Regulation of the hematopoietic stem cell pool by C-Kit-associated trogocytosis.

Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. Although fully functional, these HSCs are selectively niche-retained as opposed to stem cells lacking macrophage markers, which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), from BM-resident macrophages in mouse and human settings. Our study provides proof of concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.

Immunohistochemistry Screening of Different Tyrosine Kinase Receptors in Canine Solid Tumors-Part I: Proposal of a Receptor Panel to Predict Therapies.

The use of tyrosine kinase inhibitors (TKI) has been growing in veterinary oncology and in the past few years several TKI have been tested in dogs. However, different from human medicine, we lack strategies to select patients to be treated with each TKI. Therefore, this study aimed to screen different tumor subtypes regarding TKI target immunoexpression as a predictor strategy to personalize the canine cancer treatment. It included 18 prostatic carcinomas, 36 soft tissue sarcomas, 20 mammary gland tumors, 6 urothelial bladder carcinomas, and 7 tumors from the endocrine system. A total of 87 patients with paraffin blocks were used to perform immunohistochemistry (IHC) of human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptors 1 (EGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet derived growth factor receptor beta (PDGFR-ß), c-KIT, and extracellular signal-regulated kinase 1/2 (ERK1/ERK2). The immunohistochemical screening revealed a heterogeneous protein expression among histological types with mesenchymal tumors showing the lowest expression level and carcinomas the highest expression. We have demonstrated by IHC screening that HER2, EGFR1, VEGFR-2, PDGFR-ß and ERK1/ERK2 are commonly overexpressed in dogs with different carcinomas, and KIT expression is considered relatively low in the analyzed samples.

Transcriptomic Profile of Lin-Sca1+c-kit (LSK) cells in db/db mice with long-standing diabetes.

BACKGROUND: The Lin-Sca1+c-Kit+ (LSK) fraction of the bone marrow (BM) comprises multipotent hematopoietic stem cells (HSCs), which are vital to tissue homeostasis and vascular repair. While diabetes affects HSC homeostasis overall, the molecular signature of mRNA and miRNA transcriptomic under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored. METHODS: In this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA. RESULTS: We uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype. CONCLUSION: Our findings highlight that chronic diabetes adversely alters HSCs' homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.

Interstitial cells of the sip syncytium regulate basal membrane potential in murine gastric corpus.

Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.

c-KIT inhibitors reduce pathology and improve behavior in the Tg(SwDI) model of Alzheimer's disease.

Treatments for Alzheimer's disease have primarily focused on removing brain amyloid plaques to improve cognitive outcomes in patients. We developed small compounds, known as BK40143 and BK40197, and we hypothesize that these drugs alleviate microglial-mediated neuroinflammation and induce autophagic clearance of neurotoxic proteins to improve behavior in models of neurodegeneration. Specificity binding assays of BK40143 and BK40197 showed primary binding to c-KIT/Platelet Derived Growth Factor Receptors (PDGFR)α/ß, whereas BK40197 also differentially binds to FYVE finger-containing phosphoinositide kinase (PIKFYVE). Both compounds penetrate the CNS, and treatment with these drugs inhibited the maturation of peripheral mast cells in transgenic mice, correlating with cognitive improvements on measures of memory and anxiety. In the brain, microglial activation was profoundly attenuated and amyloid-beta and tau were reduced via autophagy. Multi-kinase inhibition, including c-KIT, exerts multifunctional effects to reduce neurodegenerative pathology via autophagy and microglial activity and may represent a potential therapeutic option for neurodegeneration.

The prognostic significance of CD117-positive mast cells and microvessel density in colorectal cancer.

