Combined Immunodeficient Mice Research Articles

Assessment of folliculogenesis in ovarian tissue from young patients with Turner syndrome using a murine xenograft model

To study the impact of aneuploid granulosa and stromal cells on folliculogenesis of small ovarian follicles from mosaic Turner syndrome patients using a murine xenograft model. Laboratory study SUBJECTS: Ovarian cortical tissue was obtained by laparoscopic surgery from 18 mosaic TS patients (aged 5-19 years) and 13 controls (aged 5-18 years). Part of each tissue fragment was used to karyotype ovarian cells in non-grafted tissue by fluorescence in situ hybridization. The remaining tissue was xenografted to severe combined immunodeficient mice for 5 months. Grafted tissue was analyzed for aneuploidy, and follicle density and morphology were determined. Expression of proliferating cell nuclear antigen and anti-Müllerian hormone were investigated by immunohistochemistry. The impact of aneuploid granulosa and stromal cells on folliculogenesis. Fluorescence in situ hybridization of ovarian tissue before grafting was performed to determine the level of aneuploidy in stromal cells, and oocytes and granulosa of small follicles. After xenografting the level of aneuploidy of the newly formed layers of granulosa cells was again determined in secondary and antral follicles. Follicle density in ovarian tissue from Turner syndrome patients was significantly lower than in controls before grafting. Fluorescence in situ hybridization analysis confirmed that 101/104 oocytes from non-grafted tissue of Turner syndrome patients showed normal X chromosome content, while granulosa and stromal cells were mainly 45,X. Fragments from 12 Turner syndrome patients contained follicles at all stages after xenografting, including secondary and antral follicles. Follicle density in Turner syndrome patients and controls decreased significantly after grafting. Moreover, a shift from high to low proportions of 45,X granulosa cells was observed during folliculogenesis. Expression of PCNA in follicles from TS patients increased significantly during grafting. Secretion of AMH was impaired before grafting in peri-/postpubertal TS girls, but recovered after grafting. Our study showed that small follicles from mosaic Turner syndrome patients undergo folliculogenesis, despite the presence of aneuploid granulosa and stromal cells. Ovarian tissue cryopreservation could therefore be a valid option to preserve fertility in young mosaic Turner syndrome patients if sufficient numbers of follicles are present, thus preferably before the age of 12.

Abstract 5190: Systematic immune-profiling of immune-deficient mouse models: A rational to select ideal host for tumor implantation

Abstract Preclinical tumor oncology research relies historically on the analysis of mouse or human tumor cell lines implanted onto severely immune-deficient mouse models carrying nude, SCID or Rag mutations on different genetical background (C57Bl/6N, BalbC, NMRI, CB17 or NOD). These lines harbor defects in several leukocyte lineages among which Tc, Bc and NKc might be affected. Humanized mice are emerging models that have been transplanted with human cells or tissues (and/or equipped with human transgenes). Currently, the most advanced strains are the nonobese diabetic, severe combined immunodeficiency (NOD-SCID) mouse with complete disruptions in the interleukin-2 (IL-2) common γ-chain (IL2R γ null) receptor (NSG) and BALB/c Rag2−/− IL2R γ null SirpaNOD mice (BRGS).1 Juvenile chimera NSG or BRGS are reconstituted with hematopoietic progenitor cell (HPCs) from human cord blood yielding robust engraftment of a human immune system (HIS).. Recent work showing the binding of human IgG4 to mouse FcgR receptor reducing clinical efficacy of therapeutic antibodies highlights the need to characterize extensively the residual immune system present in these models prior humanization in order to precisely evaluate immunotherapies. In other to fulfill this task, we genetically modified the mouse genome introducing mutations involved in Tc, Bc and NKc development. We then immuno-phenotyped over 20 lines of mouse mutants produced from the same animal house to minimize the impact of microbiota on innate immune cell populations. High content cytometry analysis of BM, spleen, thymus and peripheral blood was performed in a standardized manner. The resource obtained of these immunotypes was used to highlight similarities and differences among these lines across several genetical background. This work has shown instrumental in selecting the most appropriate model to use for humanization followed before tumor implantation. Citation Format: Hervé Luche, Lillia Hadjem, Marielle Mello, Priscilla Canavese, Fabien Angelis, Anais Joachim, Sylvie Bouilly, Frederic Guinut, Frederic Fiore, Bernard Malissen, Ana Zarubica, Erwan Corcuff. Systematic immune-profiling of immune-deficient mouse models: A rational to select ideal host for tumor implantation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5190.

