renewal rate helps us design our follow-up study to test the capability of Shh(+) cells in adult mice at different ages and how their potency changes overtime at the 'omic' level. Because this NuTRAP;Shh-Cre-ERT2 mouse model also allows us to isolate cell-type-specific DNA/RNA, we can further decipher the underlying gene/pathways which control this progenitor cell population of the adrenal gland cortex. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 1:12 p.m. - 1:17 p.m.

Single-cell RNA-sequencing uncovers compound kushen injection synergistically improves the efficacy of chemotherapy by modulating the tumor environment of breast cancer.

Due to lack of enough specific targets and the immunosuppressive tumor microenvironment (TME) of triple-negative breast cancer (TNBC), TNBC patients often cannot benefit from a single treatment option. This study aims to explore the regulatory effects of Compound kushen injection (CKI) plus chemotherapy on the TME of TNBC from a single cell level. A mouse TNBC model in BALB/c mice was established to evaluate the antitumor efficacy and toxicity of CKI combined with chemotherapy. Flow cytometry was used to observe the influence of CKI on the lymphocyte populations in the tumor bearing mice. Both bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) were applied to portray the modulation of CKI combined with chemotherapy on the TME of TNBC mice. CKI significantly enhanced the anticancer activity of chemotherapy in vivo with no obvious side effects. Flow cytometry results revealed a significantly higher activation of CD8+ T lymphocytes in the spleens and tumors of the mice with combination therapy. Bulk RNA-seq indicated that CKI could promote the cytotoxic immune cell infiltrating into tumor tissues. Meanwhile, scRNA-seq further revealed that CKI combined with chemotherapy could enhance the percentage of tumor-infiltrating CD8+ T cells, inhibit tumor-promoting signaling pathways, and promote T cell activation and positive regulation of immune response. In addition, CKI showed obvious anticancer activity against MDA-MB-231 breast tumor cells in vitro. The combination of CKI and chemotherapy might provide a higher efficiency and lower toxicity strategy than a single chemotherapy drug for TNBC. CKI potentiates the anti-TNBC effects of chemotherapy by activating anti-tumor immune response in mice.

Single-cell transcriptomics of human gut T cells identifies cytotoxic CD4+CD8A+ T cells related to mouse CD4 cytotoxic T cells.

Cytotoxic CD4+ T cells (CD4-CTLs) show the presence of cytolytic granules, which include the enzymes granzyme and perforin. The cells have a pathogenic and protective role in various diseases, including cancer, viral infection, and autoimmune disease. In mice, cytotoxic CD4+ T cells express CD8αα+ and reside in the intestine (mouse CD4+CTLs; mCD4-CTLs). The population of cytotoxic CD4+ T cells in the human intestine is currently unknown. Moreover, it is unclear how cytotoxic CD4 T cells change in patients with inflammatory bowel disease (IBD). Here, we aimed to identify cytotoxic CD4+ T cells in the human intestine and analyze the characteristics of the population in patients with IBD using single-cell RNA-seq (scRNA-seq). In CD4+ T cells, granzyme and perforin expression was high in humanMAIT (hMAIT) cells and hCD4+CD8A+ T cell cluster. Both CD4 and CD8A were expressed in hTreg, hMAIT, and hCD4+CD8A+ T cell clusters. Next we performed fast gene set enrichment analysis to identify cell populations that showed homology to mCD4CTLs. The analysis identified the hCD4+CD8A+ T cell cluster (hCTL-like population; hCD4-CTL) similar to mouse CTLs. The percentage of CD4+CD8A+ T cells among the total CD4+ T cells in the inflamed intestine of the patients with Crohn's disease was significantly reduced compared with that in the noninflamed intestine of the patients. In summary, we identified cytotoxic CD4+CD8+ T cells in the small intestine of humans. The integration of the mouse and human sc-RNA-seq data analysis highlight an approach to identify human cell populations related to mouse cell populations, which may help determine the functional properties of several human cell populations in mice.

Hematopoietic Stem Cell Identification Postirradiation.

