Cell CD4 Research Articles

Immunohistochemistry for natural killer cells, endometrial biopsy grade, and fertility in mares with repeated early embryonic death

Early embryonic death (EED) is a potential cause of infertility in mares. Causes include embryo related chromosomal defects or disturbances in the oviductal and uterine environment, but often the cause remains unknown. In humans, most of the endometrial lymphocytes are natural killer (NK) cells (CD56-positive lymphocytes), and their phenotypes, surface receptors, and cytokine production have been associated with early embryonic death and endometrial angiogenesis. (Fuchinoue et al. J Obstet Gynaecol Res. 2016;42:1541-1552). The aims of this study were to 1) describe the relationship between endometrial biopsy grade and future fertility in mares with repeated EED, and 2) to identify and localize equine natural killer cells in the endometrium of mares with repeated EED. The study included 32 formalin-fixed paraffin-embedded endometrial biopsies of mares with a history of two or more EEDs. Immunohistochemistry for the identification of NK-cells was done on endometrial biopsies, using polyclonal NK cell surface receptor antibodies NK2GD and CD56 (Thermo Fisher Scientific, MA, USA) according to manufacturer instructions. Mouse brain and spleen samples functioned as positive controls. Information on biopsies and post-biopsy foaling were collected and analysed with Fisher'sexact test to evaluate dependence between variables (age group, biopsy grade, later abortions, previous parturitions, number of EED, and live foals). A p-value of < 0.05 was considered significant. The biopsies were graded according to Kenney & Doig (In: Morrow, DA, ed. Current Therapy in Theriogenology. 1986; 723–729). Of all mares, 3.1% were grade IIA, 43.8% grade II, 53.1% grade IIB. In total, 43.8%were younger than 13 years and 56.3% 13 years or older, 71.9% had had two EEDs and 28.1% more than two. In total 71.9% of the mares had foaled previously, 68.8% were bred again of which 59.1% foaled a live foal. The age group (< 13 vs. ≥ 13), biopsy grading (grade I-II vs. IIB-III) or later abortions (abortion at over 50 days of pregnancy) were found not to have a significant effect on the later live foal rate. Previous foalings and the number of EEDs (< 3 vs. ≥ 3) seemed to affect the foaling result but failed to have statistical significance in this small study population. Natural killer cells were not identified in the endometrial samples with the selected antibodies, suggesting potential species differences in surface receptors. In conclusion, repeated EED was not related to lower future foaling rates, implying mainly incidental causes of EED in this population.

Association of peripheral memory B cell population maintenance and long term survival after perioperative chemoimmunotherapy in NSCLC (NADIM trial).

8551 Background: In the context of perioperative immunotherapy, it is crucial to personalize adjuvant treatment, identifying which patients should or should not receive post-surgical immunotherapy. Here we describe the changes in B cell peripheral blood immunophenotype during perioperative immunotherapy and its potential to predict disease progression in patients from NADIM I clinical trial (NCT03081689). Methods: Blood samples from 42 patients were obtained at pre-, post-neoadjuvant chemoimmunotherapy (pre-surgery) and at 6 and 12 months of adjuvant IO. Peripheral mononuclear cells were stained with CD19, CD20, CD38, CD24, CD27, CD25, CD10 antibody panel and analyzed by flow cytometry using FlowJo. Statistical analysis in paired samples was performed with Wilcoxon test and differences in parameters’ fold changes between disease status groups (Progression-free at 34.2 months) were determined by Mann-Whitney U tests. Variables cutoffs were stablished using ROC curve analysis. Progression-free survival (PFS) was evaluated using Kaplan-Meier curves and long-rank test. The median follow-up was 38.0 months. Results: Regarding the impact of NADIM complete treatment scheme in patient’s B cells, there was a reduction of memory B cells (CD19+CD20+CD38-CD24+CD27+; p &lt; 0.001) and CD20 levels in total B cells (CD19+CD20+; p = 0.001) (n = 17). Importantly, this decrease occurs in all patients regardless their pathological response or disease progression. However, the magnitude of the memory B cell percentage and CD20 levels reduction was significantly more pronounced in patients whose disease progress thereafter (p = 0.023, p = 0.052 at 6 and 12 months for memory B cells; and p = 0.002, p = 0.009 at 6 and 12 months for CD20 expression). Patients with a relative drop of memory B cells greater than 90% at 6 or 12 months, showed a shorter PFS (p = 0.028, p = 0.007). Likewise, patients with a relative decrease of CD20 expression on B cells higher than 60% at 6 or 12 months had shorter PFS (p = 0.016, p &lt; 0.001). The AUC ROCs to identify patients with disease progression, were 0.824 and 0.885 at 6 and 12 months for memory B cells; and 0.912 and 0.981 at 6 and 12 months for CD20 levels. Moreover, memory B cells percentage and CD20 levels decrease occur between surgery and the first 6 months of adjuvant therapy (n = 21, p &lt; 0.001, p = 0.025). After that, these levels are maintained from 6 to 12 months of adjuvant treatment (n = 20, p = 0.290, p = 0.682), thus the overall reduction is also observed between surgery and 12 months samples (n = 17, p &lt; 0.001, p = 0.001). Conclusions: Perioperative IO produces a significant reduction in memory B cells and CD20 levels. Importantly, stronger decreases might be helpful to predict progression since they are associated to shorter PFS, indicating that the maintenance of the memory B cell peripheral compartment is relevant for long-term survival of these patients.