Abstract 2238: RAS activity-related features of metastatic pancreatic cancer

Abstract Background: RAS activity might play crucial roles in the aggravation of KRAS-mutated pancreatic cancer (PC), including systemic inflammatory reactions, tumor-driven inflammation, chemo-refractoriness and anorexia. We aimed to identify RAS activity-related features in PC patients with liver metastasis (LM), to create a murine xenograft model of human PC cells representative of RAS-driven aggravation. Methods: RAS activities in 77 patients who underwent needle biopsy for LM of treatment-naïve PC were measured using an RAS signature score from a comprehensive cDNA microarray dataset of LM samples. Changes in the sum of the longest diameter of the target lesion in the first month of chemotherapy (ΔSLD), laboratory data and anorexia score at baseline were evaluated. Gene modules to be correlated with RAS activity and aggravation were evaluated using weighted co-expression network analysis (WGCNA). Human PC cells were classified as high and low activity according to in silico analysis of the DepMap CCLE expression dataset for profiling RAS activity and the best hub gene expression in the gene module. Subcutaneous xenograft models of human PC cells with high and low activity in severe combined immunodeficiency mice were compared to explore the RAS-related phenotype. Results: KRAS-mutated LM was detected in 70 of the 77 PC patients (90.9%). RAS signatures were up-regulated in patients with KRAS-mutated LM (P < 0.01), high serum level of C-reactive protein (P = 0.01) as a systemic inflammatory marker, high expression of IL-6 mRNA by the tumor (P = 0.12) as an index of tumor-driven inflammation, and high level of phospho-Erk (P = 0.03). In 58 PC patients receiving first-line chemotherapy for KRAS-mutated LM, ΔSLD in patients with a high RAS signature was increased as compared to those in patients with a low RAS signature (P = 0.045). WGCNA methods revealed that the green module highly correlated with RAS activity and aggravation. Based on in silico analysis with classification of the human PC cell lines CFPAC-1 and SUIT-2 into high and low activity cells, respectively, CFPAC-1 tumors showed desmoplasia and high expression of tumor IL-6 and liver SAA1 as compared to SUIT-2 tumors (P = 0.046 and P = 0.016, respectively). Conclusions: Tumor RAS activity is related to inflammation-related aggravation in PC patients. RAS-related inflammation can be accurately presented in a murine model of human PC cells, which might be an effective tool to test the inhibition of RAS-driven inflammation. Citation Format: Shuichi Mitsunaga, Mari Masuda, Hiroki Yoshimatsu, Yoichi Kamei, Mitsuhito Sasaki, Kazuo Watanabe, Masafumi Ikeda. RAS activity-related features of metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2238.

Carcinogenesis Observed in the Spleens of Balb/c Mice After Head-neutron Irradiation.

To investigate the long-term influence of head-neutron irradiation on mice spleens, post-radiation late effects were examined in three types of mice: Balb/c and severe combined immunodeficiency (SCID) mice, which have high radio-sensitivities, and C3H mice. Neutron irradiation was performed with the neutron beam of the Kyoto University Research Reactor. Survival fractions and the change in spleen size after head-neutron irradiation were investigated in three different types of mice. Physical condition after neutron irradiation was observed for eighteen months. The onset of primary splenic malignant lymphoma was recognized in many of the Balb/c mice 18 months after head-neutron irradiation. Eight months after head-neutron irradiation, many SCID mice developed an abscess in the part exposed to radiation and spleen swelling. The swollen spleen of SCID mice had hematopoiesis from the marrow. Low energy head-neutron irradiation damages immune organs in radiosensitive SCID and Balb/c mice. A combination of boron neutron capture therapy and immunotherapy may be less toxic than low-energy neutron-irradiation alone.