The prognostic significance of angiogenesis has been demonstrated in various types of cancer. However, in colorectal cancer (CRC), there are conflicting results regarding the relationship between angiogenesis and clinical-histopathological prognostic factors. Mast cells are immune system cells found in the inflammatory microenvironment; their role in carcinogenesis and prognosis remains unclear although they are considered to cause cancer development and progression. The present study aims to evaluate the prognostic significance of mast cell accumulation and angiogenesis assessed by microvessel density (MVD) in patients with CRC. Patients who underwent curative resection and who were not treated with neoadjuvant chemotherapy were included. The anti-CD34 antibody and anti-CD117 antibody were utilized for the immunohistochemical assessment of MVD and the mast cell count (MCC) in the tissue samples, respectively. The relationship between MCC, MVD, survival and clinical-histopathological prognostic factors were evaluated. A total of 94 patients were enrolled to the study. In a median 49-month follow-up, 65 patients (69.1%) died. The 5-year disease-free survival was 61.1% and 31.3% for the group with CD34 < 18.3% and CD34 > 18.3%, respectively (P = .001). The same groups presented 5-year overall survival rates of 77, 1% and 51, 4%, respectively (P, .012). The MVD was found to be associated with the pathological T stage, lymph node metastasis and distant metastasis (P < .05). Although the MCC was positively correlated with MVD, there was no association between the MCC and clinical-histopathological prognostic factors. MVD-assessed angiogenesis was significantly related to survival and the clinical-histopathological prognostic factors in patients diagnosed with CRC.

CD117 expression in canine ovarian tumours.

Canine ovarian cancer poses a significant diagnostic and therapeutic challenge. The heterogeneous nature of ovarian tumours makes accurate histological identification difficult, whilst treatment is limited to surgical excision. The tyrosine kinase receptor CD117 is neo-expressed in many tumours and represents a potential diagnostic and prognostic biomarker and therapeutic target. This study aimed to establish if CD117 is neoexpressed in canine ovarian tumours. Immunohistochemistry was employed to assess expression of CD117 in 29 canine ovarian tumour samples. CD117 labelling was assessed with a semiquantitative immunoreactivity score, and the location of labelling was recorded as membranous, focal cytoplasmic or diffuse cytoplasmic. Histological morphology was assessed and used to assign subgroups based on growth pattern. Cytokeratin 7 labelling was used to indicate the tumour type as epithelial or sex-cord stromal in origin. Mitotic index, percentage of necrosis and vascular invasion were also assessed and evaluated for association with CD117 expression. Overall, 81% of ovarian tumours neoexpressed CD117 and normal ovarian tissue did not express CD117. Positive immunolabelling was seen in a subset of cells in both ovarian carcinomas (n = 20) and ovarian granulosa cell tumours (n = 3). There was no association between CD117 expression and patient age, histological subtype, mitotic index, percentage of necrosis or vascular invasion. This is the largest study to identify the expression of CD117 in canine ovarian tumours, but further research is needed to elucidate its prognostic and therapeutic value.

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

An optimized method for IgE-mediated degranulation of human lung mast cells.

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

RAF1 facilitates KIT signaling and serves as a potential treatment target for gastrointestinal stromal tumor.

The aberrant activation of RAS/RAF/MEK/ERK signaling is important for KIT mutation-mediated tumorigenesis of gastrointestinal stromal tumor (GIST). In this study, we found that inhibition of RAF1 suppresses the activation of both wild-type KIT and primary KIT mutations in GIST, with primary KIT mutations showing greater sensitivity. This suggests a positive feedback loop between KIT and RAF1, wherein RAF1 facilitates KIT signaling. We further demonstrated that RAF1 associates with KIT and the kinase activity of RAF1 is necessary for its contribution to KIT activation. Accordingly, inhibition of RAF1 suppressed cell survival, proliferation, and cell cycle progression in vitro mediated by both wild-type KIT and primary KIT mutations. Inhibition of RAF1 in vivo suppressed GIST growth in a transgenic mouse model carrying germline KIT/V558A mutation, showing a similar treatment efficiency as imatinib, the first-line targeted therapeutic drug of GIST, while the combination use of imatinib and RAF1 inhibitor further suppressed tumor growth. Acquisition of drug-resistant secondary mutation of KIT is a major cause of treatment failure of GIST following targeted therapy. Like wild-type KIT and primary KIT mutations, inhibition of RAF1 suppressed the activation of secondary KIT mutation, and the cell survival, proliferation, cell cycle progression in vitro, and tumor growth in vivo mediated by secondary KIT mutation. However, the activation of secondary KIT mutation is less dependent on RAF1 compared with that of primary KIT mutations. Taken together, our results revealed that RAF1 facilitates KIT signaling and KIT mutation-mediated tumorigenesis of GIST, providing a rationale for further investigation into the use of RAF1 inhibitors alone or in combination with KIT inhibitor in the treatment of GIST, particularly in cases resistant to KIT inhibitors.