Comparative expression analysis of immune-related markers in surgically resected lung neuroendocrine neoplasms

BackgroundAlthough immunotherapy has led to a paradigm shift in the treatment of lung cancer, the therapeutic approaches for lung neuroendocrine neoplasms (LNENs) are still limited. Our aim was to explore the immunological landscape and the expression of immune checkpoint markers in LNENs. MethodsSurgically removed tumor samples of 26 atypical carcinoid (AC), 30 large cell neuroendocrine carcinoma (LCNEC) and 29 small cell lung cancer (SCLC) patients were included. The immune phenotype of each tumor type was assessed by using a panel of 15 immune-related markers. As these markers are potentially expressed by immune cells and/or tumor cells, they might serve as putative targets for immunotherapy. Expression patterns were measured by immunohistochemistry and correlated with clinicopathological parameters and prognosis. ResultsUnsupervised hierarchical clustering revealed distinct immunologic profiles across tumor types. Specifically, AC tumors were characterized by high tumor cell CD40 expression and low levels of immune infiltrates whereas SCLC samples had a high CD47 and Inducible T Cell Costimulator (ICOS) expression in tumor cells and immune cells, respectively. High CD70 and CD137 expression by tumor cells as well as elevated expression of CD27, Lymphocyte Activation Gene 3 (LAG3), and CD40 by immune cells were characteristic for LCNEC samples. Overall, SCLC and LCNEC tumors had a more immunogenic phenotype than AC samples. High tumor cell CD47 and CD40 expressions were associated with impaired and improved survival outcomes, respectively. ConclusionsBy providing insights into the widely divergent immunologic profiles of LNENs, our results might serve as a basis for the development of novel immunotherapy-related approaches in these devastating malignancies.

Inherited CD19 Deficiency Does Not Impair Plasma Cell Formation or Response to CXCL12

BackgroundThe human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear.ObjectiveUsing B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model.MethodsFlow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells.ResultsLong-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation.ConclusionCD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.

Reduction in mRNA Expression of the Neutrophil Chemoattract Factor CXCL1 in Pseudomonas aeruginosa Treated Barth Syndrome B Lymphoblasts.