Evaluation of a novel severe combined immunodeficiency mouse model for antiviral drug evaluation against Chandipura virus infection

Chandipura virus (CHPV) is a negative-sense single-stranded RNA virus known to cause fatal encephalitis outbreaks in the Indian subcontinent. The virus displays tropism towards the pediatric population and holds significant public health concerns. Currently, there is no specific, effective therapy for CHPV encephalitis. In this study, we evaluated a novel C.B-17 severe combined immunodeficiency (SCID) mouse model which can be used for pre-clinical antiviral evaluation. Inoculation of CHPV developed a lethal infection in our model. Plaque assay and immunohistochemistry detected increased viral loads and antigens in various organs, including the brain, spinal cord, adrenal glands, and whole blood. We further conducted a proof-of-concept evaluation of favipiravir in the SCID mouse model. Favipiravir treatment improved survival with pre-symptomatic (days 5–14) and post-symptomatic (days 9–18) treatment. Reduced viral loads were observed in whole blood, kidney/adrenal gland, and brain tissue with favipiravir treatment. The findings in this study demonstrate the utility of SCID mouse for in vivo drug efficacy evaluation and the potential efficacy of favipiravir against CHPV infection.

Antibodies against Poly(ethylene glycol) Activate Innate Immune Cells and Induce Hypersensitivity Reactions to PEGylated Nanomedicines.

Nanomedicines and macromolecular drugs can induce hypersensitivity reactions (HSRs) with symptoms ranging from flushing and breathing difficulties to hypothermia, hypotension, and death in the most severe cases. Because many normal individuals have pre-existing antibodies that bind to poly(ethylene glycol) (PEG), which is often present on the surface of nanomedicines and macromolecular drugs, we examined if and how anti-PEG antibodies induce HSRs to PEGylated liposomal doxorubicin (PLD). Anti-PEG IgG but not anti-PEG IgM induced symptoms of HSRs including hypothermia, altered lung function, and hypotension after PLD administration in C57BL/6 and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Hypothermia was significantly reduced by blocking FcγRII/III, by depleting basophils, monocytes, neutrophils, or mast cells, and by inhibiting secretion of histamine and platelet-activating factor. Anti-PEG IgG also induced hypothermia in mice after administration of other PEGylated liposomes, nanoparticles, or proteins. Humanized anti-PEG IgG promoted binding of PEGylated nanoparticles to human immune cells and induced secretion of histamine from human basophils in the presence of PLD. Anti-PEG IgE could also induce hypersensitivity reactions in mice after administration of PLD. Our results demonstrate an important role for IgG antibodies in induction of HSRs to PEGylated nanomedicines through interaction with Fcγ receptors on innate immune cells and provide a deeper understanding of HSRs to PEGylated nanoparticles and macromolecular drugs that may facilitate development of safer nanomedicines.

Plasma cell–derived mtDAMPs activate the macrophage STING pathway, promoting myeloma progression

AbstractMitochondrial damage–associated molecular patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context-specific immunoregulatory functions. Cell-free mitochondrial DNA (mtDNA) is recognized via pattern recognition receptors and is a potent activator of the innate immune system. Cell-free mtDNA is elevated in the circulation of trauma patients and patients with cancer; however, the functional consequences of elevated mtDNA are largely undefined. Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. Here, using in vivo models, we describe the role of MM cell–derived mtDAMPs in the protumoral BM microenvironment and the mechanism and functional consequence of mtDAMPs in myeloma disease progression. Initially, we identified elevated levels of mtDNA in the peripheral blood serum of patients with MM compared with those of healthy controls. Using the MM1S cells engrafted into nonobese diabetic severe combined immunodeficient gamma mice, we established that elevated mtDNA was derived from MM cells. We further show that BM macrophages sense and respond to mtDAMPs through the stimulator of interferon genes (STING) pathway, and inhibition of this pathway reduces MM tumor burden in the KaLwRij-5TGM1 mouse model. Moreover, we found that MM-derived mtDAMPs induced upregulation of chemokine signatures in BM macrophages, and inhibition of this signature resulted in egress of MM cells from the BM. Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signaling. We establish the functional role of these mtDAMP-activated macrophages in promoting disease progression and retaining MM cells in the protumoral BM microenvironment.

Subcutaneous transplantation of human embryonic stem cells-derived pituitary organoids.

