Expression Levels Of CD44 Research Articles

The crosstalk between lung cancer cells and platelets promotes tumor angiogenesis in vivo and in vitro.

We previously showed that the crosstalk of H1975 cells and platelets (PLTs) may promote tumor angiogenesis. This study aimed to determine whether other lung cell lines (LC) interacting with PLTs could affect tumor angiogenesis through in vivo and in vitro experiments. Cell Counting Kit-8, EdU cell proliferation, wound healing, Transwell invasion, F-actin staining, tube formation, ELISA and western blot assays were performed to investigate the properties and the expression levels of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), p-VEGFR2, PI3K, p-PI3K, Akt, p-Akt and eNOS in supernatants or HUVECs. Then, using mouse models, immunohistochemistry was applied to detect the expression levels of CD31 and VEGF. Compared with single-cultured HUVECs (EC) or incubation with either LC supernatant (EC + LC) or activated PLT supernatant (EC + PLT), incubation with SN_LCP (supernatant derived from LC cocultured with PLT, named the EC + LC + PLT group) improved the viability, proliferation, migration, invasion, and tube formation activities of HUVECs and the expression of F-actin, VEGF, VEGFR2, p-VEGFR2, p-PI3K, p-Akt and eNOS in HUVECs. Higher expression levels of CD31 and VEGF were found in the LLC + PLT (mouse model inoculated with Lewis lung cancer (LLC) cells cocultured with PLTs) group than in the LLC (mouse model inoculated with LLC cells alone) group. However, the increased angiogenic properties of HUVECs were inhibited by apatinib, an inhibitor of VEGFR2. Lung carcinoma cells interacting with PLTs may play a key role in lung carcinoma angiogenesis through the VEGF/VEGFR2 signaling pathway.

A histological study of vascular wall resident stem cells in venous malformations.

Vascular wall resident stem cells (VW-SCs) play a key role in vascular formation and remodeling under both physiological and pathological situations. They not only serve as a reservoir to supply all types of vascular cells needed, but also regulate vascular homeostasis by paracrine effects. Venous malformations (VMs) are common congenital vascular malformations which are just characterized by the deficient quantity and abnormal function of vascular cells. However, the existence and role of VW-SCs in VMs is still unclear at present. In this study, the level and distribution of VW-SCs in 22 specimens of VMs were measured by immunochemistry, double-labeling immunofluorescence, and qPCR, followed by the Spearman rank correlation test. We found that both the protein and mRNA expression levels of CD34, vWF, VEGFR2, CD44, CD90, and CD105 were significantly downregulated in VMs compared with that in normal venules. VW-SCs were sporadically distributed or even absent within and outside the endothelium of VMs. The expression of the VW-SC-related markers was positively correlated with the density of both endothelial cells and perivascular cells. All those results and established evidence indicated that VW-SCs were more sporadically distributed with fewer amounts in VMs, which possibly contributing to the deficiency of vascular cells in VMs.

Electro-acupuncture Promotes Angiogenesis via Exosomal miR-210 in the Hypoxia-induced HUVECs Mediated HIF-1α/VEGF/Notch 1 Signal Pathway.

Acupuncture has been wildly applied for cerebral ischemia treatment in China for thousands of years, while the specific mechanism remains uncertain. Recently, many studies have shown that acupuncture promotes angiogenesis after ischemia occurs. Here, we examined the effect of electro-acupuncture (EA) exosomes on angiogenesis in hypoxia-induced human umbilical vein endothelial cells (HUVECs). To investigate whether EA exosomal miR-210 promotes angiogenesis in the hypoxiainduced HUVECs via the HIF-1α/VEGF/Notch 1 signal pathway. The middle cerebral artery occlusion (MCAO) model was established and treated with EA therapy. Then, exosomes were identified and isolated from rats' plasma in the MCAO+EA group by transmission electron microscopy (TEM), surface markers expressions, and PKH26 reagent. MiR- 210 mimic, miR-210 inhibitor, and HIF-1α were transfected. Flow cytometry, CCK-8 assay, and Transwell assay were conducted to assess the migration, apoptosis, and proliferation of each group of cells. Western blot and quantitative PCR were performed to detect the CD34, HIF-1α, VEGF, Notch 1, and miR-210 expression levels in each group. MiR-210 was significantly upregulated in exosomes of the MCAO plasma, and further enhanced by EA therapy. EA-EXOs and miR-210 mimic inhibited cell apoptosis, promoted cell proliferation and cell migration in hypoxia-induced HUVECs. However, the miR-210 inhibitor reversed the proliferation and migration number induced by EA-EXOs. Besides, EA-EXOs and miR- 210 mimic further enhanced those HIF-1α, VEGF, and Notch 1 levels compared to the hypoxia treatment only. Silencing HIF-1α or miR-210 reversed the high expressions of those three angiogenic factors induced by hypoxia and EA-EXO. qPCR showed similar trends with their relative mRNAs. To analyze these associations quantificationally, Spearman's rank correlation coefficient was calculated. As revealed by results, the expression of proteins and mRNA were highly correlative with each other. These results indicated that EA-EXO miR-210 promotes angiogenesis in hypoxia conditions via HIF-1α/VEGF/Notch 1 signal pathway.