Barth Syndrome (BTHS) is a rare X-linked genetic disease caused by a mutation in the TAFAZZIN gene, which codes for the protein tafazzin involved in cardiolipin remodeling. Approximately 70% of patients with BTHS exhibit severe infections due to neutropenia. However, neutrophils from BTHS patients have been shown to exhibit normal phagocytosis and killing activity. B lymphocytes play a crucial role in the regulation of the immune system and, when activated, secrete cytokines known to attract neutrophils to sites of infection. We examined the expression of chemokine (C-X-C motif) ligand 1 (CXCL1), a known chemotactic for neutrophils, in Epstein-Barr virus transformed control and BTHS B lymphoblasts. Age-matched control and BTHS B lymphoblasts were incubated with Pseudomonas aeruginosa for 24 h and then cell viability, CD27+, CD24+, CD38+, CD138+ and PD1+ surface marker expression and CXCL1 mRNA expression determined. Cell viability was maintained in lymphoblasts incubated in a ratio of 50:1 bacteria:B cells. Surface marker expression was unaltered between control and BTHS B lymphoblasts. In contrast, CXCL1 mRNA expression was reduced approximately 70% (p < 0.05) in untreated BTHS B lymphoblasts compared to control and approximately 90% (p < 0.05) in bacterial treated BTHS B lymphoblasts compared to the control. Thus, naïve and bacterial-activated BTHS B lymphoblasts exhibit reduced mRNA expression of the neutrophil chemoattractant factor CXCL1. We suggest that impaired bacterial activation of B cells in some BTHS patients could influence neutrophil function via impairing neutrophil recruitment to sites of infection and this could potentially contribute to these infections.

Tualang and Kelulut Honey Reduced Lipopolysaccharides-stimulated Inflammatory Responses of Microglia

Microglial activity is crucial in maintaining the central nervous system (CNS) homeostasis. However, prolonged microglial activation have been implicated in the pathology of neurodegenerative diseases. Activated microglia will increase the production of inflammatory cytokines, reactive oxygen species (ROS) and alter their surface marker expression levels. This study used Malaysian honey, Tualang honey (TH), and Kelulut honey (KH) to determine lipopolysaccharide (LPS)-stimulated inflammatory responses of microglia. TH and KH at 0.1% were used in the current study as our findings showed no significant difference in the cell viability between BV2 cells treated with 0.1 % of TH and KH and control group. TH and KH reduced the ROS level significantly by 41.62±1.06% and 49.16±0.63%, respectively, and slightly reduced the expression of co-stimulatory molecules, CD40 and CD11b in LPS-activated BV2 cells. Our preliminary findings proposed an in-depth future study on the anti-inflammatory effect of TH and KH on microglial activation.

Inorganic pyrophosphatase 1 activates the phosphatidylinositol 3-kinase/Akt signaling to promote tumorigenicity and stemness properties in colorectal cancer

Colorectal cancer (CRC) is a common malignant tumor of the human digestive tract. Inorganic pyrophosphatase 1 (PPA1) plays an imperative role in the advancement of malignant tumors, but its function in CRC is ill-defined. In this study, we inspected the functions of PPA1 in CRC. The abundance of PPA1 in CRC tissues was analyzed by utilizing publicly available data from the The Cancer Genome Atlas and Human Protein Atlas project. Cell counting kit-8 assay and 5-ethynyl-2′-deoxyuridine assay were used to evaluate the viability and proliferation of CRC cells. Bioinformatics analysis was used to forecast the PPA1 related genes and signal pathways in CRC. The protein expression was examined by western blot. The xenograft model was implemented to determine the influence of PPA1 in CRC in vivo. Proliferating cell nuclear antigen, CD133, and CD44 contents in xenograft tumors were evaluated by immunohistochemistry. In the present study, we found that the PPA1 content was heightened in CRC, and the diagnostic value of PPA1 in CRC was enormous. Overexpression of PPA1 enhanced cell proliferation and stemness properties in CRC cells, while downregulation of PPA1 had the opposite effects. PPA1 promoted the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Activation of the PI3K/Akt signaling reversed the effect of PPA1 silencing on cell proliferation and stemness properties in CRC cells. Silencing of PPA1 reduced xenograft tumor growth via modulating the PI3K/Akt signaling pathway in vivo. In conclusion, PPA1 promoted cell proliferation and stemness properties in CRC by activating the PI3K/Akt signaling pathway.