Radiation exposure is particularly damaging to cells of the hematopoietic system, inducing pancytopenia and bone marrow failure. The study of these processes, as well as the development of treatments to prevent hematopoietic damage or enhance recovery after radiation exposure, often require analysis of bone marrow cells early after irradiation. While flow cytometry methods are well characterized for identification and analysis of bone marrow populations in the nonirradiated setting, multiple complications arise when dealing with irradiated tissues. Among these complications is a radiation-induced loss of c-Kit, a central marker for conventional gating of primitive hematopoietic populations in mice. These include hematopoietic stem cells (HSCs), which are central to blood reconstitution and life-long bone marrow function, and are important targets of analysis in these studies. This chapter outlines techniques for HSC identification and analysis from mouse bone marrow postirradiation.

Pro-opiomelanocortin neurons in the nucleus of the solitary tract mediate endorphinergic endogenous analgesia in mice.

The nucleus of the solitary tract (NTS) contains pro-opiomelanocortin (POMC) neurons that are 1 of the 2 major sources of β-endorphin in the brain. The functional role of these NTS POMC neurons in nociceptive and cardiorespiratory function is debated. We have shown that NTS POMC optogenetic activation produces bradycardia and transient apnoea in a working heart-brainstem preparation and chemogenetic activation with an engineered ion channel (PSAM) produced opioidergic analgesia in vivo. To better define the role of the NTS POMC neurons in behaving animals, we adopted in vivo optogenetics (ChrimsonR) and excitatory/inhibitory chemogenetic DREADD (hM3Dq/hM4Di) strategies in POMC-Cre mice. We show that optogenetic activation of NTS POMC neurons produces time-locked, graded, transient bradycardia and bradypnoea in anaesthetised mice that is naloxone sensitive (1 mg/kg, i.p.), suggesting a role of β-endorphin. Both optogenetic and chemogenetic activation of NTS POMC neurons produces sustained thermal analgesia in behaving mice that can be blocked by naloxone. It also produced analgesia in an inflammatory pain model (carrageenan) but not in a neuropathic pain model (tibial nerve transection). Inhibiting NTS POMC neurons does not produce any effect on basal nociception but inhibits stress-induced analgesia (unlike inhibition of arcuate POMC neurons). Activation of NTS POMC neuronal populations in conscious mice did not cause respiratory depression, anxiety, or locomotor deficit (in open field) or affective preference. These findings indicate that NTS POMC neurons play a key role in the generation of endorphinergic endogenous analgesia and can also regulate cardiorespiratory function.

Integrated single-cell analysis-based classification of vascular mononuclear phagocytes in mouse and human atherosclerosis.

Accumulation of mononuclear phagocytes [monocytes, macrophages, and dendritic cells (DCs)] in the vessel wall is a hallmark of atherosclerosis. Using integrated single-cell analysis of mouse and human atherosclerosis, we here aimed to refine the nomenclature of mononuclear phagocytes in atherosclerotic vessels and to compare their transcriptomic profiles in mouse and human disease. We integrated 12 single-cell RNA-sequencing (scRNA-seq) datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and data from 11 patients (n = 4 coronary vessels, n = 7 carotid endarterectomy specimens) from two studies. Integration of mouse data identified subpopulations with discrete transcriptomic signatures within previously described populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ Type 1 classical DCs (cDC1), Cd209a+ cDC2, and mature DCs (Ccr7, Fscn1) with a 'mreg-DC' signature were detected. In humans, we uncovered macrophage and DC populations with gene expression patterns similar to those observed in mice. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Comparison of mouse and human data and direct cross-species data integration suggested transcriptionally similar macrophage and DC populations in mice and humans. We refined the nomenclature of mononuclear phagocytes in mouse atherosclerotic vessels, and show conserved transcriptomic features of macrophages and DCs in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.

Thymosin β4 and prothymosin α promote cardiac regeneration post-ischaemic injury in mice.