CD147 protein expression and Temozolomide resistance in glioma cells: an Ex vivo and In vivo study

It has been noted that temozolomide resistance occurs in a number of malignancies, including glioma, although the underlying cause of this is unknown. The goal of the studyin vivo investigation to show that increased CD147 expression in glioma cells is a factor in their resistance to the chemotherapy drug temozolomide. Proliferation assays, TUNEL assays, reactive oxygen species assays, protein degradation assays, immunohistochemistry, Western blotting, quantitative polymerase chain reactions, and tumorigenicity assays were all carried out. Using the human protein atlas databases, the expression levels of CD147 in different kinds of malignancies were examined. For immunohistochemistry, a total of 7, 12, 19, 15, and 16 glioma samples were taken from para-carcinoma tissue, representing stage I, stage II, stage III, and stage IV gliomas, respectively. The expression of CD147 proteins is correlated with the tumor's aggressiveness. Cell development was slowed by suppressing the expression of the CD147 protein. The expression of the CD147 protein contributed to the emergence of temozolomide resistance. Expression of the CD147 protein reduced mRNA expression. The growth-inhibitory impact of temozolomide on glioma cells was enhanced by the suppression of CD147 protein.Nuclear factor E2-related factor 2 expression and CD147 protein expression showed a significant reciprocal connection with each other (p 0.0001, r2 = 0.3254). In glioma, resistance to temozolomide is due to overexpression of CD147 protein and induction of nuclear factor E2-related factor 2.

CD47 Expression in Non-Melanoma Skin Cancers and Its Clinicopathological Implications.

CD47 is a cell surface molecule and regulates diverse cellular responses. CD47 is highly expressed in cancer cells and has potential as a therapeutic target and prognostic factor in cancer patients. The expression patterns of CD47 in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and its precursor lesions, and its clinicopathological significance were investigated. CD47 expression was evaluated by immunohistochemistry in 152 cases of BCC and 71 cases of SCC. For comparison of CD47 expression, actinic keratosis (AK), squamous cell carcinoma in situ (SCCIS), keratoacanthoma (KA), and normal skin (NS) tissue were used. CD47 expression in BCC was significantly lower than that of SCC (p < 0.001). CD47 expression levels in SCC and KA were significantly higher than those of NS and AK (p < 0.05). High CD47 expression was significantly associated with the presence of ulceration (p = 0.005) and a deeper level of invasion (p = 0.011) in BCC. In addition, high CD47 expression was significantly associated with the presence of ulceration (p = 0.019) and larger tumor size (p = 0.004) in SCC. CD47 expression was associated with tumorigenesis and tumor progression in non-melanoma skin cancers.

Содержание CD4+-клеток с экспрессией эктонуклеотидаз CD39/CD73 у детей с воспалительными заболеваниями кишечника

The regulation of TNF inhibitor therapy-associated immune responses in inflammatory bowel diseases (IBD) in children remains an urgent problem. The study aimed at analyzing the expression of CD39/CD73 endonucleotidases by different subsets of peripheral blood T cells in children with IBD including Crohn's disease (n = 34) and ulcerative colitis (n = 33) having received TNF inhibitors in comparison with conditionally healthy children (n = 45). Lymphocyte subsets including regulatory T cells (Treg, CD4+CD127lowCD25high), activated T cells (Tact, CD4+CD25+CD127high) and Th17 cells (CD4+CD161+CD3+) were studied by flow cytometry. The results are presented as medians (Me) and quartiles (Q25–Q75). In children with IBD the highest and the lowest relative counts of CD39+ cells were found in Treg and Tact subsets — 31% (15–38) and 4% (1–7), respectively. The highest relative counts of CD73+ cells were found in Tact — 13% (8–21). The CD39 and CD73 expression ratio in patients with IBD, and in the control group as well, depended on particular subset. CD39 expression in Treg, Tact and Th17 of patients with IBD was not age-dependent. Patients with acute Crohn's disease revealed decreased expression of CD39 in Treg compared with the control group (12% (9–23) vs 35% (28–39), respectively; р = 10–6). Patients with Crohn's disease in remission revealed increased expression of CD39 in Treg compared with the acute of the disease (31% (27–40) vs 12% (9–23); р = 9.4 × 10–5). Patients with Crohn's disease in remission revealed no significant differences with the control group apart from reduced expression of CD73 by Treg in Crohn's disease. The results indicate significant association of CD39 and CD73 expression levels in particular subsets of CD4+ cells with the phase of the disease (acute vs remission) and, accordingly, with the anti-TNF regimen efficacy.