The pituitary gland, regulating various hormones, is central in the endocrine system. As spontaneous recovery from hypopituitarism is rare, and exogenous-hormone substitution is clumsy, pituitary replacement via regenerative medicine, using pluripotent stem cells, is desirable. We have developed a differentiation method that in mice yields pituitary organoids (POs) derived from human embryonic stem cells (hESC). Efficacy of these POs, transplanted subcutaneously into hypopituitary mice, in reversing hypopituitarism was studied. hESC-derived POs were transplanted into inguinal subcutaneous white adipose tissue (ISWAT) and beneath dorsal skin, a relatively avascular region (AR), of hypophysectomized severe combined immunodeficient (SCID) mice. Pituitary function was evaluated thereafter for ¾ 6mo, assaying basal plasma ACTH and ACTH response to corticotropin-releasing hormone (CRH) stimulation. Histopathologic examination of organoids 150d after transplantation assessed engraftment. Some mice received an inhibitor of vascular endothelial growth factor (VEGF) to permit assessment of how angiogenesis contributed to subcutaneous engraftment. During follow-up, both basal and CRH-stimulated plasma ACTH levels were significantly higher in the ISWAT group (p < 0.001 - 0.05 and 0.001 - 0.005, respectively) than in a sham-operated group. ACTH secretion also was higher in the ISWAT group than in the AR group. Histopathologic study found ACTH-producing human pituitary-cell clusters in both groups of allografts, which had acquired a microvasculature. POs qPCR showed expression of angiogenetic factors. Plasma ACTH levels decreased with VEGF-inhibitor administration. Subcutaneous transplantation of hESC-derived POs into hypopituitary SCID mice efficaciously renders recipients ACTH-sufficient.

Synthesis and Preclinical Evaluation of a Novel Fluorine-18-Labeled Tracer for Positron Emission Tomography Imaging of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a target for treating B-cell malignancies and autoimmune diseases. To aid in the discovery and development of BTK inhibitors and improve clinical diagnoses, we have developed a positron emission tomography (PET) radiotracer based on a selective BTK inhibitor, remibrutinib. [18F]PTBTK3 is an aromatic, 18F-labeled tracer that was synthesized in 3 steps with a 14.8 ± 2.4% decay-corrected radiochemical yield and ≥99% radiochemical purity. The cellular uptake of [18F]PTBTK3 was blocked up to 97% in JeKo-1 cells using remibrutinib or non-radioactive PTBTK3. [18F]PTBTK3 exhibited renal and hepatobiliary clearance in NOD SCID (non-obese diabetic/severe combined immunodeficiency) mice, and the tumor uptake of [18F]PTBTK3 in BTK-positive JeKo-1 xenografts (1.23 ± 0.30% ID/cc) was significantly greater at 60 min post injection compared to the tumor uptake in BTK-negative U87MG xenografts (0.41 ± 0.11% ID/cc). In the JeKo-1 xenografts, tumor uptake was blocked up to 62% by remibrutinib, indicating the BTK-dependent uptake of [18F]PTBTK3 in tumors.

Peanut allergen inhibition prevents anaphylaxis in a humanized mouse model.

Peanut-induced allergy is an immunoglobulin E (IgE)-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. Although there are treatments for symptoms in patients with allergies resulting from allergen exposure, there are few preventive therapies other than strict dietary avoidance or oral immunotherapy, neither of which are successful in all patients. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, here, we developed a humanized mouse model to test cHBI efficacy in vivo. Nonobese diabetic-severe combined immunodeficient γc-deficient mice expressing transgenes for human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 developed mature functional human mast cells in multiple tissues and displayed robust anaphylactic reactions when passively sensitized with patient-derived IgE monoclonal antibodies specific for peanut Arachis hypogaea 2 (Ara h 2). The allergic response in humanized mice was IgE dose dependent and was mediated by human mast cells. Using this humanized mouse model, we showed that cHBI prevented allergic reactions for more than 2 weeks when administered before allergen exposure. cHBI also prevented fatal anaphylaxis and attenuated allergic reactions when administered shortly after the onset of symptoms. cHBI impaired mast cell degranulation in vivo in an allergen-specific manner. cHBI rescued the mice from lethal anaphylactic responses during oral Ara h 2 allergen-induced anaphylaxis. Together, these findings suggest that cHBI has the potential to be an effective preventative for peanut-specific allergic responses in patients.

Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.