The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene programme that promotes cell division within this population of cardiomyocytes is not fully understood. In this study, we aimed to determine the gene expression profile of proliferating adult cardiomyocytes in the mammalian heart after myocardial ischaemia, to identify factors to can promote cardiac regeneration. Here, we demonstrate increased 5-ethynyl-2'deoxyuridine incorporation in cardiomyocytes 3 days post-myocardial infarction in mice. By applying multi-colour lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the borderzone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing data from cardiomyocytes at 3 days post ischaemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischaemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. This study reveals the transcriptional profile of proliferating cardiomyocytes in the ischaemic heart and shows that overexpression of the two identified factors, TMSB4 and PTMA, can promote cardiac regeneration. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is a key for regeneration to occur.

The Anti-Seizure Effect of Liraglutide on Ptz-Induced Convulsions Through its Anti-Oxidant and Anti-Inflammatory Properties.

Epilepsy is a prevalent and frequently devastating neurological disorder defined by recurring spontaneous seizures caused by aberrant electrical activity in the brain. Over ten million people worldwide suffer from drug-resistant epilepsy. This severe condition requires novel treatment approaches. Both oxidative and nitrosative stress are thought to have a role in the etiology of epilepsy. Liraglutide is a glucagon-like peptide-1 (GLP-1) analogue that is used to treat type-2 diabetes mellitus. According to recent studies, Liraglutide also shows neuroprotective properties, improving memory retention and total hippocampus pyramidal neuronal population in mice. The purpose of this investigation was to determine the anti-seizure and anti-oxidative effects of liraglutide in a pentylenetetrazole (PTZ)-induced rat model of epilepsy. 48 rats were randomly assigned to two groups: those who had electroencephalography (EEG) recordings and those who underwent behavioral assessment. Rats received either intraperitoneal (IP) liraglutide at two different dosages (3-6mg/kg) or a placebo, followed by pentylenetetrazole (IP). To determine if liraglutide has anti-seizure characteristics, we examined seizure activity in rats using EEG, the Racine convulsion scale (RCS), the time of first myoclonic jerk (FMJ), and MDA, SOD, TNF-α, IL-1β and GAD-67 levels. The mean EEG spike wave percentage score was reduced from 75.8% (placebo) to 59.4% (lower-dose) and 41.5% (higher-dose). FMJ had increased from a mean of 70.6s (placebo) to 181.2s (lower-dose) and 205.2s (higher-dose). RCS was reduced from a mean of 5.5 (placebo) to 2.7 (lower-dose) and 2.4 (higher-dose). Liraglutide (3 and 6mg/kg i.p.) successfully decreased the spike percentages and RCS associated with PTZ induced epilepsy, as well as considerably decreased MDA, TNF-α, IL-1β and elevated SOD, GAD-67 levels in rat brain. Liraglutide significantly decreased seizure activity at both dosages when compared to control, most likely due to its anti-oxidant and anti-inflammatory properties. The potential clinical role of liraglutide as an anti-seizure medication should be further explored.

Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia.

SummaryIntestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5−ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures.

Sex-dependent differences on the impact of minocycline administration in the development of coronary artery disease in a murine model of accelerated atherosclerosis

Background and Aims : Cardiovascular risk considerably increases in women after menopause, even surpassing that of men, suggesting an effect of age and sex. We seek to compare sex-dependent differences of minocycline administration on survival, inflammation, oxidative stress and monocyte population in young male and female mice, using a diet-induced model of atherosclerosis.

