Severe Combined Immunodeficient Mice Research Articles

Ultrasound Imaging of Pancreatic Perfusion Dynamics Predicts Therapeutic Prevention of Diabetes in Preclinical Models of Type 1 Diabetes.

In type 1 diabetes (T1D), immune-cell infiltration into islets of Langerhans (insulitis) and β-cell decline occur years before diabetes presents. There is a lack of validated clinical approaches for detecting insulitis and β-cell decline, to diagnose eventual diabetes and monitor the efficacy of therapeutic interventions. We previously determined that contrast-enhanced ultrasound measurements of pancreas perfusion dynamics predict disease progression in T1D pre-clinical models. Here, we test whether these measurements predict therapeutic prevention of T1D. We performed destruction-reperfusion measurements with size-isolated microbubbles in non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) mice receiving an adoptive transfer of diabetogenic splenocytes. Mice received vehicle control or the following treatments: (i) anti-CD3 to block T-cell activation; (ii) anti-CD4 to deplete CD4+ T cells; (iii) verapamil to reduce β-cell apoptosis; or (iv) tauroursodeoxycholic acid (TUDCA) to reduce β-cell endoplasmic reticulum stress. We compared measurements of pancreas perfusion dynamics with subsequent progression to diabetes. Anti-CD3, anti-CD4, and verapamil delayed diabetes development. Blood flow dynamics was significantly altered in treated mice with delayed/absent diabetes development compared with untreated mice. Conversely, blood flow dynamics in treated mice with unchanged diabetes development was similar to that in untreated mice. Thus, measurement of pancreas perfusion dynamics predicts the successful prevention of diabetes. This strategy may provide a clinically deployable predictive marker for therapeutic prevention in asymptomatic T1D.

O-034 Xenotransplantation of ovarian cortex tissue from young girls with Turner Syndrome in a mice model: is normal follicular development possible?

Abstract Study question Does aneuploidy of granulosa and stromal cells affect folliculogenesis of small follicles in girls with mosaic Turner Syndrome (TS)? Summary answer Our results showed that despite high level aneuploidy in granulosa cells of small follicles and stromal cells folliculogenesis was not affected in mosaic TS patients. What is known already In 2018 the TurnerFertility study was initiated to explore if ovarian tissue cryopreservation (OTC) is a viable option for TS patients to preserve their fertility at an early age. In this study, karyotyping of ovarian cells in 7 mosaic TS patients showed that the majority of oocytes had a normal X chromosomal content, while granulosa and stromal cells were aneuploid. Until now, it remains unclear if follicular development is affected, and consequently the success rate of autotransplantation after OTC in girls with TS. Study design, size, duration A collaboration project was initiated between Radboudumc, The Netherlands and UCLouvain, Belgium to assess folliculogenesis of small follicles in TS patients by using a xenograft mice model. Ovarian cortex tissue was obtained from 18 mosaic TS patients and 13 age-matched controls. Participants/materials, setting, methods After unilateral ovariectomy, one fragment of the ovarian tissue was used for research. One part of this fragment was used for fluorescence in situ hybridization (FISH) to determine the X chromosomal content of ovarian cells in non-grafted tissue. The other part was xenografted into severe combined immunodeficient mice. After 5 months, grafts were retrieved and analysed for aneuploidy by FISH. The expression of 6 proteins essential for folliculogenesis was analysed by immunohistochemistry and immunofluorescence. Main results and the role of chance The mean follicle density in ovarian tissue of TS patients before grafting was significantly lower compared to controls. FISH analysis showed that 97% of the oocytes in non-grafted tissue had a normal X chromosomal content. Follicles of TS patients contained mainly or exclusively 45,X granulosa cells, but different levels of X chromosome mosaicism between TS patients and between follicles of the same patient were observed. In total, 12/18 grafts contained follicles after 5 months xenografting. Follicle density of both TS patients and controls decreased significantly after xenotransplantation. Despite the presence of high level aneuploidy in granulosa and stromal cells in the tissue before grafting, secondary and even antral follicles were observed after xenotransplantation. Remarkably, a shift from high to low percentage of 45,X granulosa cells was observed during folliculogenesis. Immunohistochemistry showed that proliferating cell nuclear antigen (PCNA) positive follicles from TS patients increased during grafting to almost 100%. Secretion of anti-Müllerian hormone by granulosa cells was impaired before grafting in peri/postpubertal TS girls, but recovered after grafting. Expression of c-kit receptor and bone morphogenetic protein 15 (BMP15) in peri/postpubertal TS patients remained abnormal after xenotransplantation, while secretion of growth differentiation factor 9 (GDF9) and kit ligand was similar to controls. Limitations, reasons for caution In this study, only ovarian tissue of mosaic TS patients was examined, because the chances of finding ovarian follicles in this subgroup is considerably higher than in other TS patients. Subtle effects of reduced expression of c-kit receptor and BMP15 on folliculogenesis might have gone unnoticed. Wider implications of the findings Small follicles of mosaic TS patients are able to grow to secondary and antral follicles, despite the presence of aneuploid granulosa and stromal cells before grafting. Therefore, OTC could be a realistic option for young mosaic TS patients to preserve their fertility provided that sufficient numbers of follicles are present. Trial registration number NCT03381300