Quantifying in vitrocomplement-dependent cytotoxicity (CDC) using advanced flow cytometry

Abstract Complement dependent cytotoxicity (CDC) is a key Fc mediated function of monoclonal antibody (mAb) therapeutics. mAb binding to antigen triggers a complex molecular cascade with sequential recruitment of serum proteins, eventuating in target cell lysis. Here we demonstrate CDC quantification in a streamlined, in vitro, advanced flow cytometry assay. Anti-CD20 mAbs were incubated with target cell lines in 96- or 384-well plates. Human serum (15%) was then added to induce CDC. Cells were labeled with iQue® Cell Membrane Integrity (R/Red) Dye to enable assessment of cell death using the iQue® Advanced Flow Cytometry Platform. Induction of CDC by anti-CD20 mAbs correlated with target cell CD20 expression, with most cell death observed with high-CD20 expressing Ramos cells. Maximal Ramos cell death induced by Rituximab and Truxima® was 68% and 70%, respectively. Comparatively, cell death of mid-CD20 expressing Raji cells was lower at 34% and 24%. No CDC was induced with CD20 negative Jurkat cells. Another anti-CD20-IgG1 mAb was compared against two isotype mutants: IgG1fut (non-fucosylated) and IgG1NQ (non-glycosylated). Their activity was profiled towards three previously described effector functions: antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP) and CDC. CDC induction was comparable between the three mAbs, however their ADCC and ADCP activity differed. The IgG1NQ did not exert ADCP or ADCC activity, whilst the IgG1fut showed more ADCC and less ADCP than the native. These data exemplify the use of advanced flow cytometry to quantify Fc function of mAbs towards a range of effector mechanisms, highlighting the potential to profile libraries of novel therapeutics in minimal time.

Lack of B-cell Depletion with Ocrelizumab (P7-3.006)

Lack of B-cell Depletion with Ocrelizumab (P7-3.006)

Abstract LB163: A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer death in the US by 2030. Besides the late detection and lack of effective treatments, the greatest treatment hurdle to PDAC is the ineffectiveness of patient-derived tumor models. Precision medicine-based treatment decisions require the generation of consistent and trackable complex organoid models that is not achievable by tissue biopsies. Liquid biopsies such as blood, instead, contain circulating biomarkers released by the tumor, among which especially interested in for this study are circulating tumor cells (CTCs). This work develops microfluidic 3D organoids from CTCs to be used as a precision medicine tool to predict and guide the drug response of early stage I-IIB (PDAC) patients. Experimental Methods CTCs are isolated from PDAC patient blood samples using microfluidic Labyrinth following removal of red blood cells with 6% dextran. Collected cells post-isolation are immunofluorescence (IF) stained with DAPI (nuclei), CD45 (leukocyte), pancytokeratin (type I/II cytokeratin 1-8, 10, 14- 16 and 19), Vimentin, and Epithelial cell adhesion molecule (EpCAM) to identify CTCs and characterize their phenotypes. The mold of the microfluidic platform for CTC organoids is fabricated with the standard SU-8 photolithography. The PDMS-based device is tested with a low seeding number of a pancreatic cancer CTC cell line labeled fluorescently. Decellularization of cancer associated fibroblasts (CAFs) generates the in vitro matrix that supplies amino-acid precursors for PDAC metabolism, promoting CTC organoid formation. Serial images of cell growth are obtained throughout the course using a Ti-20 microscope. After each 7-day culture, organoids are dissociated from the device and IF stained using a similar panel as above (with Ki67 marker instead of CD45 for cell proliferation). Preliminary gemcitabine dosage-performance analysis is performed on-chip. Results CTC count from the tested patient panel (n=16) averages 17.0±8.4/mL of blood. CAFs are successfully decellularized on-chip and the embedded pancreatic CTC cell line growth curve is generated. IF staining of the dissociated cells from the organoids confirms the proliferation of the loaded pseudo-CTCs. Conclusion The work above demonstrates the successful isolation of CTCs from PDAC patient blood samples and an engineered microfluidic platform to expand low abundance of cells into organoids. It has a potential to be tested and employed to expand CTCs from PDAC into organoids for drug screening. Citation Format: Yu Zhang, Scott Smith, Minal Nenwani, Valerie Gunchick, Vaibhav Sahai, Sunitha Nagrath, Deepak Nagrath. A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB163.

Extracellular Vesicles in Chronic Lymphocytic Leukemia: Tumor Microenvironment Messengers as a Basis for New Targeted Therapies?