Extract of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae] Suppresses Hepatocellular Carcinoma by Inhibiting Angiogenesis.

The extract of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae] (MTE) has shown a significant anti-cancer effect on hepatocellular carcinoma (HCC), but its mechanism remains unclear. In this study, we used transcriptomics methods to investigate the underlying mechanism of MTE against HCC. Both MHCC97H and HepG2 cell lines were treated with MTE. The cell viability and migration were measured using the cell counting kit-8 assay and transwell assay. RNA-sequencing was used to identify differentially expressed genes (DEGs) between HepG2 cells treated with and without MTE. The expression levels of selected DEGs—vascular endothelial growth factor-A (VEGFA), platelet-derived growth factor receptor-β (PDGFRB), and von Willebrand factor (VWF)—were verified by RT-PCR and Western blot. The effect of conditioned medium from HCC cells with MTE treatment (CM-MTE) on blood vessels was observed by tube formation assay of HUVECs and chick chorioallantoic membrane (CAM) assay. A mouse model of HCC patient-derived tumor xenograft (PDX) was established and treated with MTE. The effect of MTE on the growth and angiogenesis of HCC-PDX was analyzed. The results demonstrated that MTE inhibited the viability and migration of HCC cells. RNA-seq showed that MTE treatment downregulated multiple genes associated with metabolism and angiogenesis. The expression levels of VEGFA, VWF, PDGFB, and PDGFRB in HCC cells were significantly suppressed by MTE. Meanwhile, MTE effectively inhibited the tube-forming capability of HUVECs and the angiogenesis of chick CAM. In vivo experiments revealed that the extract reduced tumor volume, inhibited the proliferation of HCC cells, and expanded the necrotic area of the tumor. Immunohistochemical results showed that the expression levels of CD31, PDGFB, VEGF, VWF, and PDGFRB in the HCC-PDX tumor tissues were all downregulated by MTE in a dose-dependent manner. Taken together, MTE could inhibit angiogenesis by repressing the expression of VEGF, VWF, PDGF, and PDGFRB in HCC cells, a mechanism that may enable MTE to counter HCC development.

CD73 (NT5E) Promotes the Proliferation and Metastasis of Lung Adenocarcinoma through the EGFR/AKT/mTOR Pathway.

Introduction Lung cancer is the most common malignant tumor and the main cause of tumor-related death globally. As the 5-year survival rate of lung adenocarcinoma (LUAD) remains low, it is necessary to investigate novel molecular markers and therapeutic targets for LUAD. Materials and Methods The protein expression of CD73 (NT5E) in LUAD specimens was analyzed using immunohistochemistry. Reverse transcription-quantitative PCR and western blot analysis were used to analyze the mRNA and protein expression levels of several genes in LUAD cells. The proliferation of LUAD cells was evaluated using proliferation and colony formation assays and apoptosis analysis. Wound healing and Transwell invasion assays were used to analyze the migration and invasion of the A549 cells, respectively. In addition, overexpression plasmids and small interfering RNAs were used to overexpress or knockdown the expression levels of CD73 in the A549 cell line, respectively. Finally, the interaction between CD73 and EGFR in the A549 cell line was analyzed using immunoprecipitation. Results Our research emphasized the importance of CD73 in the prognosis of LUAD and highlighted it as a potential therapeutic target. We also found that the mRNA and protein expression levels of CD73 are increased in LUAD specimens and cell lines and were associated with a poor prognosis in patients with LUAD. Furthermore, it was revealed that CD73 may promote the proliferation, migration, and invasion of the A549 cell line. Finally, we demonstrated that CD73 could bind epidermal growth factor receptor (EGFR) to further regulate the activation of the AKT/mTOR signaling pathway. Conclusions CD73 promotes LUAD proliferation and metastasis through EGFR/AKT/mTOR axis.

Mechanism of ischemic brain injury repair by endothelial progenitor cell‑derived exosomes.