A general toxicity and biodistribution study of human natural killer cells by single or repeated intravenous dose in severe combined immune deficient mice

Natural killer (NK) cells are a part of the innate immune system and represent the first line of defense against infections and tumors. NK cells can eliminate tumor cells without major histocompatibility restriction and are independent of the expression of tumor-associated antigens. Therefore, they are considered an emerging tool for cancer immunotherapy. However, the general toxicity and biodistribution of NK cells after transplantation remain to be understood. This study was conducted to evaluate the general toxicity and biodistribution of human NK cells after single or repeated intravenous dosing in severely combined immunodeficient (SCID) mice. There were no test item-related toxicological changes in single and repeated administration groups. The no observed adverse effect level of human NK cells was 2 × 107 cells/head for both male and female SCID mice. Results from the biodistribution study showed that human NK cells were mainly distributed in the lungs, and a small number of the cells were detected in the liver, heart, spleen, and kidney of SCID mice, in both the single and repeated dose groups. Additionally, human NK cells were completely eliminated from all organs of the mice in the single dose group on day 7, while the cells persisted in mice in the repeated dose group until day 64. In conclusion, transplantation of human NK cells in SCID mice had no toxic effects. The cells were mainly distributed in the lungs and completely disappeared from the body over time after single or repeated intravenous administration.

Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells

BackgroundHuman-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost.MethodsIn this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down’s syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2.ResultsWe found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers.ConclusionsOur xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.

Clinical Dose Preparation of [177Lu]Lu-DOTA-Pertuzumab Using Medium Specific Activity [177Lu]LuCl3 for Radioimmunotherapy of Breast and Epithelial Ovarian Cancers, with HER2 Receptor Overexpression.

Background: The overexpression of human epidermal growth factor receptor 2 (HER2) is commonly associated with metastatic breast cancer and epithelial ovarian cancer. The U.S. Food and Drug Administration (FDA) has approved Trastuzumab as an anti-HER2 agent for the metastatic breast and epithelial ovarian cancer. However, Trastuzumab has severe limitations in the treatment of metastatic breast cancer associated with ligand-dependent dimerization of HER2 receptor at the extracellular domain-II (ECD-II) region. The therapeutic approach in combination of pertuzumab and trastuzumab is found to be effective in preventing HER2 dimerization at the ECD-II region. The radioimmunotherapeutic approach, utilizing both these anti-HER2 agents (trastuzumab/pertuzumab), radiolabeled with [177Lu]Lu3+, has proved to be clinically efficacious with promising potential. Toward this, the formulation for clinical doses of [177Lu]Lu-DOTA-pertuzumab has been optimized using medium specific activity (0.81 GBq/μg) [177Lu]LuCl3. Materials and Methods: Preconcentrated pertuzumab was conjugated with p-NCS-benzyl-DOTA. Purified DOTA-benzyl-pertuzumab conjugate was radiolabeled with carrier-added [177Lu]LuCl3. Quality control parameters were evaluated for the [177Lu]Lu-DOTA-pertuzumab. In vivo biodistribution was carried out at different time points postadministration. Specific cell binding, immunoreactivity, and internalization were investigated by using SKOV3 and SKBR3 cells. Results: In this study, the authors reported a consistent and reproducible protocol for clinical dose formulations of [177Lu]Lu-DOTA-pertuzumab, with a radiochemical yield of 86.67% ± 1.03% and radiochemical purity (RCP) of 99.36% ± 0.36% (n = 10). Preclinical cell binding studies of [177Lu]Lu-DOTA-pertuzumab revealed specific binding with SKOV3 and SKBR3 cells up to 24.4% ± 1.4% and 23.2% ± 0.8%, respectively. The uptakes in SKOV3- and SKBR3-xenografted tumor in severe combined immunodeficiency mice were observed to be 25.9% ± 0.8% and 25.2% ± 1.2% ID/g at 48 and 120 h postinjection, respectively. Conclusions: A protocol was optimized for the preparation of ready-to-use clinical dose of [177Lu]Lu-DOTA-pertuzumab, in hospital radiopharmacy settings. The retention of RCP of the radiopharmaceutical, on storage in saline and serum, at -20°C, up to 120 h postradiolabeling, confirmed its in vitro stability.

Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV.

Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

Human acute myeloid leukemia stem cells: evolution of concept.

The history of human acute myeloid leukemia stem cells (AMLSCs) began in a seminal study performed by Lapidot and Dick, proving that only CD34+CD38- human primary acute myeloid leukemia (AML) cells can repopulate in severe combined immunodeficient mice. The concept of leukemic stem cells (LSCs) has impeded a huge change in the treatment strategy against AML from killing proliferating leukemic cells to eradicating quiescent/dormant LSCs. As next-generation sequencing technologies have developed, multiple and recurrent genetic mutations have been discovered in large cohorts of patients with AML, and the updated understanding of leukemogenesis has improved the old concept of LSC to a revised version of a serial developmental model of LSC; that is, pre-LSCs are generated as seeds by the first hit on epigenetic regulators, and then, leukemia-initiating LSCs emerge from seeds by the second hits on genes involved in transcription and signaling. Dreams for universal and targetable AMLSC biomarker sparing healthy hematopoietic stem cells have weakened after the confrontation of significant heterogeneity of AMLSCs from genomic and immunophenotypic viewpoints. However, there is still hope for effective targets for AMLSCs since there is evidence that grouped gene signatures, such as 17-gene LSC score, and common epigenetic signatures, such as HOXA clusters, independent of various gene mutations, exist. Recently, the LSC niche in the bone marrow has been actively investigated and has expanded our knowledge of the physiology and vulnerability of AMLSCs. Presently, an applicable treatment that always works in AMLSCs is lacking. However, we will find a way, we always have.

Is Cryptosporidium a hijacker able to drive cancer cell proliferation?

The pathophysiological mechanisms of Cryptosporidium infection are multifactorial and not completely understood. Some advances achieved recently revealed that the infection by Cryptosporidium parvum induces cytoskeleton remodeling and actin reorganization through the implication of several intracellular signals involving, for example, PI3K, Src, Cdc42 and GTPases. It has also been reported that the infection by C. parvum leads to the activation of NF-κβ, known to induce anti-apoptotic mechanisms and to transmit oncogenic signals to epithelial cells. Despite the growing evidence about the hijacking of cellular pathways, potentially being involved in cancer onset, this information has rarely been linked to the tumorigenic potential of the parasite. However, several evidences support an association between Cryptosporidium infection and the development of digestive neoplasia. To explore the dynamics of Cryptosporidium infection, an animal model of cryptosporidiosis using corticoid dexamethasone-treated adult SCID (severe combined immunodeficiency) mice, orally infected with C. parvum or Cryptosporidium muris oocysts was implemented. C. parvum-infected animals developed digestive adenocarcinoma. When mechanisms involved in this neoplastic process were explored, the pivotal role of the Wnt pathway together with the alteration of the cytoskeleton was confirmed. Recently, a microarray assay allowed the detection of cancer-promoting genes and pathways highly up regulated in the group of C. parvum infected animals when compared to non-infected controls. Moreover, different human cases/control studies reported significant higher prevalence of Cryptosporidium infection among patients with recently diagnosed colon cancer before any treatment when compared to the control group (patients without colon neoplasia but with persistent digestive symptoms). These results suggest that Cryptosporidium is a potential oncogenic agent involved in cancer development beyond the usual suspects. If Cryptosporidium is able to hijack signal transduction, then is very likely that this contributes to transformation of its host cell. More research in the field is required in order to identify mechanisms and molecular factors involved in this process and to develop effective treatment interventions.

