Levels Of CD44 Research Articles

High levels of soluble CD73 unveil resistance to BRAF inhibitors in melanoma cells

Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment.

Gene expression profiling in porocarcinoma indicates heterogeneous tumor development and substantiates poromas as precursor lesions.

Malignant sweat gland tumors are rare, with the most common being eccrine porocarcinoma (EP). Approximately 18% of benign eccrine poroma (EPO) transit to EP. Previous research has provided first insights into the mutational landscape of EP. However, only few studies have performed gene expression analyses. This leaves a gap in the understanding of EP biology and potential drivers of malignant transformation from EPO to EP. Transcriptome profiling of 23 samples of primary EP and normal skin (NS). Findings from the EP samples were then tested in 17 samples of EPO. Transcriptome profiling revealed diversity in gene expression and indicated biologically heterogeneous sub-entities as well as widespread gene downregulation in EP. Downregulated genes included CD74, NDGR1, SRRM2, CDC42, ANXA2, KFL9 and NOP53. Expression levels of CD74, NDGR1, SRRM2, ANXA2, and NOP53 showed a stepwise-reduction in expression from NS via EPO to EP, thus supporting the hypothesis that EPO represents a transitional state in EP development. We demonstrated that EP is molecularly complex and that evolutionary trajectories correspond to tumor initiation and progression. Our results provide further evidence implicating the p53 axis and the EGFR pathway. Larger samples are warranted to confirm our findings.

IMMU-20. INNATE IMMUNE EVASION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cell surface proteome analysis of Group3-MB cell lines after direct MYC inhibition to identify changes in surface proteins and sensitivity to macrophage-mediated phagocytosis. METHODS We had previously demonstrated the preferential activity of HDACi (CI994) in MYC-driven medulloblastoma. CI-994, showed significant cell viability reduction mediated by reduction in the MYC (mRNA and protein), induction of apoptosis in MYC-driven medulloblastoma. In this study, we directly inhibited MYC using MYCi975 and OMO-MYC and carried out a cell surface proteomic analysis on Group3-MB cell lines to reveal CD24 and CD59 as potential immune evasion markers. While CD24 is a potential don’t eat me signal, CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that acts as an inhibitor to the membrane attack complex and protects cells from CDC. In certain tumors, CD59 expression is enhanced, posing a significant obstacle to treatment by hindering effective monoclonal antibody-induced CDC. We use inhibitors to CD24 and CD59 to confirm their role as innate immune evasion proteins in Group 3MB. RESULTS Meta-analysis of published datasets reveals CD24 and CD59 as strong prognostic indicators in Group3-MB as well as within the Group3g-subtype. Inhibition of MYC reduces surface levels of CD24, increasing macrophage-mediated phagocytosis. The use of a blocking anti-CD24 mAb significantly enhanced macrophage-mediated phagocytosis. Furthermore, a significant reduction in surface expression was observed in CD59. Blocking CD59 mAb significantly enhanced anti-CD47 mAb mediated phagocytosis. Combined treatment of anti-CD24 and anti-CD47 significantly enhanced the survival of Grp3-MB tumor-bearing mice.

Comparative characterisation of an ecto-5'-nucleotidase (CD73) in non-tumoral MCF10-A breast cells and triple-negative MDA-MB-231 breast cancer cells.

Ecto-5'-nucleotidase (CD73) hydrolyses 5'AMP to adenosine and inorganic phosphate. Breast cancer cells (MDA-MB-231) express high CD73 levels, and this enzyme has been found to play a tumour-promoting role in breast cancer. However, no studies have sought to investigate whether CD73 has differential affinity or substrate preferences between noncancerous and cancerous breast cells. In the present study, we aimed to biochemically characterise ecto-5'-nucleotidase in breast cancer cell lines and assess whether its catalytic function and tumour progression are correlated in breast cancer cells. The results showed that compared to nontumoral breast MCF-10A cells, triple-negative breast cancer MDA-MB-231 cells had a higher ecto-5'-nucleotidase expression leveland enzymatic activity. Although ecto-5'-nucleotidase activity in the MDA-MB-231 cell line showed no selectivity among monophosphorylated substrates, 5'AMP was preferred by the MCF-10A cell line. Compared to the MCF-10A cell line, the MDA-MB-231 cell line has better hydrolytic ability, lower substrate affinity, and high inhibitory potential after treatment with a specific CD73 inhibitor α,β‑methylene ADP (APCP). Therefore, we demonstrated that a specific inhibitor of the ecto-5-nucleotidase significantly reduced the migratory and invasive capacity of MDA-MB-231 cells, suggesting that ecto-5-nucleotidase activity might play an important role in metastatic progression.