Diagnostic and prognostic significance of mast cell markers in HIV/AIDS: Current insights and future directions.

Human immunodeficiency virus (HIV) infection continues to pose significant global health challenges, necessitating advancements in diagnostic and prognostic approaches to optimize disease management. While primarily recognized for their roles in allergic responses, mast cells have emerged as potential markers with diagnostic and prognostic significance in the context of HIV/AIDS. This paper aims to synthesize current insights and delineate future directions regarding the utility of mast cell markers in diagnosing HIV infection, predicting disease progression, and guiding therapeutic strategies. Mast cells, equipped with distinct markers such as tryptase, chymase, carboxypeptidase A3, and c-kit/CD117 receptors, exhibit tissue-specific expression patterns that offer potential as diagnostic indicators for HIV infection. Understanding the dynamics of these markers in different tissues and body fluids holds promise for accurate HIV diagnosis, disease staging, and monitoring treatment responses. Moreover, the prognostic significance of mast cell markers in HIV/AIDS lies in their potential to predict disease progression, immune dysregulation, and clinical outcomes. The integration of mast cell markers into clinical applications offers promising avenues for refining diagnostic assays, patient monitoring protocols, and therapeutic strategies in HIV/AIDS. Future research directions involve the development of novel diagnostic tools and targeted therapies based on mast cell-specific markers, potentially revolutionizing clinical practice and enhancing patient care in the management of HIV/AIDS. Continued investigations into mast cell markers' diagnostic and prognostic implications hold immense potential to advance our understanding and improve outcomes in HIV/AIDS management.

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene.

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between the Kit and Kdr genes have a phenotypic manifestation of the mutation - a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

Therapeutic modulation of KIT ligand in melanocytic disorders with implications for mast cell diseases.

KIT ligand and its associated receptor KIT serve as a master regulatory system for both melanocytes and mast cells controlling survival, migration, proliferation and activation. Blockade of this pathway results in cell depletion, while overactivation leads to mastocytosis or melanoma. Expression defects are associated with pigmentary and mast cell disorders. KIT ligand regulation is complex but efficient targeting of this system would be of significant benefit to those suffering from melanocytic or mast cell disorders. Herein, we review the known associations of this pathway with cutaneous diseases and the regulators of this system both in skin and in the more well-studied germ cell system. Exogenous agents modulating this pathway will also be presented. Ultimately, we will review potential therapeutic opportunities to help our patients with melanocytic and mast cell disease processes potentially including vitiligo, hair greying, melasma, urticaria, mastocytosis and melanoma.

SCF/C-kit drives spermatogenesis disorder induced by abscopal effects of cranial irradiation in mice.

Cranial radiotherapy is a major treatment for leukemia and brain tumors. Our previous study found abscopal effects of cranial irradiation could cause spermatogenesis disorder in mice. However, the exact mechanisms are not yet fully understood. In the study, adult male C57BL/6 mice were administrated with 20 Gy X-ray cranial irradiation (5 Gy per day for 4 days consecutively) and sacrificed at 1, 2 and 4 weeks. Tandem Mass Tag (TMT) quantitative proteomics of testis was combined with bioinformatics analysis to identify key molecules and signal pathways related to spermatogenesis at 4 weeks after cranial irradiation. GO analysis showed that spermatogenesis was closely related to oxidative stress and inflammation. Severe oxidative stress occurred in testis, serum and brain, while serious inflammation also occurred in testis and serum. Additionally, the sex hormones related to hypothalamic-pituitary-gonadal (HPG) axis were disrupted. PI3K/Akt pathway was activated in testis, which upstream molecule SCF/C-Kit was significantly elevated. Furthermore, the proliferation and differentiation ability of spermatogonial stem cells (SSCs) were altered. These findings suggest that cranial irradiation can cause spermatogenesis disorder through brain-blood-testicular cascade oxidative stress, inflammation and the secretory dysfunction of HPG axis, and SCF/C-kit drive this process through activating PI3K/Akt pathway.