Abstract 1593: Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer

Abstract Patients affected by colorectal cancer (CRC) with microsatellite instability (MSI), which is caused by DNA mismatch repair deficiency (MMRd), are eligible for therapies based on immune checkpoint inhibitors (CPI), while microsatellite stable (MSS) tumors are not. However, a subset of MSS CRCs contains variable fractions of MMRd cells. How the presence of MMRd cells in tumors classified as MSS impacts cancer immune surveillance is largely unknown. It is also unclear whether the pharmacological modulation of MMRd cells percentage in MMR heterogeneous tumor can occur and if this might result in the improvement of tumor immune control.To shed light on these aspects we studied isogenic mismatch repair proficient and deficient mouse tumor cells, generated by genetic inactivation of MLH1, a key component of the MMR machinery. We mixed MLH1+/+and MLH1-/-cells at different ratios, injecting the resulting heterogeneous populations in mice. In the presence of a competent immune system, the tumorigenic potential and immune surveillance of MSS/MSI (MLH1+/+/MLH1-/-) heterogeneous tumors was dependent upon the MMRd fraction. Tumor rejection was observed when at least 50% of the cells were MMRd, but tumor growth delay was also evident when as low as 20% of MMRd cells were present in the mixed population and this was paralleled by immune infiltration of the tumor. Molecular profiles of samples from MSS/MSI heterogeneous tumors that evaded immune control, showed enrichment of the MSS fraction. Treatment of MSS/MSI mixed populations with the antimetabolite 6-Thioguanine (6TG) greatly enriched the MMRd fraction and improved immune response. Overall, these results suggest that genetic and/or pharmacological modulation of the DNA mismatch repair machinery can foster immune surveillance of MMR heterogeneous tumors and modulate the cancer immune environment. Citation Format: Vito Amodio, Giovanni Germano, Benedetta Mussolin, Simona Lamba, Rosaria Chilà, Giuseppe Rospo, Caterina Marchiò, Silvia Marsoni, Gianluca Mauri, Federica Di Nicolantonio, Alberto Bardelli. Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1593.

IMMU-01. Combining CD28 and 4-1BB costimulationin trans enhances the anti-glioma efficacy and persistence of B7-H3 CAR T cells in immune-competent brain tumor models

We and others have demonstrated that B7-H3 CAR T-cells have potent antitumor responses in xenograft models for brain tumors; however, these models do not recapitulate the immunosuppressive tumor microenvironment (TME) in patients with high-grade glioma. To evaluate the safety and efficacy of antigen-specific CAR T-cells, we adapted the immune-competent GL261 glioma model which recapitulates human disease and host immune barriers. We generated a library of B7-H3 CARs with different transmembrane (CD8, CD28), costimulatory (CD28, 4-1BB), and activation (ζ, mutζ) domains. We then compared their cytolytic activity, expansion, and anti-tumor activity. Results show that B7-H3 CARs with CD28 transmembrane and costimulatory domains have superior efficacy compared to CARs with CD8 and 4-1BB domains. Additionally, CARs with mutated ζ activation domain have better overall persistence. However, providing costimulation signals through CD28 or 4-1BB alone does not induce superior anti-glioma efficacy of B7-H3 CAR T-cells in vivo. Thus, we next investigated whether incorporating 4-1BB signaling into CD28-based CARs using in trans design enhances the therapeutic efficacy of B7-H3 CAR T-cells. We found that in repeat stimulation assays, surface expression of 4-1BBL enhanced expansion of B7H3 CAR T-cells at least 300-folds more than T-cells with CD28 or 4-1BB costimulatory domains alone. Additionally, 4-1BBL expression significantly enhanced the sequential killing capacity compared to CD28- or 41BB-based B7-H3 CAR T-cells. High dimensional flow cytometry analysis of GL261 tumors post CAR T-cell injection revealed unique immune clusters including dendritic cells and lymphoid predominant populations in mice treated with 4-1BBL expressing CARs. Thus, expression of 4-1BBL on CD28-based CARs reshaped the TME and enhanced persistence and anti-glioma efficacy of B7-H3 CAR T-cells. Studies examining transcriptional and epigenetic programs, and TME/CAR T-cell interactions are in progress. Results will define pathways that dictate CAR T-cell performance and will identify unique mechanisms for further improvements utilizing other members of TNF-superfamily.

SOLVx therapeutics vaccine - Activate T-cell immunity using broad surveillance epitope strategy against mutant strains SARS-COV2.