Functional multiomics reveals genetic and pharmacologic regulation of surface CD38 in multiple myeloma

AbstractCD38 is a surface ectoenzyme expressed at high levels on myeloma plasma cells and is the target for the monoclonal antibodies (mAbs) daratumumab and isatuximab. Pretreatment CD38 density on tumor cells is an important determinant of mAb efficacy. Several small molecules have been found to increase tumor surface CD38, with the goal of boosting mAb efficacy in a cotreatment strategy. Numerous other CD38-targeting therapeutics are currently in preclinical or clinical development. Here, we sought to extend our currently limited insight into CD38 surface expression by using a multiomics approach. Genome-wide CRISPR interference screens integrated with patient–centered epigenetic analysis confirmed known regulators of CD38, such as RARA, while revealing XBP1 and SPI1 as other key transcription factors governing surface CD38 levels. CD38 knockdown followed by cell surface proteomics demonstrated no significant remodeling of the myeloma “surfaceome” after genetically induced loss of this antigen. Integrated transcriptome and surface proteome data confirmed high specificity of all-trans retinoic acid in upregulating CD38, in contrast to the broader effects of azacytidine and panobinostat. Finally, unbiased phosphoproteomics identified inhibition of MAP kinase pathway signaling in tumor cells after daratumumab treatment. Our work provides a resource to design strategies to enhance efficacy of CD38-targeting immunotherapies in myeloma.

Fibulin-4 as a potential extracellular vesicle marker of fibrosis in patients with cirrhosis.

Chronic liver injury leads to decreased liver function and increased fibrosis. Fibrosis is not only associated with the development of portal hypertension and carcinogenesis, but with the occurrence of events and a poor prognosis, highlighting the importance of non-invasive fibrosis assessment in patients. In the present study, we searched for markers related to liver fibrosis via proteomic analysis of small extracellular vesicles (sEVs). In the discovery cohort, proteomic analysis was carried out in the sEVs extracted from the sera of 5 patients with decompensated cirrhosis, 5 patients with compensated cirrhosis, and 5 controls without liver disease. Interestingly, in this cohort, fibulin-4 was significantly associated with cirrhosis while in the validation cohort [formed by 191 patients: 7 patients without disease, 16 patients without liver disease (other diseases), 38 patients with chronic liver disease (CLD), 75 patients with cirrhosis of Child-Pugh class A (36 without hepatocellular carcinoma [HCC], 29 with HCC), and 65 patients with cirrhosis of Child-Pugh class B-C (39 without HCC, 26 with HCC)], fibulin-4/CD9 levels increased with cirrhosis progression. Furthermore, the fibulin-4/CD9 ratio was significantly higher in patients with varices. Immunostaining also revealed strong fibulin-4 expression in cholangiocytes within the fibrous areas and mesothelial cells in liver tissue blood vessels. Taken together, our results suggest that fibulin-4, essential for lysyl oxidase activation, might be a new liver fibrosis marker found in the sEVs of patients with cirrhosis.