Comparison of the Concentration of Encephalitozoon cuniculi Genotypes I and III in Inflammatory Foci Under Experimental Conditions.

BackgroundMicrosporidia of the genus Encephalitozoon are usually associated with severe infections in immunodeficient hosts while, in immunocompetent ones, microsporidiosis produces minimal clinically apparent disease. Despite their microscopic size, microsporidia are capable of causing systemic infection within a few days. However, the mechanisms by which microsporidia reach target tissues during acute infection remain unclear. Out of four genotypes of Encephalitozoon cuniculi, only three are available for experimental studies, with E. cuniculi genotype II being the best characterized.MethodsIn the present study, we tested the association between inflammation induction in immunocompetent and immunodeficient mice and the presence of spores of E. cuniculi genotypes I and III in selected organs using molecular methods and compared the results with previously published data on E. cuniculi genotype II.ResultsWe reported the positive connection between inflammation induction and the significant increase of E. cuniculi genotypes I and III occurrence in inflammatory foci in both immunocompetent BALB/c and immunodeficient severe combined immunodeficient (SCID) mice in the acute phase of infection. The induction of inflammation resulted in increased concentration of E. cuniculi of both genotypes in the site of inflammation, as previously reported for E. cuniculi genotype II. Moreover, our study extended the spectrum of differences among E. cuniculi genotypes by the variations in dispersal rate within host bodies after experimentally induced inflammation.ConclusionThe results imply possible involvement of immune cells serving as vehicles transporting E. cuniculi towards inflammation foci. The elucidation of possible connection with pro-inflammatory immune responses represents an important challenge with implications for human health and the development of therapeutic strategies.

CGAS/STING signaling in the regulation of rheumatoid synovial aggression.

BackgroundFibroblast-like synoviocytes (FLSs) play a critical role in promoting synovial aggression and joint destruction in rheumatoid arthritis (RA). Cyclic GMP‐AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling plays an important role in controlling a series of cellular biological processes. However, it is still unclear whether cGAS/STING signaling regulates rheumatoid synovial aggression.MethodsCell migration and invasion were detected using a Transwell chamber. Gene expression was measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and protein expression was detected by western blotting. Reactive oxygen species (ROS) levels were measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. F-actin staining and immunofluorescence assays were used to investigate lamellipodia formation and nuclear translocation, respectively. A severe combined immunodeficiency (SCID) mouse model was established to observe the migration and invasion of RA FLSs in vivo.ResultsOur results showed that cytosolic double-stranded DNA (dsDNA)-induced cGAS/STING activation promoted the in vitro migration and invasion of RA FLSs. Moreover, RA FLSs treated with cGAS or STING short hairpin RNA (shRNA) exhibited reduced invasion into cartilage in the SCID model. Mechanistically, we determined that cGAS/STING activation leads to increased mitochondrial ROS levels, and thereby increases phosphorylation of mammalian sterile 20-like kinase 1 (MST1), a core component of the Hippo pathway, subsequently promoting activation of forkhead box1 (FOXO1). MST1 and FOXO1 knockdown also diminished the migration and invasion of RA FLSs.ConclusionsOur findings suggest that cGAS/STING signaling has an important role in regulating rheumatoid synovial aggression and that targeting cGAS/STING may represent a novel potential therapy for RA.

Enhancing Antigen Retrieval to Unmask Signaling Phosphoproteins in Formalin-fixed Archival Tissues.

The introduction of targeted therapy has revolutionized cancer treatment. Nonetheless, for this approach to succeed, it is crucial to identify the targets, particularly when activated, in tumor tissues. Phosphorylation is a posttranslational modification that causes activation of numerous oncogenic protein kinases and transcription regulators. Hence, phosphoproteins is a class of biomarkers that has therapeutic and prognostic implications directly relevant to cancer patients' management. Despite the progress in histopathology methodology, analysis of the expression of phosphoproteins in tumor tissues still represents a challenge owing to preanalytical and analytical factors that include antigen retrieval strategies. In this study, we tested the hypothesis that optimizing antigen retrieval methods will improve phosphoproteins unmasking and enhance their immunohistochemical staining signal. We screened 4 antigen retrieval methods by using antibodies specific for 3 oncogenic phosphoproteins to stain human lymphoma tumors that were developed in severe combined immunodeficiency mice and subsequently fixed in formalin for 2 years. Then, we used antibodies specific for 15 survival phosphoproteins to compare the most effective method identified from our screening experiment to the antigen retrieval method that is most commonly utilized. Using the antigen retrieval buffer Tris-EDTA at pH 9.0 and heating for 45 minutes at 97°C unmasked and significantly enhanced the staining of 9 of the 15 phosphoproteins (P<0.0001). Our antigen retrieval approach is cost effective and feasible for clinical and research settings. We anticipate that combining this approach with the newly proposed methods to improve tissue fixation will further improve unmasking of phosphoproteins in human and animal tissues.