Highly mutable Coronavirus-19 continuously reconstructs its genome and renders prophylactic vaccines ineffective. The objective of the present study was to demonstrate the anti-viral efficacy and safety of the SOLVx therapeutics vaccine. The peptides were designed with Neo7Logix R&D and synthesized with Genescript GLP laboratory with 95 % purity. BALB/C mice were used to develop the HCoV-229E mutant coronavirus model and viral mRNA confirmation in the lung tissue was assessed with qPCR. Mice were euthanized and effects of treatment on various parameters (Viral mRNA in lungs, cytokine levels, PBMC differentiation, hematological and biochemical) were assessed with respective biological samples. Immuno-typing analysis of PBMCs by flowcytometry showed marked increase in T cell subsets, % of B cells and NK cell population in mice treated with SOLVx (Series 1) in a dose dependent manner. Serum immunoglobulin G, and M levels were increased significantly (P < 0.001). In the peptide treatment groups, there was a dose dependent statistically significant decrease in IL-6, IL-10 and TNF-α levels (P < 0.001). IFN-γ was elevated in treatment group significantly (P < 0.001). In conclusion, the qPCR results suggested that the SOLVx vaccine (Series 1) reduced the SARS-COV2 virus infectivity in a dose dependent manner. The humoral, cellular and functional activity of the SOLVx showed that it worked through multi-mechanistic targeting the virus evolution, offering immune response, defense and eradication of the SARS-COV2 virus.

Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

Anti-CD19 chimeric antigen receptor (CAR)-T therapy for B cell malignancies has shown clinical success, but a major limitation is the logistical complexity and high cost of manufacturing autologous cell products. If engineered for improved safety, direct infusion of viral gene transfer vectors to initiate in vivo CAR-T transduction, expansion, and anti-tumor activity could provide an alternative, universal approach. To explore this approach we administered approximately 20 million replication-incompetent vesicular stomatitis virus G protein (VSV-G) lentiviral particles carrying an anti-CD19CAR-2A-GFP transgene comprising either an FMC63 (human) or 1D3 (murine) anti-CD19 binding domain, or a GFP-only control transgene, to wild-type C57BL/6 mice by tail vein infusion. The dynamics of immune cell subsets isolated from peripheral blood were monitored at weekly intervals. We saw emergence of a persistent CAR-transduced CD3+ T cell population beginning week 3–4 that reaching a maximum of 13.5% ± 0.58% (mean ± SD) and 7.8% ± 0.76% of the peripheral blood CD3+ T cell population in mice infused with ID3-CAR or FMC63-CAR lentivector, respectively, followed by a rapid decline in each case of the B cell content of peripheral blood. Complete B cell aplasia was apparent by week 5 and was sustained until the end of the protocol (week 8). No significant CAR-positive populations were observed within other immune cell subsets or other tissues. These results indicate that direct intravenous infusion of conventional VSV-G-pseudotyped lentiviral particles carrying a CD19 CAR transgene can transduce T cells that then fully ablate endogenous B cells in wild-type mice.

Spi1 -14 Kb upstream regulatory element (URE) is not required for maintenance of PU.1 expression in macrophages

Background: Previous work suggested an upstream regulatory element (URE) of Spi1 was required to maintain constant expression of the PU.1 transcription factor in bone marrow and foetal liver cells. PU.1, encoded by Spi1, is essential for development and maintenance of myeloid and B-lymphocyte populations in mice. Deletion of this (-14 Kb) URE potentially reduces expression of PU.1 and therefore provides a way to investigate its role in myeloid populations in development and disease. This study aimed to examine the impact of removal of the -14 Kb Spi1 URE in Cx3cr1+ cells on the myeloid lineage formation and maintenance. Methods: B6;129-Spi1tm1.2Dgt/J mice, whose -14 Kb Spi1 URE mice is flanked by LoxP sites (‘floxed’), were bred to a strain with constitutively active Cre expressed under the Cx3cr1 promoter (B6J.B6N(Cg)-Cx3cr1tm1.1(cre)Jung/J) to delete the Spi1 URE in myeloid cells. The floxed mice were also bred to mice with a tamoxifen-inducible Cre expressed under the Cx3cr1 promoter (B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J) to be used as URE intact controls and to permit temporally-controlled deletion of the URE if required. PU.1 protein expression was measured in the peritoneal macrophages and microglia by flow cytometry. Additionally, a Cre-encoding lentiviral vector was used to assess the impact on PU.1 expression in bone-marrow derived macrophages from these mice in vitro. Results: Expression of the PU.1 transcription factor was not significantly altered in the peritoneal macrophages or microglia in mice lacking the -14 Kb Spi1 URE. Moreover, initial experiments utilising Cre encoding lentivirus did not reduce PU.1 protein in bone-marrow derived macrophages differentiated from the -14 Kb Spi1 URE floxed mice. Conclusions: These observations suggest that the -14 Kb URE does not play a major role in PU.1 protein expression in either mature peritoneal macrophages or microglia.