In addition to intrinsic genomic and nongenomic alterations, tumor progression is also dependent on the tumor microenvironment (TME, mainly composed of the extracellular matrix (ECM), secreted factors, and bystander immune and stromal cells). In chronic lymphocytic leukemia (CLL), B cells have a defect in cell death; contact with the TME in secondary lymphoid organs dramatically increases the B cells' survival via the activation of various molecular pathways, including the B cell receptor and CD40 signaling. Conversely, CLL cells increase the permissiveness of the TME by inducing changes in the ECM, secreted factors, and bystander cells. Recently, the extracellular vesicles (EVs) released into the TME have emerged as key arbiters of cross-talk with tumor cells. The EVs' cargo can contain various bioactive substances (including metabolites, proteins, RNA, and DNA); upon delivery to target cells, these substances can induce intracellular signaling and drive tumor progression. Here, we review recent research on the biology of EVs in CLL. EVs have diagnostic/prognostic significance and clearly influence the clinical outcome of CLL; hence, from the perspective of blocking CLL-TME interactions, EVs are therapeutic targets. The identification of novel EV inhibitors might pave the way to the development of novel combination treatments for CLL and the optimization of currently available treatments (including immunotherapy).

Neutrophil-Membrane-Coated Biomineralized Metal-Organic Framework Nanoparticles for Atherosclerosis Treatment by Targeting Gene Silencing.

Antisense oligonucleotides (ASOs) are promising tools for gene silencing and have been exploited as therapeutics for human disease. However, delivery of therapeutic ASOs to diseased tissues or cells and subsequent escape from the endosomes and release of ASO in the cytosol remain a challenge. Here, we reported a neutrophil-membrane-coated zeolitic imidazolate framework-8 (ZIF-8) nanodelivery platform (AM@ZIF@NM) for the targeted transportation of ASOs against microRNA-155 (anti-miRNA-155) to the endothelial cells in atherosclerotic lesions. Neutrophil membrane could improve plaque endothelial cells targeting through the interaction between neutrophil membrane protein CD18 and endothelial cell membrane protein intercellular adhesion molecule-1 (ICAM-1). The ZIF-8 "core" provided high loading capacity and efficient endolysosomal escaping ability. Delivery of anti-miR-155 effectively downregulated miR-155 expression and also saved the expression of its target gene BCL6. Moreover, RELA expression and the expression of its downstream target genes CCL2 and ICAM-1 were correspondingly reduced. Consequently, this anti-miR-155 nanotherapy can inhibit the inflammation of atherosclerotic lesions and alleviate atherosclerosis. Our study shows that the designed biomimetic nanodelivery system has great application prospects in the treatment of other chronic diseases.

Abstract 606: Cis-B7:CD28 interactions at invaginated synaptic membranes activate CD28 and promote T cell function

Abstract Absence of adequate CD28 costimulation is a common feature of dysfunctional T cells in cancer. However, although augmentation of CD28 signaling is well known to be essential for restoring dysfunctional T cell immune responses upon immune checkpoint blockade treatment, it is unclear how CD28 in tumor-infiltrated T cells is activated in the absence of corresponding ligands on cancer. In current dogma, the T cell CD28 can be activated in trans by CD80 (B7-1) or CD86 (B7-2) expressed on professional antigen presenting cells (APCs) enriched in secondary lymphoid organs. Notably, besides professional APCs, B7 ligands are also displayed by T cells, but their functions are unclear. Here we report that B7 ligands expressed on T cells interact with CD28 in cis at membrane invaginations of the immunological synapse, as a result of phosphoinositide-3-kinase (PI3K) mediated membrane remodeling. Cis-B7:CD28 interactions triggered CD28 signaling through protein-kinase-C-theta (PKCθ) and promoted T cell survival, migration and cytokine production. In a B7 deficient tumor model grafted with primed CD8+ T cells, blockade of T cell intrinsic B7:CD28 interactions accelerated tumor growth and decreased intratumoral T cells. Thus, B7 ligands on T cells can evoke cell autonomous CD28 costimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality. Citation Format: Yunlong Zhao, Christine Caron, Ya-Yuan Chan, Calvin K. Lee, Xiaozheng Xu, Jack D. Bui, Enfu Hui. Cis-B7:CD28 interactions at invaginated synaptic membranes activate CD28 and promote T cell function [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 606.