Ischemic stroke is a refractory disease that seriously endangers human health and life. The main treatment aim of stroke is to alleviate brain injury. The present study aimed to investigate the effects and mechanisms of endothelial progenitor cell (EPC)‑derived exosomes in repairing ischemic brain injury. Sprague‑Dawley rat models of cerebral ischemia‑reperfusion (IR) injury were established by middle cerebral artery occlusion. The IR model rats were then treated with PBS, EPC or exosomes; untreated and Sham rats were used as control. EPCs were obtained from tibias and femurs, and exosomes were isolated from the EPCs and characterized. To measure brain injury, 2,3,5‑triphenyltetrazolium chloride staining was used to measure the infarct area, neurological deficit was scored, hematoxylin and eosin staining was used to examine pathological changes and TUNEL staining was used to quantify apoptosis. Immunofluorescence staining and reverse transcription‑quantitative PCR were used to determine CD31 and VEGF protein and mRNA expressions, respectively, and western blot analysis was used out to measure the protein expression levels of Wnt3a, GSK‑3β and phosphorylated (p)‑GSK‑3β. Compared with rats in the Control and Sham groups, in IR model rats the nerve fibers were slightly necrotic and swollen and the number of nerve cells was reduced. Following EPC treatment, the brain tissue exhibited mild liquefaction and degeneration in the small focus area with mild edema in the stroma. The numbers of nerve cells decreased, and the distribution of nerve cells was not very uniform; proliferation of glial cells was observed. Following treatment with exosomes, the distribution of nerve cells was more uniform with less degeneration and necrosis; the proliferation of glial cells was remarkable. Compared with the Control group, the infarct size, neurological defect score, percentage of apoptotic cells, expression of CD31, VEGF, Wnt3a, and p‑GSK‑3β were significantly higher in the IR model (P<0.05). After EPC and exosome treatments, the infarct size, neurological defect score, percentage of apoptotic cells, expression of Wnt3a, and p‑GSK‑3β were significantly reduced (P<0.05), whereas the mRNA and protein expression levels of CD31 and VEGF were significantly increased (P<0.05). Results from the present study demonstrated that EPC‑derived exosomes may alleviate ischemic injury by inhibiting apoptosis and promoting angiogenesis. These findings suggested that exosomes may have a protective role for nerve cells and may be a potentially effective option for treating stroke. However, human clinical studies are needed to validate these findings from animals.

Peripheral blood derived endothelial colony forming cells as suitable cell source for pre-endothelialization of arterial vascular grafts under dynamic flow conditions

In regenerative medicine, autologous peripheral blood derived endothelial colony forming cells (PB-derived ECFC) represent a promising source of endothelial cells (EC) for pre-endothelialization of arterial tissue engineered vascular grafts (TEVG) since they are readily attainable, can easily be isolated and possess a high proliferation potential. The aim of this study was to compare the phenotype of PB-derived ECFC with arterial and venous model cells such as human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) under dynamic cell culture conditions to find a suitable cell source of EC for pre-endothelialization. In this study PB-derived ECFC were cultivated over 24 h under a high pulsatile shear stress (20 dyn/cm2, 1 Hz) and subsequently analyzed. ECFC oriented and elongated in the direction of flow and expressed similar anti-thrombotic and endothelial differentiation markers compared to HAEC. There were significant differences observable in gene expression levels of CD31, CD34 and NOTCH4 between ECFC and HUVEC. These results therefore suggest an arterial phenotype for PB-derived ECFC both under static and flow conditions, and this was supported by NOTCH4 protein expression profiles. ECFC also significantly up-regulated gene expression levels of anti-thrombotic genes such as krueppel-like factor 2, endothelial nitric oxide synthase 3 and thrombomodulin under shear stress cultivation as compared to static conditions. Dynamically cultured PB-derived ECFC therefore may be a promising cell source for pre-endothelialization of arterial TEVGs.

Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration.

Disruption of the neurovascular unit (NVU) underlies the pathophysiology of various CNS diseases. One strategy to repair NVU dysfunction uses stem/progenitor cells to provide trophic support to the NVU’s functionally coupled and interdependent vasculature and surrounding CNS parenchyma. A subset of endothelial progenitor cells, endothelial colony-forming cells (ECFCs) with high expression of the CD44 hyaluronan receptor (CD44hi), provides such neurovasculotrophic support via a paracrine mechanism. Here, we report that bioactive extracellular vesicles from CD44hi ECFCs (EVshi) are paracrine mediators, recapitulating the effects of intact cell therapy in murine models of ischemic/neurodegenerative retinopathy; vesicles from ECFCs with low expression levels of CD44 (EVslo) were ineffective. Small RNA sequencing comparing the microRNA cargo from EVshi and EVslo identified candidate microRNAs that contribute to these effects. EVshi may be used to repair NVU dysfunction through multiple mechanisms to stabilize hypoxic vasculature, promote vascular growth, and support neural cells.