Attenuated Toxoplasma gondii enhances the antitumor efficacy of anti-PD1 antibody by altering the tumor microenvironment in a pancreatic cancer mouse model.

To investigate whether attenuated Toxoplasma is efficacious against solid tumors of pancreatic cancer and whether attenuated Toxoplasma improves the antitumor activity of αPD-1 antibody on pancreatic cancer. The therapeutic effects of attenuated Toxoplasma NRTUA strain monotherapy and combination therapy of NRTUA with anti-PD-1 antibody on PDAC tumor volume and tumor weight of Pan02 tumor-bearing mice were investigated. We characterized the effects of combination therapy of NRTUA with anti-PD-1 antibody on tumor-infiltrating lymphocytes and tumor-specific IFN-γ by using immunohistochemistry, flow cytometry and ELISA. The antitumor mechanisms of combination therapy of NRTUA with anti-PD-1 antibody were investigated via depletion of CD8+ T cells and IL-12. NRTUA strain treatment inhibited tumor growth in a subcutaneous mouse model of PDAC through activating dendritic cells and increasing CD8+ T cell infiltration in the tumor microenvironment. More importantly, combination therapy of NRTUA with anti-PD-1 antibody elicited a significant antitumor immune response and synergistically controlled tumor growth in Pan02 tumor-bearing mice. Specifically, the combination treatment led to elevation of CD8+ T cell infiltration mediated by dendritic cell-secreted IL-12 and to tumor-specific IFN-γ production in the PDAC tumor microenvironment. Also, the combination treatment markedly reduced the immunosuppressive myeloid-derived suppressor cell population in PDAC mice. These findings could provide a novel immunotherapy approach to treating solid tumors of PDAC and overcoming resistance to anti-PD-1 agents in PDAC tumors.

A 24‐Hour Fast Increases Liver Cytotoxic T Cell Populations in Genetically Obese Mice: A Preliminary Study

BACKGROUNDRecent data suggests that regulated fasting protocols can improve metabolic parameters associated with obesity. However, questions remain regarding the appropriate fasting duration needed to attenuate obesity‐associated complications such as glucose intolerance, arterial stiffness, and immune cell dysfunction. Therefore, the primary purpose of this study was to examine if an acute 24‐hour fast improves detrimental consequences of obesity and alters tissue‐specific immune cell counts in obese mice.METHODSTen 12‐week‐old male and female genetically obese mice (Ob; n=20) and wild‐type littermates (WT; n=20) were fed a standard diet and randomized to either ad libitum feeding or a 24‐hour fast (WT‐CON, WT‐Fast, Ob‐CON, Ob‐Fast). Mouse weight was measured before and after feeding protocols. Following feeding protocols, arterial stiffness was assessed via arterial pulse wave velocity (aPWV); liver and mesenteric adipose tissue (MAT) immune cell populations were assessed via spectral flow cytometry and normalized by tissue weight. For statistical analyses, sex‐adjusted regression models were fit to account for the confounding effects of sex.RESULTSThere was not a significant interaction between genotype and intervention on changes in body weight, plasma glucose concentrations, or aPWV (p=0.16; p=0.33; p=0.37, respectively). After adjusting for genotype, acute fasting did not significantly alter changes in plasma glucose concentrations or aPWV (p=0.95; p=0.22). However, after adjusting for genotype, acute fasting promoted significant weight loss (CON 0.4g ± 0.56; Fast ‐2.6g ± 0.72; p&lt;0.01). Compared to Ob‐CON, Ob‐Fast mice had significant increases in liver total immune and cytotoxic T cells (p&lt;0.01) yet no significant changes in either liver regulatory T or helper T cells nor in MAT total immune, cytotoxic T, helper T, and regulatory T cell populations. WT‐Fast mice had significant reductions in liver total immune, cytotoxic T, helper T, and regulatory T cell populations while these same cell populations were increased in MAT when compared to WT‐CON mice.CONCLUSIONSAn acute 24 hr. fast in mice was insufficient to significantly modulate glucose intolerance and aPWV in Ob mice compared to WT. However, fasting differentially affected liver and MAT T cell populations in mice. These results suggest acute fasting could be a robust mediator of T cell populations with a particular emphasis on expanding liver cytotoxic T cell communities in obese mice.