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Abstract Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies. Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturation in vitro. In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test. Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became mature in vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile. Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse's body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 μM) and achieves “artemisinin-like” kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

Abstract P4-04-01: β2-adrenergic receptor signaling regulates metabolic pathways critical for the function of myeloid-derived suppressor cells

Abstract Summary Nerve-driven chronic adrenergic stress can suppress anti-tumor immunity. However, details are still missing or poorly understood. Catecholamines (epinephrine, norepinephrine) released by sympathetic nerves can activate adrenergic receptors present on nearly every cell type including myeloid derived suppressor cells (MDSCs). MDSCs are increasingly recognized as critical players in tumor immunology because, in addition to their fundamental roles in suppressing effector NK cells, CD8+ T cells and CD4+ T cells, they also play a direct role in tumor growth, differentiation and metastasis. Recently, we have shown that β2 adrenergic receptor signaling activated by chronic stress increases the immunosuppressive function of MDSCs, but the precise mechanisms remain unresolved. Building upon our earlier work, we now have discovered that the beta-2 adrenergic receptor (β2-AR) stress pathway drives the immune suppressive activity of MDSCs through altering their metabolism. First, we found that the expression of surface β2-AR on MDSCs increases with tumor growth in 4T1 breast cancer model and patients with breast cancer. We also found that β2-AR signaling decreases glycolysis, while increasing oxidative phosphorylation and fatty acid oxidation (FAO). β2-AR signaling also increased mitochondrial intensity and mitochondrial potential of MDSCs both in vitro and in vivo. β2-AR signaling in MDSCs also increased the expression of Carnitine Palmitoyltransferase 1A (CPT1A) necessary for the FAO-mediated immunosuppressive function of MDSCs. Blocking CPT1A using etomoxir decreased tumor growth and immunosuppressive function of MDSCs in Wild Type (WT) mice but not β2-AR-/- mice or severe combined immunodeficient mice (SCID) mice. Moreover, we found that β2-AR signaling increases autophagy and activates the arachidonic acid cycle, both required for increasing the release of the immunosuppressive mediator, PGE2 in both mouse MDSCs and. PBMCs derived MDSCs. Together, our data reveal that stress-driven β2-AR signaling is an important physiological determinant that regulates key metabolic pathways in MDSCs enabling their immunosuppressive function. These data reveal a major, physiologically relevant role for nervous activity and adrenergic stress in MDSC-tumor biology. Keywords: β2 adrenergic receptor, stress, MDSC, Metabolism, Breast Cancer, Citation Format: Hemn Mohammadpour. β2-adrenergic receptor signaling regulates metabolic pathways critical for the function of myeloid-derived suppressor cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-01.

Xenograft cancer vaccines prepared from immunodeficient mice increase tumor antigen diversity and host T cell efficiency against colorectal cancers.

Autologous cancer vaccines (ACVs) are a desirable approach for personalized medicine, but the efficiency of ACVs remains unsatisfactory due to their low immunogenicity. This study developed a platform that can enhance the immunogenicity of ACVs by transplanting the tumors into immunodeficient mice. The CT26cell line was inoculated into severe combined immunodeficient mice (SCID) for vaccine preparation where escalates tumor development, subsequently diversifying the tumor antigenic topology. CT26/SCID cancer vaccines significantly inhibited tumor growth, increased the amount of tumor infiltrating lymphocytes, and triggered Th-1 predominant immune responses. Tumor antigenic profiles of CT26/SCID cells were further analyzed by liquid chromatography-tandem mass spectrometry. Compared to CT26 parental cells, a total of 428 differentially expressed proteins (DEPs) were detected. These DEPs revealed that CT26/SCID cells overexpressed several novel therapeutic targets, including KNG1, apoA-I and, β2-GPI, which can trigger cytotoxic T cells towards Th-1 predominant immune responses and directly suppress proliferation in tumors. CT26/SCID cancer vaccines can be easily manufactured, while traits of triggering stronger antigen-specific Th-1 immune activity against tumors, are retained. Results of this study provide an effective proof-of-concept of an ACV for personalized cancer immunotherapy.