Four and a half LIM domains 2 (FHL2) attenuates tumorigenesis of gastrointestinal stromal tumors (GISTs) by negatively regulating KIT signaling.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn't alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

Delivery of Stem Cells and BMP-2 With Functionalized Self-Assembling Peptide Enhances Regeneration of Infarcted Myocardium.

Stem cell transplantation is a promising therapeutic strategy for myocardial infarction (MI). However, engraftment, survival and differentiation of the transplanted stem cells in ischemic and inflammatory microenvironment are poor. We designed a novel self-assembly peptide (SAP) by modifying the peptide RADA16 with cell-adhesive motif and BMP-2 (bone morphogenetic protein-2)-binding motif. Effects of the functionalized SAP on adhesion, survival and differentiation of c-kit+ MSCs (mesenchymal stem cells) were examined. Myocardial regeneration, neovascularization and cardiac function were assessed after transplantation of the SAP loading c-kit+ MSCs and BMP-2 in rat MI models. The SAP could spontaneously assemble into well-ordered nanofibrous scaffolds. The cells adhered to the SAP scaffolds and spread well. The SAP protected the cells in the condition of hypoxia and serum deprivation. Following degradation of the SAP, BMP-2 was released sustainedly and induced c-kit+ MSCs to differentiate into cardiomyocytes. At four weeks after transplantation of the SAP loading c-kit+ MSCs and BMP-2, myocardial regeneration and angiogenesis were enhanced, and cardiac function was improved significantly. The cardiomyocytes differentiated from the engrafted c-kit+ MSCs were increased markedly. The differentiated cells connected with recipient cardiomyocytes to form gap junctions. Collagen volume was decreased dramatically. These results suggest that the functionalized SAP promotes engraftment, survival and differentiation of stem cells effectively. Local sustained release of BMP-2 with SAP is a viable strategy to enhance differentiation of the engrafted stem cells and repair of the infarcted myocardium.

[Salivary carcinoma showing thymus-like differentiation: clinicopathological analysis of 7 cases].

Objective: To analyze the clinicopathological features of salivary carcinoma showing thymus-like differentiation(CASTLE). Methods: Cases diagnosed with salivary CASTLE from January 2020 to December 2023 were collected and selected from the Department of Oral Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. A total of 7 cases of salivary CASTLE were identified. All the cases originated from parotid. There were 3 males and 4 females. The patients' age range was 11-70 years.The clinical, microscopic, immunohistochemical and prognostic features of these cases were analyzed. Results: The duration of disease ranged from 1 month to 1 year, and 1 patient had facial numbness and 1 with swelling sensation occasionally. Radiographically, 4 cases showed malignant signs. Microscopically, 4 cases involved in parotid gland, and all the tumors had different degrees of lymphoid tissue background. The tumor cells arranged in nests, 5 cases with lymphoepithelial carcinoma-like and 2 cases with squamous cell carcinoma morphology. The tumor cells expressed CD5 and CD117 proteins diffusely in lymphoepithelial carcinoma-like cases. However, the tumor cells expressed CD5 diffusely and CD117 focally in cases with squamous cell carcinoma morphology. All the cases had no Epstein-Barr virus infection. Among the 6 patients with follow-up information, all of them underwent postoperative radiotherapy, and none of them had local recurrence and lymph node metastasis. Conclusions: Salivary CASTLE is a rare tumor, it should be distinguished from lymphoepithelial carcinoma and squamous cell carcinoma. The patients often have better prognosis and CD5 protein expression has a valuable role in the differential diagnosis.

Efficient generation of cloned cats with altered coat colour by editing of the KIT gene.

Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.

Stem cell factor and cKIT modulate endothelial glycolysis in hypoxia.

AIMS: In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. METHODS AND RESULTS: SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. CONCLUSION: We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.