The ErbB3 Receptor Restricts Bmi1 to Regulate Paneth Cells

ErbB3 is a RTK family member known to regulate proliferation and growth in the intestinal epithelium. Previous work by our lab determined that targeted Erbb3 deletion from the intestinal epithelium resulted in precocious Paneth cell development and an expanded population in mice. This was associated with increased expression of intestinal stem cell (ISC) markers. Conversely, ileal enteroids treated with the ErbB3 ligand neuregulin (NRG)‐1β had significantly reduced expression of ISC markers, including Lgr5 and Bmi1. These data suggest that ErbB3 restricts secretory cell differentiation and stemness in the intestinal crypt. However, the underlying mechanisms are largely unknown. BMI1 is a proto‐oncogene and member of the PRC complex. Bmi1+ intestinal epithelial cells represent a slow‐cycling ISC population that can regenerate the epithelium following injury and loss of Lgr5+ ISCs. Recent evidence determined that Bmi1+ ISCs are enriched in enteroendocrine markers and can give rise to goblet cells, suggesting BMI1 may be critical for differentiation of secretory lineages. We hypothesized that ErbB3 restricts secretory cell differentiation through regulation of BMI1.AimsTo define ErbB3‐dependent regulation of Bmi1 and determine if ErbB3 restriction of Paneth cell development and differentiation is through suppression of Bmi1.MethodsMucosal scrapings were collected from Vil‐Cre;Erbb3flox/flox mice and Erbb3flox/flox littermate controls for protein and gene expression analysis. Ileal enteroids and HT‐29 colorectal adenocarcinoma cells were treated with vehicle DMSO, the PI3K inhibitor LY294002 (10 μM), the MEK inhibitor U0126 (5 μM), the BMI1 inhibitor PTC‐209 (1 μM), and/or recombinant NRG‐1β (10 ng/mL). Enteroids and cell monolayers were harvested and prepared for gene expression analysis. RT‐qPCR for Bmi1 and Lyz1 was performed relative to Hprt as a loading control. Statistical analysis by one‐way ANOVA was conducted using Prism 9.ResultsConsistent with previous data, BMI1 protein and gene expression were significantly increased in small and large intestinal mucosal scrapings from Vil‐Cre;Erbb3flox/flox mice versus littermate controls. To determine if Bmi1 expression is sensitive to PI3K/Akt or MAPK signaling downstream of ErbB3, HT‐29 cells were treated with inhibitors followed by NRG‐1β after 1 hour, then harvested 24 hours later. PI3K and MAPK inhibition significantly increased Bmi1 expression. NRG‐1β treatment was able to overcome MAPK inhibition to reduce BMI1 expression. Similarly, in ileal enteroids, PI3K and MAPK blockade increased Bmi1 expression and NRG‐1β treatment reversed this effect. To determine whether BMI1 regulates expression of the Paneth cell marker LYZ1, HT‐29 cells were treated with the BMI1 inhibitor PTC‐209. BMI1 inhibition decreased LYZ1 expression.ConclusionsOur data demonstrate that ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling in both human and mouse intestinal cells. Furthermore, BMI1 activity promotes LYZ1 expression. Together, these results support our hypothesis that ErbB3 regulates secretory cell differentiation through BMI1 in the intestinal epithelium.