The potential mechanism of HIF-1α and CD147 in the development of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis, and the outcomes of common therapy were not favorable. The samples of 84 patients with TNBC and 40 patients with breast fibroadenoma were collected in the pathology department specimen library of our hospital. The prognosis of patients was obtained through outpatient follow-up information, telephone and WeChat contacts, and medical records. The mRNA expression was analyzed using bioinformation and quantitative real-time polymerase chain reaction (qPCR). The protein expression was determined by hematoxylin-eosin staining and immunohistochemical staining. The results of survival analysis were visualized using Kaplan-Meier curves. The immunohistochemical staining showed that hypoxia-inducible factor-1alpha (HIF-1α) was mainly distributed in the nucleus and cytoplasm, while CD147 is mainly distributed in cell membrane and cytoplasm. The qPCR results exhibited that the expression level of HIF-1α and CD147 in TNBC tissue was significantly higher than that in breast fibroadenoma tissue. The expression of HIF-1α was related to the histological grade and lymph node metastasis in TNBC, and the expression of CD147 was related to Ki-67, histological grade and lymph node metastasis. There was a positive relationship between the expression of CD147 and HIF-1α. The upregulated expression of CD147 was closely related to the poor prognosis of OS in TNBC. CD147 could be a biomarker for the prognosis of TNBC and closely related to the expression of HIF-1α.

Human-derived Tumor-On-Chip model to study the heterogeneity of breast cancer tissue

One of the leading causes that complicate the treatment of some malignancies, including breast cancer, is tumor heterogeneity. In addition to inter-heterogeneity and intra-heterogeneity of tumors that reflect the differences between cancer cell characteristics, heterogeneity in the tumor microenvironment plays a critical role in tumor progression and could be considered an overlooked and a proper target for the effective selection of therapeutic approaches. Due to the difficulty of completely capturing tumor heterogeneity in conventional detection methods, Tumor-on-Chip (TOC) devices with culturing patient-derived spheroids could be an appropriate alternative. In this research, human-derived spheroids from breast cancer individuals were cultured for 6 days in microfluidic devices. To compare TOC data with conventional detection methods, immunohistochemistry (IHC) and ITRAQ data were employed, and various protein expressions were validated using the transcriptomic databases. The behavior of the spheroids in the collagen matrix and the cell viability were monitored over 6 days of culture. IHC and immunocytochemistry (ICC) results revealed that inter and intra-heterogeneity of tumor spheroids are associated with HER2/ER expression. HER2 expression levels revealed a more important biomarker associated with invasion in the 3D culturing of spheroids. The expression levels of CD163 (as a marker for Ma2 macrophages) and CD44 (a marker for cancer stem cells (CSCs)) were also evaluated. Interestingly, the levels of M2a macrophages and CSCs were higher in triple-negative specimens and samples that showed higher migration and invasion. Cell density and extracellular matrix (ECM) stiffness were also important factors affecting the migration and invasion of the spheroids through the matrix. Among these, rigid ECM revealed a more crucial role than cell density. To sum up, these research findings demonstrated that human-derived spheroids from breast cancer specimens in microfluidic devices provide a dynamic condition for predicting tumor heterogeneity in patients, which can help move the field forward for better and more accurate therapeutic strategies.

Autologous Platelet-Rich Gel Accelerates Diabetic Wound Healing Through Inhibition of Ferritinophagy.

Aims: The objective was to examine the efficacy of autologous platelet-rich gel (APG) in treating diabetic wound and investigate the association between APG and ferritinophagy. Methods: A total of 32 patients with diabetic foot (DF) and Wagner grade 1 to 2 were included. Within the APG group, individuals with DF received weekly APG treatment. In the non-APG group, DF patients received daily dressing changes. Flow cytometry quantified the proportion of endothelial progenitor cells (EPCs) in peripheral blood on days 0 and 10. The diabetic rat model was induced using Streptozotocin. Two circular skin wounds were created on the backs of rats. The normal glucose group received daily dressing changes on the wound. In the diabetic group, the left wound underwent daily dressing changes, whereas the right wound was treated with APG once a week. CD34 levels were tested 7 days after the skin damage. The levels of glutathione peroxidase 4 (GPX4), Nuclear Receptor Coactivator 4 (NCOA4), Light chain 3 (LC3), and Masson staining were quantified on 14 days. The wound area and wound healing rate were separately measured at 0 and 14 days after the injury, regardless of DF patients or diabetic rats. Results: The wound healing rate was higher in the APG group than in the non-APG group, regardless of DF patients or diabetic rats. The APG group had a greater ΔEPCs% in DF patients than the non-APG group. Regarding rat experiment, the APG group exhibited lower levels of NCOA4, and LC3 expressions and a shorter wound healing time. However, the APG group showed higher levels of CD34 expression, GPX4 protein, and collagen fibers than the non-APG group. Conclusions: Autologous platelet-rich gel accelerated the wound healing rate in diabetic populations and rats. Autologous platelet-rich gel promoted EPCs counts, collagen fiber volume, and vessel numbers. Autologous platelet-rich gel decreased LC3 and NCOA4 expression, but increased GPX4 protein expression. The possible mechanism was the inhibition of ferritinophagy.