Abstract 2103: Immunophenotyping of extracellular vesicles isolated from lymphatic exudate identifies CD24 as a marker for lymph node metastases in oral cavity squamous cell carcinoma

Abstract Background: Squamous cell carcinoma of the oral cavity (OSCC) is debilitating and fatal with a high risk of lymph node metastases. One of the key problems is that currently there is no robust molecular biomarker to determine whether the tumor has spread or is at risk of spreading to the regional lymph nodes, the first echelon that OSCC spreads to. Extracellular vesicles (EVs) are functional nanoparticles that are bound by natural protein-lipid bilayers. They are deliberately packaged and proactively secreted by all types of cells and serve as intercellular communication vehicles. Tumors release an excess of EVs which are loaded with the molecular and genetic cargo of the parental tumor cells. Methods: Following ethical approval and written informed consent, pre-surgical plasma as well as matched post-surgical lymphatic drainage fluid were collected from 20 OSCC patients (nodal disease n=6, no node n=14), and control plasma was also collected from healthy donors. EVs were isolated by ultracentrifugation, quantified and characterized by nanoparticle tracking analysis, transmission electron microscopy, and western blot. Immune profiling of 37 EV surface markers was determined using flow cytometry with the MACSPlex Exosome kit. Results: Lymphatic exudate was greatly enriched in EVs in comparison to plasma. Common EV tetraspanins, platelet markers, cancer-related proteins, as well as characteristic markers of immune cells were detected in both plasma and lymphatic exudate EVs based on normalized median fluorescence intensity (nMFI). The presence of OSCC led to an altered composition of blood cell-derived EVs. Elevated levels of CD3, CD25, and CD86, and cancer stem cell marker CD133 were observed in the plasma EVs. However, there were no significant changes in EV markers in plasma between patients with and without lymph node metastasis. Notably, EVs isolated from plasma and lymphatic exudate revealed completely divergent signatures of EV surface markers. Among them, cancer-related protein CD24 was found to be the most highly expressed EV marker in lymphatic exudate from patients with nodal disease (nMFI 1.28±0.50 versus 0.84±0.32, p&amp;lt;0.05) and likewise correlated with advanced disease stages (nMFI 1.17±0.40 versus 0.70±0.27, p&amp;lt;0.05). Conclusions: Plasma-derived EVs are promising non-invasive biomarkers for cancer detection. This study shows that lymphatic exudate is a better source of EV biomarkers due to its high abundance of tumor-related factors directly from draining lymph nodes. In addition, CD24, as reported, is indicative of an immunosuppressive tumor microenvironment and it may serve as a predictor for lymph node metastases and progression of OSCC. These findings shed new light on the understanding of OSCC metastases and provide impetus for consideration of CD24-based liquid biopsy testing and possibly novel immunotherapy. Citation Format: Xinyu Qu, Leanne Lee Leung, Cherrie Wing Kei Ng, Hiu Ching Chan, Zigui Chen, Katie Meehan, Xianhai Zeng, Jason Ying Kuen Chan. Immunophenotyping of extracellular vesicles isolated from lymphatic exudate identifies CD24 as a marker for lymph node metastases in oral cavity squamous cell carcinoma [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 2103.