Targeted silencing of the MCL-1 gene using multi-layered dendrimer-based nanoconstructs achieves efficient tumor regression in xenografted mice models

The setback in the practical clinical use of RNA interference (RNAi)-based cancer treatment stems from the lack of targeted small interfering RNA (siRNA) delivery. Here, we show that luteinizing hormone-releasing hormone(LHRH) analog-tethered multi-layered polyamidoamine (PAMAM) nanoconstructs silence the anti-apoptotic MCL-1 gene in LHRH receptor overexpressing human breast (MCF-7) and prostate cancer (LNCaP) cells with 70.91 % and 74.10 % efficiency, respectively. These results were confirmed by RT-PCR. The Acridine orange/Ethidium bromide (AO/EB) dual staining revealed that the silencing of MCL-1 induced apoptosis in both the cell lines. In vivo tumor regression studies performed using MCF-7 and LNCaP xenografted severe combined immunodeficiency(SCID) mice demonstrated highly improved tumor regression in groups treated with targeted nanoconstructs complexed with MCL-1 siRNA (T + siMCL-1) compared to the other treatment groups. The quantitative RT-PCR results of tumor tissues demonstrated significant MCL-1 gene silencing, i.e., 73.76 % and 92.63 % in breast and prostate tumors, respectively, after T + siMCL-1 treatment. Reduction in MCL-1 protein expression as assessed by immunohistochemistry further confirmed these results. Furthermore, the caspase 3/7 assay demonstrated apoptosis in the MCL-1 silenced tissues. The study strongly suggests that targeted delivery of siRNAs using multi-layered dendrimer nanostructures could be an effective therapy for LHRH overexpressing cancers.

Halogen Replacement on the Lysine Side Chain of Lys-Urea-Glu-Based PSMA Inhibitors Leads to Significant Changes in Targeting Properties.

Investigate the impact of various halogens on pharmacokinetics, biodistribution, and micro positron emission tomography/computed tomography (PET/CT) imaging of Glu-urea-Lys-based prostate-specific membrane antigen (PSMA) inhibitors. Based on the modification of SC691, a small molecule inhibitor of PSMA previously developed by our group, we synthesized 68Ga-labeled compounds by modifying the lysine terminal amino with different halogenated phenyl substituents. After complete characterization, in vitro and in vivo properties were studied. The [68Ga]Ga-DOTA-SC691-R possesses a high radiochemical yield (98-99%). The internalization values of [68Ga]Ga-DOTA-SC691-H, [68Ga]Ga-DOTA-SC691-Cl, and [68Ga]Ga-DOTA-SC691-Br in LNCaP cells all displayed time-dependent pattern enhanced with time. The results of in vitro competitive inhibition assay showed that the affinity of natGa-DOTA-SC691-R for PSMA had a trend of H < F < Cl < Br < I. The blocking imaging and dynamic imaging on micro-PET/CT of male non-obese diabetic/severe combined immunodeficiency mice with LNCaP tumors showed the rapid tumor targeting properties of [68Ga]Ga-DOTA-SC691-R with specificity for PSMA. Static imaging of micro-PEC/CT of these compounds could rapidly localize LNCaP tumors with decent image quality (except for [68Ga]Ga-DOTA-SC691-H). Biodistribution data showed that [68Ga]Ga-DOTA-SC691-R were metabolized via the kidney and tumor accumulation followed the order of H ≈ F ≈ Cl < I < Br uptake values at 1 h. [68Ga]Ga-DOTA-SC691-Br showed the highest tumor accumulation and retention (15.21 ± 5.57%ID/g at 30 min, 20.39 ± 4.38%ID/g at 60 min, and 13.30 ± 4.39%ID/g at 120 min), which is consistent with the results of the competitive inhibition assay and cell binding assay. It was demonstrated that the halogen substituent on the lysine terminal amino group on the Glu-urea-Lys backbone did positively affect the binding of [68Ga]Ga-DOTA-SC691-R to PSMA. The bulkier and less electronegative Br (or I) elements are preferred for structural modifications here.

Cardiac muscle patches containing four types of cardiac cells derived from human pluripotent stem cells improve recovery from cardiac injury in mice.