Mitophagy and cancer: role of BNIP3/BNIP3L as energetic drivers of stemness features, ATP production, proliferation, and cell migration.

Mitophagy is a selective form of autophagy which permits the removal of dysfunctional or excess mitochondria. This occurs as an adaptative response to physiological stressors, such as hypoxia, nutrient deprivation, or DNA damage. Mitophagy is promoted by specific mitochondrial outer membrane receptors, among which are BNIP3 and BNIP3L. The role of mitophagy in cancer is being widely studied, and more specifically in the maintenance of cancer stem cell (CSC) properties, such as self-renewal. Given that CSCs are responsible for treatment failure and metastatic capacity, targeting mitophagy could be an interesting approach for CSC elimination. Herein, we describe a new model system to enrich sub-populations of cancer cells with high basal levels of mitophagy, based on the functional transcriptional activity of BNIP3 and BNIP3L. Briefly, we employed a BNIP3(L)-promoter-eGFP-reporter system to isolate cancer cells with high BNIP3/BNIP3L transcriptional activity by flow cytometry (FACS). The model was validated by using complementary lysosomal and mitophagy-specific probes, as well as the mitochondrially-targeted red fluorescent protein (RFP), namely mt-Keima. High BNIP3/BNIP3L transcriptional activity was accompanied by increases in i) BNIP3/BNIP3L protein levels, ii) lysosomal mass, and iii) basal mitophagy activity. Furthermore, cancer cells with increased BNIP3/BNIP3L transcriptional activity exhibited CSC features, such as greater mammosphere-forming ability and high CD44 levels. To further explore the model, we also analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, directly demonstrating that BNIP3(L)-high cells were more metabolically active, proliferative, migratory, and drug-resistant, with elevated anti-oxidant capacity. Therefore, high levels of basal mitophagy appear to enhance CSC features.

Effect of AURKA targeting on nuclear PD-L1 and the efficacy of immune checkpoint inhibitors in triple-negative breast cancer.