Skin and heart allograft rejection solely by long-lived alloreactive TRM cells in skin of severe combined immunodeficient mice.

Whether induced tissue-resident memory T (TRM) cells in nonlymphoid organs alone can mediate allograft rejection is unknown. By grafting alloskin or heart into severe combined immunodeficient or Rag2KO mice in which a piece of induced CD4+ and/or CD8+ TRM cell–containing MHC-matched or syngeneic skin was transplanted in advance, we addressed this issue. The induced CD4+ TRM cells in the skin alone acutely rejected alloskin or heart grafts. RNA-seq analysis showed that induced CD4+ TRM cells in skin favorably differentiated into TH17-like polarization during the secondary immune response. Inhibition of the key TH17 signaling molecule RORγt attenuated TRM cell–mediated graft rejection. Thus, we offer a unique mouse model to specifically study TRM cell–mediated allograft rejection without the involvement of lymphocytes in lymphoid organs and tissues. Our study provides strong evidence supporting the hypothesis that long-lived alloreactive TRM cells resident in other organs/tissues substantially contribute to organ allograft rejection.

Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans

Aberrant protein glycosylation is a hallmark of cancer, but few drugs targeting cancer glycobiomarkers are currently available. Here, we showed that a lectibody consisting of the high-mannose glycan-binding lectin Avaren and human immunoglobulin G1 (IgG1) Fc (AvFc) selectively recognizes a range of cell lines derived from lung, breast, colon, and blood cancers at nanomolar concentrations. Binding of AvFc to the non-small cell lung cancer (NSCLC) cell lines A549 and H460 was characterized in detail. Co-immunoprecipitation proteomics analysis revealed that epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) are among the lectibody's common targets in these cells. AvFc blocked the activation of EGFR and IGF1R by their respective ligands in A549 cells and inhibited the migration of A549 and H460 cells upon stimulation with EGF and IGF1. Furthermore, AvFc induced potent Fc-mediated cytotoxic effects and significantly restricted A549 and H460 tumor growth in severe combined immunodeficiency (SCID) mice. Immunohistochemistry analysis of primary lung tissues from NSCLC patients demonstrated that AvFc preferentially binds to tumors over adjacent non-tumor tissues. Our findings provide evidence that increased abundance of high-mannose glycans in the glycocalyx of cancer cells can be a druggable target, and AvFc may provide a new tool to probe and target this tumor-associated glycobiomarker.

Focused Ultrasound Stimulation of Microbubbles in Combination With Radiotherapy for Acute Damage of Breast Cancer Xenograft Model.

Objective: Several studies have focused on the use of ultrasound-stimulated microbubbles (USMB) to induce vascular damage in order to enhance tumor response to radiation. Methods: In this study, power Doppler imaging was used along with immunohistochemistry to investigate the effects of combining radiation therapy (XRT) and USMB using an ultrasound-guided focused ultrasound (FUS) therapy system in a breast cancer xenograft model. Specifically, MDA-MB-231 breast cancer xenograft tumors were induced in severe combined immuno-deficient female mice. The mice were treated with FUS alone, ultrasound and microbubbles (FUS + MB) alone, 8 Gy XRT alone, or a combined treatment consisting of ultrasound, microbubbles, and XRT (FUS + MB + XRT). Power Doppler imaging was conducted before and 24 h after treatment, at which time mice were sacrificed and tumors assessed histologically. The immunohistochemical analysis included terminal deoxynucleotidyl transferase dUTP nick end labeling, hematoxylin and eosin, cluster of differentiation-31 (CD31), Ki-67, carbonic anhydrase (CA-9), and ceramide labeling. Results: Tumors receiving treatment of FUS + MB combined with XRT demonstrated significant increase in cell death (p = 0.0006) compared to control group. Furthermore, CD31 and Power Doppler analysis revealed reduced tumor vascularization with combined treatment indicating (P < .0001) and (P = .0001), respectively compared to the control group. Additionally, lesser number of proliferating cells with enhanced tumor hypoxia, and ceramide content were also reported in group receiving a treatment of FUS + MB + XRT. Conclusion: The study results demonstrate that the combination of USMB with XRT enhances treatment outcomes.