We have shown that human cardiac muscle patches (hCMPs) containing three different types of cardiac cells-cardiomyocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs), all of which were differentiated from human pluripotent stem cells (hPSCs)-significantly improved cardiac function, infarct size, and hypertrophy in a pig model of myocardial infarction (MI). However, hPSC-derived CMs (hPSC-CMs) are phenotypically immature, which may lead to arrhythmogenic concerns; thus, since hPSC-derived cardiac fibroblasts (hPSC-CFs) appear to enhance the maturity of hPSC-CMs, we compared hCMPs containing hPSC-CMs, -SMCs, -ECs, and -CFs (4TCC-hCMPs) with a second hCMP construct that lacked hPSC-CFs but was otherwise identical [hCMP containing hPSC-CMs, -AECs, and -SMCs (3TCC-hCMPs)]. hCMPs were generated in a fibrin scaffold. MI was induced in severe combined immunodeficiency (SCID) mice through permanent coronary artery (left anterior descending) ligation, followed by treatment with cardiac muscle patches. Animal groups included: MI heart treated with 3TCC-hCMP; with 4TCC-hCMP; MI heart treated with no patch (MI group) and sham group. Cardiac function was evaluated using echocardiography, and cell engraftment rate and infarct size were evaluated histologically at 4 weeks after patch transplantation. The results from experiments in cultured hCMPs demonstrate that the inclusion of cardiac fibroblast in 4TCC-hCMPs had (i) better organized sarcomeres; (ii) abundant structural, metabolic, and ion-channel markers of CM maturation; and (iii) greater conduction velocities (31 ± 3.23 cm/s, P < 0.005) and action-potential durations (APD50 = 365 ms ± 2.649, P < 0.0001; APD = 408 ms ± 2.757, P < 0.0001) than those (velocity and APD time) in 3TCC-hCMPs. Furthermore, 4TCC-hCMPs transplantation resulted in better cardiac function [ejection fraction (EF) = 49.18% ± 0.86, P < 0.05], reduced infarct size (22.72% ± 0.98, P < 0.05), and better engraftment (15.99% ± 1.56, P < 0.05) when compared with 3TCC-hCMPs (EF = 41.55 ± 0.92%, infarct size = 39.23 ± 4.28%, and engraftment = 8.56 ± 1.79%, respectively). Collectively, these observations suggest that the inclusion of hPSC-CFs during hCMP manufacture promotes hPSC-CM maturation and increases the potency of implanted hCMPs for improving cardiac recovery in mice model of MI.

Establishment of hyperoxic cell culture system for predicting drug-induced liver injury: reducing accumulated lipids in hepatocytes derived from chimeric mice with humanized liver.

Drug-induced liver injury (DILI) is a major adverse reaction. Species-specific differences between humans and laboratory animals make it difficult to establish evaluation models that can accurately predict DILI in the preclinical phase. Chimeric mice with humanized liver are potential predictive models for understanding DILI. Chimeric mice generated by transplanting human hepatocytes into urokinase-type plasminogen activator/severe combined immunodeficient mice are known to develop fatty liver and show lipid accumulation in isolated hepatocytes. It is speculated that the lipids accumulated in hepatocytes may interfere with DILI assessment. It is known that normal 20% oxygen culture conditions do not meet oxygen demand because oxygen consumption rate is higher than the oxygen supply rate. Therefore, we predicted that hyperoxic cultures could induce hepatocyte function and reduce accumulated lipids. A culture of chimeric mouse hepatocytes in 40% oxygen showed reduced intracellular lipid and triglyceride levels compared to those cultured in 20% oxygen on days 7 and 10. In addition, fatty acid β-oxidation (FAO) activity increased from day 7 under 40% oxygen conditions. On the other hand, FAO activity increased on day 10 under 20% conditions. Microarray and Ingenuity Pathway Analysis showed that lipid metabolism-related pathways were downregulated under 40% oxygen conditions for 7 days, suggesting the involvement of several mechanisms in decreasing lipid levels and increasing FAO. Furthermore, some pathways related to cellular function and maintenance were upregulated under 40% oxygen conditions for 7 days. In conclusion, chimeric mouse hepatocytes cultured under hyperoxic conditions may be useful for predicting DILI.

Quantitative Ultrasound for Evaluation of Tumour Response to Ultrasound-Microbubbles and Hyperthermia.

Objectives: Prior study has demonstrated the implementation of quantitative ultrasound (QUS) for determining the therapy response in breast tumour patients. Several QUS parameters quantified from the tumour region showed a significant correlation with the patient's clinical and pathological response. In this study, we aim to identify if there exists such a link between QUS parameters and changes in tumour morphology due to combined ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) using the breast xenograft model (MDA-MB-231). Method: Tumours grown in the hind leg of severe combined immuno-deficient mice were treated with permutations of USMB and HT. Ultrasound radiofrequency data were collected using a 25 MHz array transducer, from breast tumour-bearing mice prior and post-24-hour treatment. Result: Our result demonstrated an increase in the QUS parameters the mid-band fit and spectral 0-MHz intercept with an increase in HT duration combined with USMB which was found to be reflective of tissue structural changes and cell death detected using haematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labelling stain. A significant decrease in QUS spectral parameters was observed at an HT duration of 60 minutes, which is possibly due to loss of nuclei by the majority of cells as confirmed using histology analysis. Morphological alterations within the tumour might have contributed to the decrease in backscatter parameters. Conclusion: The work here uses the QUS technique to assess the efficacy of cancer therapy and demonstrates that the changes in ultrasound backscatters mirrored changes in tissue morphology.