e13153 Background: Aurora-A mitotic kinase (AURKA) plays a role in Triple Negative Breast Cancer (TNBC) progression through activation of epithelial to mesenchymal transition (EMT)-mediated cancer cell plasticity. Cancer cells that undergo EMT acquire a CD44high/CD24low and/or ALDHhigh cancer stem-like phenotype (Cancer Stem Cells) characterized by intrinsic drug resistance. Cancer Stem Cells (CSCs) shows high PD-L1 expression that induces tumor immune escape through CD8+ T-cells exhaustion. Significantly, “undraggable” nuclear PD-L1 may play a key role in induction of immune therapy resistance and cancer cell plasticity. Methods: (1) TNBC Models: MDA-MB 231, unique MDA-MB 231/LM Durvalumab resistant (DUV-R) and TNBC-M40 cells established from metastatic PDXs; (2) In Vitro Studies: TNBC cells were treated with the AURKA inhibitor alisertib (50nM). PD-L1 expression was evaluated by immunoblotting. Apoptosis was measured by immunofluorescence using Cleaved-PARP antibodies. To assess stemness capacity, TNBC cells were cultured under non-adherent conditions to form mammospheres. ALDH activity (functional stemness marker) was measured using the Aldefluor Kit. (3) In Vivo Studies: 1x106 MDA-MB 231/LM cells were injected into the mammary fat pad of humanized NSG-CD34+ female mice. Animals were treated with anti-PD-L1 ICI atezolizumab (20 mg/Kg) and alisertib (25 mg/Kg) as monotherapy or in combination. 1x106 MDA-MB 231/LM cells were also injected into the mammary fat pad of humanized NSG-CD34+ female mice and treated with durvalumab (20 mg/Kg) to develop durvalumab-resistant models. PD-L1, CD44, Vimentin expression and tumor infiltration of CD8+ T-cells were assessed by immunofluorescence. Results: Treatment of MDA-MB 231/LM xenografts with alisertib decreased the levels of CD44 and PD-L1 expression and increased CD8+ T-cells tumor infiltration. Because PD-L1 genetic targeting enhanced in vitro alisertib-induced apoptosis, MDA-MB 231/LM xenografts were treated with alisertib and the anti-PD-L1 ICI atezolizumab. Com- bination of atezolizumab and alisertib resulted in the reduced expression of the EMT marker vimentin that was linked to lack of organ metastasis. Combination of atezolizumab and alisertib also induced a significant reduction of ALDHHigh CSC in ex-vivo 3D-Organoids established from TNBC-M40 cells. MDA-MB 231/LM DUV-R cells showed increased nuclear PD-L1 compared to parental cells. The combination of alisertib with Immune Checkpoint Inhibitors (ICIs) induced apoptosis of MDA-MB 231/LM DUV-R cells through down-regulation of intra-tumoral nuclear PD-L1 expression. Conclusions: This study provides innovative pre-clinical rationale for combining AURKA inhibitors with ICIs to reduce “ undruggable” nuclear PD-L1 expression and impair cancer cell plasticity of metastatic TNBCs that currently have limited effective therapeutic options.

Graves-PCD: protocol for a randomised, dose-finding, adaptive trial of the plasma cell-depleting agent daratumumab in severe Graves’ disease

IntroductionSevere Graves’ disease is a life-changing condition with poor outcomes from currently available treatments. It is caused by directly pathogenic thyroid-stimulating hormone receptor-stimulating antibodies (TRAb), which are secreted from plasma...

Using cell-free RNA in monitoring immune system and the demonstration of significant systemic deficiency in lymphoid and myeloid biomarkers in patients with cancer.

3048 Background: Immune system plays a major role in the clinical course of cancer. Evaluating the local interaction between tumor cells and the immune cells (microenvironment) at the cancer site provides important information. However, currently little is known about the immune system as a whole in patients with cancer. Since cell-free circulating RNA (cfRNA) may reflect the entire body, we hypothesized that cfRNA might provide important information on the health and the status of the immune system. To simplify our approach, we evaluated only cellular biomarkers characteristic for lymphoid and myeloid cells using only the expression of 55 genes typically used in flow cytometry evaluation of hematologic cells. We compared findings between patients with cancer, patients with CHIP (clonal hematopoiesis of indeterminate potential), and normal individuals. Methods: cfRNA was extracted from plasma samples of 681 patients with various types of solid tumors, 113 patients with CHIP, and 34 normal individuals. cfRNA was sequenced via a hybrid capture-based panel targeting 55 genes reflecting immune cells including T-cells, B-cells, histiocytes, monocytes, and myeloid cells. The RNA was quantified using TPM (transcript per million). Results: There was significant difference (P<0.0001) between normal individuals and patients with cancers in the levels of circulating biomarkers specific for immune cells. Surprisingly, expression of B-cell, T-cell, monocytic/histiocytic genes were significantly lower in patients with solid tumors when compared to normal individuals. This included CD19, CD20, CD2, CD22, CD3D, CD3E, CD3G, CD4, CD52, CD7, CD79A, CD79B, CD8A, CD8B,CD33, FCER2(CD23), IL2RA(CD25), ITGAM(CD11B), and ITGAX(CD11C). In contrast, there was no significant difference between CHIP and normal for B- or T-cell markers. After adjusting for multiple testing, no deficiency in immune stimulatory markers was present in patients with CHIP. Patients with CHIP showed significantly (P<0.001) lower levels of CD38, CD58, CD16, CD15, CD25, and CD123 mRNA as compared with normal. Furthermore, using 35 immune cell biomarkers in a machine learning algorithm using 2/3 of samples for training and 1/3 for testing predicted the presence of cancer vs no cancer (AUC =0.820), and CHIP from cancer (AUC =0.0830) and CHIP vs normal (AUC =0.871). Conclusions: Immune related biomarkers using cfRNA provide important information on the immune system that can be used to monitor patients and to predict the presence of cancer or CHIP. The demonstration that patients with cancer have deficiency in overall systemic immune elements suggests that further studies are needed in monitoring the immune system and exploring means to boost systemic immunity to prevent the development of overt cancers.