Abstract 6132: Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells

Abstract Stomach cancer ranks fifth and fourth for the incidence and mortality rate for cancer worldwide, respectively. In recent years, several inhibitors with specificity to one or more members of human epidermal growth factor receptor (HER) family have been approved for the treatment of patients with different cancer types. Of these, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients and with tumor heterogeneity being one contributing factor. In this study, we investigated the effect of various types of targeted agents on the growth in vitro of a panel of human stomach cancer cells (HSCCLs) and the impact of stomach cancer proliferation rate on the anti-tumor activities of these agents. Moreover, we investigated the cell surface expression of the HER family members, other biomarkers such as C-Met, Alk-7 and cancer stem cell makers CD44 and CD133 by flow cytometry and the effect various targeted agents on tumor migration using Incucyte. Of the 18 agents examined, the CDK 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at IC50 values between 1nM to 9nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual EGFR/HER2 TKI lapatinib and the EGFR specific TKI erlotinib in inhibiting the growth of HSCCLs. Interestingly, the HER-2 overexpressing cells lines NCI-N87 (Mean Fluorescence Intensity =596) was most sensitive to the HER inhibitors. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs and with the IC50 values ranging from 2nM to 7uM. Interestingly, many of these agents were more effective in inhibiting the growth of HSCCLs when proliferating at slower rate. Of the agent examined, neratinib, afatinib, dinacicilib, dasatinib, stattic and miransertib also inhibited the migration of stomach cancer cells. Finally, treatment with a combination of afatinib with dinaciclib, capmatinib, dasatinib, stattic, ponatinib or miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. As stomach cancer cells are heterogenous in nature, our results support further research on the therapeutic potential of the CDK dinaciclib in combination with a pan-HER inhibitor in stomach cancer. Citation Format: Tina Al-Janaby, Narmin Nahi, Said A. Khelwatty, Alan Seddon, Izhar Bagwan, Helmout Modjtahedi. Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells. [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 6132.

NF-kB and the CLL microenvironment.

Chronic lymphocytic leukemia (CLL) is the most prevalent type of leukemia in the western world. Despite the positive clinical effects of new targeted therapies, CLL still remains an incurable and refractory disease and resistance to treatments are commonly encountered. The Nuclear Factor-Kappa B (NF-κB) transcription factor has been implicated in the pathology of CLL, with high levels of NF-κB associated with disease progression and drug resistance. This aberrant NF-κB activation can be caused by genetic mutations in the tumor cells and microenvironmental factors, which promote NF-κB signaling. Activation can be induced via two distinct pathways, the canonical and non-canonical pathway, which result in tumor cell proliferation, survival and drug resistance. Therefore, understanding how the CLL microenvironment drives NF-κB activation is important for deciphering how CLL cells evade treatment and may aid the development of novel targeting therapeutics. The CLL microenvironment is comprised of various cells, including nurse like cells, mesenchymal stromal cells, follicular dendritic cells and CD4+ T cells. By activating different receptors, including the B cell receptor and CD40, these cells cause overactivity of the canonical and non-canonical NF-κB pathways. Within this review, we will explore the different components of the CLL microenvironment that drive the NF-κB pathway, investigating how this knowledge is being translated in the development of new therapeutics.

Collagen XII regulates stromal wound closure

The role of collagen XII in regulating injury repair and reestablishment of corneal function is unknown. This manuscript aims to investigate the role(s) of collagen XII in the repair of incisional and debridement injuries in an adult mouse model. Two different types of injury in wild type and Col12a1−/− corneas were created to investigate the effects of collagen XII -in wound repair and scar formation-by using clinical photographs, immunohistology, second harmonic generation imaging and electron microscopy. Results showed that collagen XII is a regulator of wound closure after incisional injuries. Absence of collagen XII retarded wound closure and the wound healing process. These findings show that collagen XII regulates fibrillogenesis, CD68 cell lineage infiltration, and myofibroblast survival following injury. In vitro studies suggest that collagen XII regulates deposition of an early and provisional matrix by interacting with two proteins regulating early matrix deposition: fibronectin and LTBP1(latent transforming growth factor β binding protein 1). In conclusion, collagen XII regulates tissue repair in corneal incisional wounds. Understanding the function of collagen XII during wound healing has significant translational value.

Profiling of Circulating Tumor Cells for Screening of Selective Inhibitors of Tumor-Initiating Stem-Like Cells.