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention.

Therapeutic targeting of hepatocellular carcinoma cells with antrocinol, a novel, dual-specificity, small-molecule inhibitor of the KRAS and ERK oncogenic signaling pathways

Until recently, sorafenib has been the only treatment approved by the U.S. Food and Drug Administration for patients with advanced hepatocellular carcinoma (HCC). Some patients, however, exhibit resistance to this treatment and subsequently experience cancer progression, recurrence, or death. Therefore, identifying a new alternative treatment for patients with little or no response to sorafenib treatment is vital. In this study, we explored the therapeutic potential and underlying molecular mechanism of antrocinol ((3aS,4R,6aS,10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphtho[1,8a-c]furan-1-one) in patients with HCC. The results indicated that antrocinol was more therapeutically effective than antrocin, Stivarga, and sorafenib against HCC cell lines. Antrocinol also substantially suppressed the expression of KRAS-GTP, p-MEK1/2, p-ERK1/2, and p-AKT in the Huh7 cell line. Additionally, antrocinol-induced apoptosis in the Huh7 cell line, inhibited the formation of tumorspheres, and suppressed the expression of cancer stem cell markers CD133, KLF4, CD44, OCT4, SOX2, and c-Myc. Animal studies revealed that antrocinol alone considerably suppressed tumor growth in nonobese diabetic/severe combined immunodeficient mice inoculated with Huh7 tumorspheres. It also synergistically enhanced the anticancer effect of sorafenib, resulting in enhanced suppression of tumor growth (p < 0.001) and tumorsphere formation (p < 0.001). In tumor samples resected from mice treated with antrocinol alone or in combination with sorafenib, immunohistochemical analysis revealed an increase in BAX expression and a decrease in ERK and AKT protein expression. To the best of our knowledge, this is the first report of the anti-HCC activity of antrocinol. With its higher therapeutic efficacy than that of sorafenib, antrocinol is a candidate drug for patients with HCC who demonstrate little or no response to sorafenib treatment.

Experimental model of micronodular thymic neoplasm with lymphoid stroma.

Micronodular thymic neoplasm with lymphoid stroma (MNT), a subtype of thymic tumor, is histopathologically characterized by micronodular thymic epithelial cell nests with lymphoid stroma. Despite the distinct histopathology of MNT, its pathogenesis remains unclear. In this study, we aimed to examine a thymic tumor harboring thymic epithelial and lymphoid cells in a nonobese diabetic/severe combined immunodeficiency mouse. The excised tumor cells were cultured in vitro and comprised epithelial tumor cells and lymphoid cells. During a three-dimensional cell culture, the epithelial tumor cells formed micronodular cell nests surrounded by lymphoid stroma. Notably, the lymphoid cells underwent apoptosis when they were separated from the epithelial tumor cells. Cutaneous transplantation of the cultured epithelial cells with splenocytes from BALB/c mice led to tumor formation, and these cells demonstrated a histopathology similar to that of human MNT in a nonobese diabetic/severe combined immunodeficiency mouse. Given its overlapping features with human MNT, the transplanted tumor could serve as an experimental model of this disease.

BI-907828, a novel potent MDM2 inhibitor, inhibits glioblastoma brain tumor stem cells in vitro and prolongs survival in orthotopic xenograft mouse models.

The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including glioblastoma (GBM). In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification statuses of MDM2, in vitro and in orthotopic xenograft models. In vitro growth inhibition and on-target efficacy of BI-907828 were assessed by cell viability, co-immunoprecipitation assays, and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry, and in intracranial xenograft models. BI-907828 decreases viability and induces cell death at picomolar concentrations in both MDM2 amplified and normal copy number TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. Pharmacokinetic-pharmacodynamic studies in orthotopic tumor-bearing severe combined immunodeficiency (SCID) mice demonstrated that a single 50mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.