Label-free optical fiber biosensor for the detection of CD44-expressing breast cancer cells

Increased level of CD44, transmembrane glycoprotein, is observed in several cancers such as breast, colorectal, head and neck, gastric, and ovarian cancer. Current methods for analyzing CD44 expressing cells are time-consuming, semi-quantitative, use labeled reagents for analysis or are not suitable for in situ analysis. Therefore exploring novel methods which are label-free and fast with the in situ detection capability for the quantification of these cells is of importance.Using optical fiber as a sensing element in biosensors offers low cost, high sensitivity, chemical inertness, and immunity to electromagnetic interference, and possibility of being used in in situ applications. This study reports the first label-free optical fiber biosensor for detection of CD44-expressing cancer cells. A fiber-optic ball resonator (BR) sensor was fabricated and then functionalized with CD44 antibodies. Different concentrations of the cells were incubated with the biosensor to measure the reflected light using optical backscatter reflectometer. The biosensor was able to detect cancer cells with a limit of detection of 335 cells/mL. The presence of the cells on the sensor surface was demonstrated by fluorescent and scanning electron microscope analysis. The work is the first proof-of-concept biosensor based on optical fibers for CD44 cancer cells and acts as a promising tool for in situ detection of cancer cells.

Synthetic abnormal mast cell particles successfully mimic neoplastic mast cells by flow cytometry.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.

The role of serotonin receptor 7 signalling in macrophages for the inflammatory response after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background The hormone and neurotransmitter serotonin regulates multiple processes, including immune responses and inflammatory signalling. After a cardiac injury, such as myocardial infarction (MI), a precise regulation of the local inflammation is determining the healing process and therefore functional outcome. Immune cells invading the infarcted tissue can be critical modulators, and various immune cells, including macrophages, express different serotonin receptors. Purpose We characterized the importance of serotonin receptor 7 (5-HT7R) signalling in the regulation of macrophage function, which infiltrate the heart after MI and thus can determine the course of cardiac remodelling processes. Methods For investigation of 5-HT7R signalling on a cellular level, we used two in vitro models: primary murine bone marrow-derived macrophages (BMDM) and secondary human THP-1-derived macrophages. To validate the impact of macrophages on intact cardiac tissue, we pre-treated pro-inflammatory M1-like BMDMs with a selective 5-HT7R agonist and then co-cultured them with living cardiac rat slices for up to 48 h (ex vivo) followed by functional analysis. Results We detected 5-HT7R expression in both models during all stages of macrophage polarization at mRNA as well as protein levels. Pro-inflammatory THP-1-derived macrophages displayed significantly enhanced mRNA expression levels of IL-1β and CD80, while pro-inflammatory differentiated M1-like BMDMs showed increased levels of CD38 and TNFα. Pharmacological activation of 5-HT7R with LP-211 reduced phagocytic activity up to 4-fold in THP-1-derived pro-inflammatory macrophages, altered their cytokine secretion profile and inhibited their migration ability (recovered area in scratch assay 1.3 % after LP-211 treatment compared to 6.1 % in control). A reduction in phagocytic activity was also verified in pro-inflammatory BMDMs. Our results also indicate reduced ability of LP-211 treated M1-like BMDMs to infiltrate living cardiac slices 3, 12, 24 and 48 h after seeding. Conclusion 5-HT7R signalling affects the functional properties of pro-inflammatory macrophages, including phagocytic activity, motility and secretion profile. Targeted manipulation of 5-HT7R activity in macrophages represents thus a possibility to modulate their inflammatory profile, which might be a reasonable approach to guide recovery post MI.