IHC characteristics of stem cell markers in PDAC with different degrees of differentiation

Introduction. Cancer stem cells (CSCs) play a key role in the development, progression and metastasis of cancer. Cell surface markers, in particular CD44 and EpCAM, are used to identify CSCs. Marker expression patterns depend on the stage of progression and differentiation. Materials and methods. Histopathological and IHC studies of 49 cases of the pancreas ductal adenocarcinoma (PDAC) of G2, G3 degrees of differentiation. Results of IHC reactions with antibodies against CD44 and EpCAM were estimated by photo digital morphometry and were expressed in immunostained cells relative area (%). Results. Adenocarinoma of the pancreas is characterized by a moderate level of expression of cell surface markers CSC - CD44 and EpCAM. The expression level of CD44 in the І gr. - Me = 34.11% (Q1 = 31.03; Q3 = 38.39), in the II gr. - Me = 43.17% (Q1 = 39.83; Q3 = 47.04), significantly higher than in the I gr. (Kruskal-Wallis test p&lt;0.05). The expression level of EpCAM in the I gr. - Ме=26,10% (Q1=20,42; Q3=31,89), in the II gr. - Ме=31,07% (Q1=27,03; Q3=34,00) (p&gt;0,05). In both groups, the relative area of EpCAM was significantly lower than CD44 (Kruskal-Wallis test p&lt;0.05). Correlation analysis (Pearson's ratio) showed a weak negative relationship between markers at G2 (r = -0.17, p&gt;0.05), at G3 - a weak positive (r = + 0.06, p&gt;0.05).

Abstract CT011: NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC)

Abstract Background: Neoadjuvant anti-PD-(L)1 therapy may offer clinical benefit in resectable, early-stage NSCLC. NeoCOAST (NCT03794544) is a global, randomized phase 2 study of the anti-PD-L1 monoclonal antibody (mAb) durvalumab (D) alone or combined with the anti-CD73 mAb oleclumab (O), the anti-NKG2A mAb monalizumab (M), or the anti-STAT3 antisense oligonucleotide danvatirsen (Da) as neoadjuvant therapy. Methods: Pts with previously untreated, cytologically/histologically documented, resectable, Stage I [&amp;gt;2 cm]-IIIA NSCLC and ECOG PS 0-1 were randomized 1:1:1:1 (stratified by lymph node involvement) to receive D 1500 mg IV alone Q4W or combined with O 3000 mg IV Q2W, M 750 mg IV Q2W, or Da 200 mg IV QW, for one 28-day cycle, followed by surgery. Da monotherapy was also given on days 1, 3 and 5 of the week prior to combination treatment. The primary endpoint was investigator-assessed major pathological response (MPR; ≤10% residual viable tumor cells at tumor site and nodes, at surgery). Secondary endpoints were pathological complete response (pCR; no viable tumor cells), safety and tolerability, feasibility of surgery, PK and immunogenicity. Exploratory endpoints included tumor and microbiome biomarkers and blood mRNA signatures. Results: From March 2019 to Sept 2020, 84 pts were randomized and 83 received D (n=26), D+O (n=21), D+M (n=20), or D+Da (n=16). MPR occurred in 11.1% (95% CI, 2.4-29.2), 19.0% (95% CI, 5.4-41.9), 30.0% (95% CI, 11.9-54.3), and 31.3% (95% CI, 11.0-58.7%), respectively. pCR occurred in 3.7% (95% CI, 0.1-19.0), 9.5% (95% CI, 1.2-30.4), 10.0% (95% CI, 1.2-31.7%) and 12.5% (95% CI, 1.6-38.3), respectively. Rates of treatment-related AEs were 34.6% with D, 57.1% with D+O, 50.0% with D+M, and 43.8% with D+Da (grade ≥3 in 0%, 4.8%, 0% and 6.3%, respectively). Most pts (76/83; 91.6%) completed surgery with no significant delay; of the 7 pts unable to, 5 had progressive or stage IV disease. Across all arms, MPR was more common in pts with baseline tumor PD-L1 expression ≥1% vs those with &amp;lt;1%. With D, CD73 expression was correlated with greater residual viable tumor cells at surgery; however, with D+O, high CD73 (≥10% tumor cells) was associated with reduced viable tumor cells. Transcriptome analysis of post-treatment and baseline blood samples showed upregulation of genes involved in B cell activation and antigen presentation in pts with MPR in the D+O arm, and upregulation of genes associated with Tregs in pts with MPR in the D+M arm. Conclusions: One cycle of D + O, M or Da improved MPR and pCR rates vs D alone, with no new safety signals. Responses were associated with baseline tumor PD-L1 and CD73 expression levels. Pts with MPR receiving D+O or D+M had peripheral transcriptomic signatures related to immune cell function. These data warrant further investigation of these agents in resectable NSCLC. Citation Format: Tina Cascone, Rosario García-Campelo, Jonathan Spicer, Walter Weder, Davey Daniel, David Spigel, Maen Hussein, Julien Mazieres, Julio Oliveira, Edwin Yau, Alexander Spira, Raymond Mager, Oday Hamid, Lin-Yang Cheng, Ying Zheng, Jorge Blando, Lara McGrath, Italia Grenga, Yee Soo-Hoo, Rakesh Kumar, Patrick Forde. NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT011.