Breast Cancer Xenograft Murine Models.

Mice are used as model organisms to understand the pathological basis of a variety of human diseases, including breast cancer. Both immunocompetent and immunocompromised mouse models are used depending on the scope of the study. Immunocompetent models allow the study of the impact of the immune system in murine models of mammary cancer, while immunodeficient mice serve as ideal host organisms to understand the behavior of human breast cancers within a biological system. Xenografting of human breast cancer cells into immunocompromised mouse models continues to be the most used fundamental animal model in preclinical breast cancer research. These in vivo models allow critical understanding of tumor biology and assessment of novel treatments, a necessary prelude to testing new drugs in the clinic. In this chapter, we provide detailed methodology for the use of non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice in several breast cancer xenografting procedures, including established cell lines and patient-derived xenografts (PDXs).

Orelabrutinib, a Selective Bruton's Tyrosine Kinase (BTK) Inhibitor in the Treatment of Primary Immune Thrombocytopenia (ITP)

Background:Primary immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disorder characterized by decreased platelet count. increased risk of bleeding, and poor quality of life. Only about 70% patients response to first-line treatments, some patients are still refractory or relapsed after combined therapies, therefore it is necessary to explore new therapeutic targets. Bruton's tytosine kinase (BTK) is a non-receptor tyrosine kinase of Tec family, which is widely expressed in hematopoietic cells including B cells, monocytes/macrophages, and others. BTK participates in a variety of signaling pathways of innate and adaptive immunity, and plays an important role in cell survival and maturation. Platelet destruction mediated by anti-platelet glycoprotein antibodies is considered to be the main cause of ITP. B cells differentiate into plasma cells and produce autoantibodies due to the intolerance to autoantigens, which are important effectors in the pathogenesis of ITP. We speculated that inhibition of BTK may reduce platelet destruction by inhibiting B cell activation and autoantibody production. Orelabrutinib is a new generation of BTK inhibitor which has been used in hematological malignancies, this is the first study to explore the mechanisms of BTK inhibitor in the treatment of ITP.Methods:The concentrations of orelabrutinib were set at 1 nM, 10 nM, 100 nM and 1 μM in the in vitro study. Peripheral blood mononuclear cells (PBMCs) were isolated from active ITP patients and healthy controls and cultured for 72 hours, the apoptosis rate of PBMCs in each group was measured by Annexin V/PI double staining. CD19 + B cells of ITP patients were sorted by magnetic beads and stimulated with anti-human IgM to evaluate the activation of B-cell receptor (BCR) pathway and differentiation of plasma cells, respectively. Further, we transfused the splenocytes of immunized CD61-KO mice (C57BL/6) into the severe combined immunodeficient (SCID) mice to establish the active ITP murine models. Orelabrutinib was administered intragastrically at 10mg/kg, once a day. The control group was treated with 0.5% CMC at the same volume and frequency. Platelet count was measured weekly, the peripheral blood was collected and the B cell subsets in spleen were detected by flow cytometry at days 28 after splenocyte transfusion.Results:The proportion of early apoptotic cells (Annexin V +PI -) in PBMCs from both ITP patients and healthy controls was increased by orelabrutinib at 1μM,but there was no statistical difference. Orelabrutinib significantly inhibited the expression of CD69 in a dose-dependent manner at the concentrations of 10nM, 100nM and 1μM. Another early activation marker of BCR signaling pathway, CD86, was also found to be inhibited by orelabrutinib at 100nM and 1μM. The number of CD138 + plasma cells was decreased after treated with orelabrutinib at 10nM, 100nM and 1μM without dose-dependent manner. In the murine models, mice administered with orelabrutinib had significantly higher platelet count than the control mice at days 7, 14, 28 after splenocyte transfusion. The frequency of total B cells in peripheral blood leukocytes, the proportion of GL-7 + germinal center B cells and plasma cells in splenocytes were all determined to be lower in mice treated with orelabrutinib than the control group, though did not reach the statistical significance.Conculsion:Orelabrutinib could effectively suppress the activation and differentiation of B cells in vitro and in vivo, thus alleviate the thrombocytopenia in active ITP murine models. It could be the new treatment strategy for refractory/relapsed ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.