A Four-Gene Panel in Rectal Swab Samples as a Biomarker for Colorectal Cancer Screening.

The dysregulation of gene expression is one of the key molecular features of colorectal cancer (CRC) development. This study aimed to investigate whether such dysregulation is reflected in rectal swab specimens of CRC patients and to evaluate its potential as a non-invasive approach for screening. We compared the expression level of 14 CRC-associated genes in tumor and adjacent non-tumor tissue of CRC patients and examined the correlation of their levels in tissue with paired rectal swab specimens. The level of these 14 genes in rectal swab specimens was compared among patients with CRC or polyp and control subjects, and the diagnostic potential of each dysregulated gene and the gene panel were evaluated. The expression of CXCR2, SAA, COX1, PPARδ, PPARγ, Groγ, IL8, p21, c-myc, CD44 and CSF1 was significantly higher in CRC, and there was a significant correlation in the levels of most of them between the CRC and rectal swab specimens. In the training study, we showed that CD44, IL8, CXCR2 and c-myc levels were significantly higher in the rectal swab specimens of the CRC patients. Such result was confirmed in the validation study. A panel of these four genes was developed, and ROC analysis showed that this four-gene panel could identify CRC patients with an AUC value of 0.83 and identify overall polyp and precancerous adenoma patients with AUC values of 0.6522 and 0.7322, respectively. Finally, the predictive study showed that the four-gene panel demonstrated sensitivities of 63.6%, 76.9% and 88.9% in identifying overall polyp, precancerous adenoma and CRC patients, respectively, whereas the specificity for normal subjects was 72.2%. The expression of CRC-associated genes in rectal swab specimens reflects the dysregulation status in colorectal tissue, and the four-gene panel is a potential non-invasive biomarker for early precancerous adenoma and CRC screening.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal ‘M1-like’ phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Human serum albumin-based drug-free macromolecular therapeutics induce apoptosis in chronic lymphocytic leukemia patient cells by crosslinking of CD20 and/or CD38 receptors.

This study explores the efficacy of human serum albumin (HSA)-based Drug-Free Macromolecular Therapeutics (DFMT) in treating Chronic Lymphocytic Leukemia (CLL), a prevalent adult leukemia subtype. DFMT, a novel strategy, employs biomimetic crosslinking of CD20 and CD38 receptors on malignant B cells without the need for low molecular weight drugs. Apoptosis is initiated via a two-step process: i) Recognition of a bispecific engager, Fab' fragment conjugated with morpholino oligonucleotide (Fab'-MORF1), by a cell surface antigen; followed by ii) crosslinking of the MORF1-decorated cells with a multivalent effector, HSA holding multiple copies of complementary MORF2, HSA-(MORF2)x. Herein we evaluated the efficacy of HSA-based DFMT in the treatment of 56 samples isolated from patients diagnosed with CLL. Fab' fragments from Obinutuzumab (OBN) and Isatuximab (ISA) were employed in the synthesis of anti-CD20 (Fab'OBN-MORF1) and anti-CD38 (Fab'ISA-MORF1) bispecific engagers. The efficacy of DFMT was significantly influenced by the expression levels of CD20 and CD38 receptors. Dual-targeting DFMT strategies (CD20 + CD38) were more effective than single-target approaches, particularly in samples with elevated receptor expression. Pretreatment of patient cells with gemcitabine or ricolinostat markedly increased cell surface CD20 and CD38 expression, respectively. Apoptosis was effectively initiated in 62.5% of CD20-targeted samples and in 42.9% of CD38-targeted samples. Our findings demonstrate DFMT's potential in personalized CLL therapy. Further research is needed to validate these outcomes in a larger number of patient samples and to explore DFMT's applicability to other malignancies.