Abstract 5080: Age- and pregnancy-related impact on soluble immune checkpoint protein profiles in mice as analyzed by a novel multiplex immunoassay

Abstract Immune checkpoint proteins are key regulators of the immune system and drug targets of cancer immunotherapy. There is extensive literature on the use of various types of mouse models for biomarker discovery and drug testing. Only a few studies have examined the association between soluble immune checkpoint proteins and the immune status. Following the development of multiplex immunoassay panels for profiling human co-inhibitory and co-stimulatory immune checkpoint proteins, here we report the development of a novel magnetic bead-based Luminex multiplex assay for the quantitative detection of 28 mouse immune checkpoint proteins and immune regulators in 25 μL of sample (PD-1, PD-L1, PD-L2, CTLA-4/CD152, TIM-3, LAG-3, B7-H2/ICOSL, B7-H3/CD276, BTLA, GITR, HVEM, CD27, CD40, CD80/B7-1, CD226/DNAM-1, 4-1BBL/TNFSF9, CD137/4-1BB, 5’-NT/CD73, BCA-1/CXCL13, CD25/IL-2Rα, Granzyme B, E-cadherin, Galectin-1, Galectin-3, IFNγ, IL-10, TLR-2, and TNFα). Immune function declines with age, while the incidence of cancer rises. In this study, we examined age-related serum samples by quantitative profiling soluble checkpoint proteins from young (age of 8-month-old, n=8) and old-aged mice (48-month-old, n=8) of C57BL/6 strain. A significant age effect was observed on the levels of soluble checkpoint proteins in mouse serum samples tested. Comparing with the young mice, decreased levels of circulating HVEM, Granzyme B, LAG-3, PD-L1, CD73, B7-H2, CD25, and CD27; and increased levels of circulating TIM-3, PD-1, PD-L2, CD40, Galectin-3, IL-10, BCA-1, and TLR-2 were observed in aged C57BL/6 mice (Mann Whitney test, P value &amp;lt;0.05). The immune system also changes during pregnancy. We used the similar multiplex array approach to profile the soluble checkpoint proteins in non-pregnant (n=16) and pregnant CD-1 mice (n=9). Significant decreased levels of soluble LAG-3, B7-H2, B7-H3, CD25, CD27, and Galectin-1; and significant increase of soluble CD137, Galectin-3, and CD73 expression levels were detected in the serum samples of pregnant mice, as compared to the samples from the non-pregnant mice (Mann Whitney test, P value &amp;lt;0.05). Altogether, using bead-based quantitative multiplex analysis, our results demonstrate that the changes on circulating immune checkpoint protein profiles occur in the immunologic adaptations with age and during pregnancy in mice. Citation Format: Wen-Rong Lie, Christine Kornmeier, Ling Zeng, James Hoberg. Age- and pregnancy-related impact on soluble immune checkpoint protein profiles in mice as analyzed by a novel multiplex immunoassay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5080.

The prognostic effect on childhood acute lymphoblastic leukemia of CD34+CD38− expressed in leukemia cells