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

Exosomes from CD133+ human urine-derived stem cells combined adhesive hydrogel facilitate rotator cuff healing by mediating bone marrow mesenchymal stem cells

The inadequate regeneration of natural tissue (mainly fibrocartilage) between tendon and bone during rotator cuff (RC) repair results in an unsatisfactory quality of RC healing. Cell-free therapy based on stem cell exosomes is a safer and more promising approach for tissue regeneration. Here, we investigated the effect of exosomes from human urine-derived stem cells (USCs) and their subpopulations (CD133+USCs) on RC healing. USCs were isolated from urine and sorted by flow cytometry to obtain CD133+ urine-derived stem cells (CD133+ USCs). Urine-derived stem cell exosomes (USC-Exos) and CD133+ urine-derived stem cell exosomes (CD133+ USC-Exos) were subsequently isolated from the cell supernatant and identified by transmission electron microscopy (TEM), particle size analysis, and Western blot. We performed in vitro functional assays to evaluate the effects of USC-Exos and CD133+ USC-Exos on human bone marrow mesenchymal stem cells (BMSCs) proliferation, migration, osteogenic differentiation, and chondrogenic differentiation. In vivo experiments were performed by local injection of exosome-hydrogel complexes for the treatment of RC injury. The effects of CD133+ USC-Exos and USC-Exos on RC healing were assessed from imaging, histological, and biomechanical tests. CD133+ USCs were positive for CD29, CD44, CD73, CD90, CD133, but negative for CD34 and CD45. Differentiation ability test results showed that both USCs and CD133+ USCs had the potential for osteogenic, chondrogenic, and adipogenic differentiation, but CD133+ USCs had stronger chondrogenic differentiation ability. CD133+ USC-Exos and USC-Exos could be efficiently taken up by BMSCs and promote their migration, osteogenic and chondrogenic differentiation. However, CD133+ USC-Exos could promote the chondrogenic differentiation of BMSCs more than USC-Exos. Compared with USC-Exos, CD133+ USC-Exos could promote the healing of bone-tendon interface (BTI) more effectively, which might be related to its ability to promote the differentiation of BMSCs into chondroblasts. Although the two exosomes exhibited the same effect in promoting subchondral bone repair in BTI, the CD133+ USC-Exos group had higher histological scores and stronger biomechanical properties. CD133+ USC-Exos hydrogel complex may become a promising therapeutic approach for RC healing based on stem cell exosomes. This is the first study to assess the specific role of CD133+ USC-Exos in RC healing which may be related to the activation of BMSCs by CD133+ USC-Exos towards chondrogenic differentiation. Further, our study provides a reference for possible future treatment of BTI by applying CD133+ USC-Exos hydrogel complex.

Exposure to Secreted Bacterial Factors Promotes HIV-1 Replication in CD4+ T Cells.

Microbial translocation is associated with systemic immune activation in HIV-1 disease. Circulating T cells can encounter microbial products in the bloodstream and lymph nodes, where viral replication takes place. The mechanisms by which bacteria contribute to HIV-associated pathogenesis are not completely deciphered. Here, we examined how bacteria may impact T cell function and viral replication. We established cocultures between a panel of live bacteria and uninfected or HIV-1-infected activated peripheral blood CD4-positive (CD4+) T cells. We show that some bacteria, such as Escherichia coli and Acinetobacter baumannii, sustain lymphocyte activation and enhance HIV-1 replication. Bacteria secrete soluble factors that upregulate CD25 and ICAM-1 cell surface levels and activate NF-κB nuclear translocation. Our data also demonstrate that CD25 polarizes at the virological synapse, suggesting a previously unappreciated role of CD25 during viral replication. These findings highlight how interactions between bacterial factors and T cells may promote T cell activation and HIV-1 replication. IMPORTANCE People living with HIV suffer from chronic immune activation despite effective antiretroviral therapy. Early after infection, HIV-1 actively replicates in the gut, causing the breakage of the intestinal epithelial barrier and microbial translocation. Microbial translocation and chronic immune activation have been proven linked; however, gaps in our knowledge on how bacteria contribute to the development of HIV-related diseases remain. Whether T cells in the peripheral blood react to bacterial products and how this affects viral replication are unknown. We show that some bacteria enriched in people living with HIV activate T cells and favor HIV-1's spread. Bacteria release soluble factors that cause the overexpression of cellular molecules related to their activation state. T cells overexpressing these molecules also replicate HIV-1 more efficiently. These results help us learn more about how HIV-1, T cells, and bacteria interact with each other, as well as the mechanisms behind chronic immune activation.