The Role of Ubiquitin E3 Ligase, FBOX21, in Regulating Healthy and Malignant Hematopoietic Stem and Progenitor Cells (HSPC)

Hematopoietic stem cells allow for the formation of all cell types in the blood and maintain these populations throughout a person’s life. Hematopoietic cell fate decisions such as self‐renewal and differentiation are highly regulated through multiple molecular pathways and dysregulation can lead to the initiation of malignant hematopoietic disorders like acute myeloid leukemia (AML), immune deficiencies, and anemia. One pathway that regulates hematopoietic cell fate decisions is the ubiquitin proteasome system (UPS). The UPS controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component, the protein that decides which substrate will be chosen for degradation, of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21was highly expressed in the hematopoietic stem and progenitor (HSPC) population, and was more highly expressed in this population when compared to other Fbox genes suggesting a possible role in maintaining the HSPC population. Likewise, Western blot confirmed high levels of FBXO21 in HSPCs and revealed low to no protein in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self‐renewal, and differentiation, we generated shRNAs against both Fbxo21 and FBXO21. We found that silencing Fbxo21/FBXO21 in healthy HSPCs and in human AML cell lines lead to a loss in colony formation, a decrease in proliferation, and an increase in differentiation towards the mature myeloid lineage. While overexpression of FBXO21 lead to an increase in colony formation and proliferation. This leads us to hypothesize that the ubiquitin E3 ligase, FBXO21, maintains the hematopoietic stem cell population while loss of Fbxo21/FBXO21 leads to a promotion in myeloid differentiation.We then generated the first conditional Fbxo21 knockout (KO) mouse model to delete Fbxo21 in hematopoietic cells. Our Fbxo21 KO mice exhibited no significant difference in primary sites of hematopoietic development, but display changes in hematopoietic cell populations in both young and aged mice. To further identify the substrates and pathway of FBXO21, we performed TMT Mass Spectrometry to analyze changes in protein levels upon knockdown of Fbxo21/FBXO21 in healthy HSPC and human AML cell lines. We found that both populations displayed an increase in proteins involved in the inflammatory response, the immune response primarily maintained by the myeloid cells through cytokine signaling. Which among both our Fbxo21 KO mice and human AML cell lines with FBXO21 knocked down, we see alterations in the cell’s response to certain cytokines, including IL‐6 and G‐CSF, upon Fbxo21/FBXO21 being knocked down. Therefore, deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.

MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER Ao Zhang, Hong Qiu, Timothy Chan, and Byron Lee Ao ZhangAo Zhang More articles by this author , Hong QiuHong Qiu More articles by this author , Timothy ChanTimothy Chan More articles by this author , and Byron LeeByron Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 6A (KDM6A) is a chromatin modifier that is frequently mutated in bladder cancer and causes epigenetic dysregulation. Recently immune check point inhibition has shown great clinical promise in bladder cancer. Because epigenetic signature has been shown to predict response to immunotherapy, here we assessed if loss of KDM6A leads to alterations of tumor immune microenvironment in a mouse bladder cancer model. METHODS: Kdm6a urothelium conditional knockout (cKO) mice were generated by crossing Kdm6aflox/flox mice with Upk3aCre mice that express Cre recombinase in the urothelium. Littermate control mice were of the same genetic background but intact Kdm6a gene. Mice at 8 weeks of age (three in each group) were fed with drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Bladders were harvested after 4 weeks and analyzed with H&E staining. Single cell RNA sequencing was performed on urothelium from control and Kdm6A cKO mice without BBN treatment. RESULTS: After treatment with BBN for 4 weeks, there was marked edema and inflammatory changes with increased infiltration of immune cells in H&E stained bladder sections in Kdm6a cKO mice compared to Kdm6a intact controls (Figure A). While urothelium from control mice maintained grossly unchanged tissue architecture, urothelium from cKO mice exhibited cytologic atypia with loss of organized cell layer. Comparison of single cell RNA sequencing profile revealed enrichment of immune cell population in cKO mice (Figure B, cluster 12), which was consistent with histological findings. CONCLUSIONS: Loss of KDM6A in the urothelium creates a distinct immune microenvironment in the BBN mouse bladder cancer model. Whether this immune cell-rich microenvironment affects response to immune check point inhibition warrants further investigation. Source of Funding: This work was supported in part by the 2021 Urology Care Foundation Residency Research Award Program and the Kahlert Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e80 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ao Zhang More articles by this author Hong Qiu More articles by this author Timothy Chan More articles by this author Byron Lee More articles by this author Expand All Advertisement PDF DownloadLoading ...