ABSTRACT Objective Acute lymphoblastic leukemia is the most common malignant disease in children. CD34 and CD38 are expressed in both normal and leukemia cells, but studies of their prognostic associations in childhood acute lymphoblastic leukemia are limited. The aim of this study was to investigate the prognostic effect of CD34 + CD38− leukemia cells in this childhood cancer. Methods From January 2014 to January 2019, children with newly diagnosed acute lymphoblastic leukemia were included in this study and followed up until July 2020. The participants were divided into CD34+ and CD34− groups according to CD34 expression level at diagnosis, and the CD34+ group was further divided into CD34 + CD38− and CD34 + CD38+ subgroups based on CD38 expression level. We tracked clinical biological features, therapeutic outcomes, and other patient data for comparisons. Results The OS and EFS did not differ significantly between the CD34+ and CD34− groups (both P > 0.05). CD34+CD38- group and CD34+CD38+ group were further compared. OS differed significantly between these two groups (χ2 = 3.89, P = 0.048), as did the recurrence rate (χ2 = 5.04, P = 0.025), but EFS did not (χ2 = 1.45, P > 0.05). Survival analysis in patients with recurrence showed a significantly higher OS for the CD34 + CD38+ group compared with the CD34 + CD38− group (χ2 = 5.08, P = 0.024). The CD34+CD38- group and CD34+CD38+ group were matched for propensity scores. When recurrence was compared in the two groups after matching, the difference was statistically significant (P < 0.001). Conclusion CD34+ and CD34− expression does not differ by prognosis in children with acute lymphoblastic leukemia, but CD34 + CD38− may indicate a poor prognosis.

Expression of HIF-1α, ANXA3, CD133 and their associations with clinicopathological parameters in human colon carcinoma.

BackgroundOur previous study found an association between the expression of hypoxia-inducible factor-1alpha (HIF-1α) and annexin A3 (ANXA3) in colon cancer. ANXA3 correlated with expansion of CD133+ tumor cells in hepatocellular carcinoma cancer stem-like cells (CSCs), for which CD133 has been recognized as a typical marker in many cancer cells, including: gastric cancer, lung cancer and colorectal cancer. But the expression and association of HIF-1α, ANXA3 and CD133 in colon cancer has not been reported. The purpose of the present study was to evaluate the correlation among the expression of HIF-1α, ANXA3 and CD133 in human colon cancer and to investigate its clinicopathological parameters.MethodsThe data for 35 patients diagnosed with colon adenocarcinoma in The First Affiliated Hospital of Chongqing Medical University and who had undergone colectomy, tumor and adjacent normal colon tissues were collected. The expressions of HIF-1α, ANXA3, and CD133 were measured by immunohistochemistry in colon cancer and surrounding non-tumor tissues and measured by using a semiquantitative score system. Finally, relationships between HIF-1α, ANXA3, and CD133 immunohistochemical staining and clinicopathologic variables were analyzed using the Fisher’s exact probability test. Associations between the expression levels of HIF-1α, ANXA3, and CD133 were analyzed by the Spearman’s rank correlation.ResultsThe positive rate of expression of HIF-1α in colon cancer and normal colon tissue was 80% (28/35) and 14% (5/35), 77% (27/35) and 20% (7/35) for ANXA3, and 71% (25/35) and 23% (8/35) for CD133, respectively. The coefficient of correlation for the association among HIF-1α, ANXA3 and CD133 showed that the expression of HIF-1α was positively related with ANXA3 and CD133 in colon cancer tissues (r1=0.408, P1=0.015, r2=0.474, P2=0.004) and a positive correlation was observed between the expression of ANXA3 and CD133 (r3=0.409, P3=0.015). Expression of HIF-1α, ANXA3 and CD133 were associated with tumor size, lymphatic metastasis and clinic stage of colon cancer (all P<0.05).ConclusionsHIF-1α, ANXA3 and CD133 were overexpressed in human colon cancer and showed positive correlations among themselves. The expression of HIF-1α, ANXA3 and CD133 were closely related to the size of the tumor, lymphatic metastasis and clinical stage of colon cancer, which indicated that they could be promising biomarkers for the study of colon CSCs and treatment of colon carcinoma.

CD47 expression patterns in thymic epithelial tumors.

8586 Background: Blockade of CD47, an immunoglobulin overexpressed on solid tumor cells that inhibits macrophage phagocytosis, is a promising anti-cancer immunotherapy which has not yet been explored in thymic epithelial tumors (TETs). TETs, including thymomas and thymic carcinomas, are rare tumors with limited immunotherapy treatment options due to the high rates of immune-related adverse effects observed with PD-1/PD-L1 checkpoint inhibitors. This study aimed to examine CD47 protein expression in TETs. Methods: A clinically annotated tissue microarray of 67 TETs consisting of 64 thymomas and 3 thymic carcinomas, as well as 14 thymic controls were included. Each sample with an average of 3 cores was stained for CD47 epithelial expression (rabbit monoclonal antibody SP279, Abcam, USA). Samples were scored for intensity as follows: 0 = none, 1 = weak, 2 = moderate, and 3 = strong. An H-score, defined as intensity x percentage of tumor involved, was also assigned and ranged from 0 to 300. Samples with an intensity score of &lt; 2 or an H-score of &lt; 150 were categorized as CD47low, while the rest as CD47high. Multivariate linear regression analysis accounted for WHO subtype, stage, resection status and presence of paraneoplastic syndrome (Prism 9, Graphpad). Results: Compared to normal thymic tissue, TETs were more frequently CD47 positive and had significantly higher levels of CD47 expression. CD47 was present in 91% of TETs, compared to 64.3% of normal thymus. Importantly, the level of expression was significantly higher in TETs by 16-fold (mean H-score 75.0 vs 4.6, p = 0.003). Among tumors, univariate analyses showed that higher CD47 expression was correlated with a lower stage (p = 0.032) and more complete resection (p = 0.058). A multivariate analysis accounting for these factors showed that CD47 expression by both H-score and intensity were each highly correlated with WHO histology subtype (p = 0.0005; p = 0.0017 respectively) with lower grade subtypes more frequently found in CD47high tumors. The most frequent subtype in CD47high, when compared to CD47low tumors, was AB (61.5% vs 13.7%) and the least frequent was B2 (0% vs 37.3%). Tumors with the highest grade (subtype C, thymic carcinomas) were exclusively CD47low. CD47high tumors were associated with an increased incidence of paraneoplastic syndromes (52.4% vs 12.0%, p = 0.0014). Conclusions: CD47 expression was found in the vast majority of TETs, and in significantly higher levels than normal thymic tissue. Among tumors, those with higher CD47 expression tended to have lower grade and stage, as well as higher frequency of paraneoplastic syndromes. This study is the first to examine CD47 expression in TETs. Given the prevalent expression of CD47 found in TETs and current available CD47 targeted agents, this study lends support for further investigation of this novel therapeutic approach.

CD44 potentiates hepatocellular carcinoma migration and extrahepatic metastases via the AKT/ERK signaling CXCR4 axis.

BackgroundCell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM).MethodsThe expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot.ResultsIn this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs.ConclusionsCD44 could represent a future therapeutic target for EHM.

Mechanism of ketotifen fumarate inhibiting renal calcium oxalate stone formation in SD rats

ObjectivesTo investigate the inhibitory effect of ketotifen fumarate (KFA), a mast cell membrane stabilizer, on renal calcium oxalate stone (CaOx) formation and its possible molecular mechanism. MethodsWe used the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for functional and pathway enrichment analyses of osteopontin (OPN), CD44 and fibronectin (FN). Blood biochemistry, reactive oxygen species ratio (ROS), mast cells, proteins (CD44, OPN and FN) and OPN receptor integrin family genes were detected by ELISA, flow cytometry, immunohistochemistry and RT-QPCR, respectively. ResultsThe crystal area of CaOx in the KFA and Control group was significantly smaller than that in the Model group. The number of activated mast cells, the expression levels of OPN and CD44 in the Control and KFA groups were significantly lower than those in the Model group, and the percentage of ROS in the KFA group was also significantly lower than that in the Model group. The mRNA expression levels of ITGB1, ITGA9, ITGAV and ITGA4 genes in the prominent OPN receptor integrin family increased significantly in the Model group. ConclusionsKetotifen can effectively inhibit the crystal formation of CaOx and reduce the inflammatory response of tissue in SD rats. The mechanism may be to reduce the infiltration and activation of mast cells in renal tissue and down-regulate the expression of OPN, CD44 and FN in renal tubules and renal interstitium. And affect the synthesis of integrins (ITGA9, ITGA4, ITGAV, ITGB1, ITGB3 and ITGB5) and ROS.

The Dibenzyl Isoquinoline Alkaloid Berbamine Ameliorates Osteoporosis by Inhibiting Bone Resorption.

Postmenopausal osteoporosis (PMOP) is a kind of primary osteoporosis that is characterized by decreased bone density and strength. Berbamine is a nonbasic quaternary benzylisoquinoline plant alkaloid that has been widely used in the clinic to treat leukopenia in China. We found that berbamine inhibited RANKL-induced osteoclastogenesis of bone marrow-derived macrophages (BMMs) in vitro, which mainly occurred in the middle phase and late phase. The gene and protein expression levels of osteoclast-related molecules, including CTSK, MMP-9, NFATc1, CD44 and DC-STAMP, were also downregulated by berbamine. In vivo, we treated PMOP mice with berbamine for 8 weeks and found that the extent of osteoporosis was alleviated significantly according to micro-CT scanning, hematoxylin-eosin staining, DC-STAMP immunohistochemical staining and TRAP immunohistochemical staining in the distal femurs of the mice. Our findings demonstrate that berbamine has an inhibitory effect on the osteoclastogenesis of BMMs and can prevent bone loss after ovariectomy in vivo. This study provides evidence that berbamine is a potential drug for the prevention and treatment of PMOP.