Fluorescence-activated Cell Sorting Analysis Research Articles

Effect of extended treatment with IAP inhibitor xevinapant post radiotherapy (RT) on efficacy and the tumor microenvironment (TME) in preclinical models.

6027 Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis, induced by RT and chemotherapy, and enhance antitumor immunity. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved 18-month locoregional control and improved 3-year progression-free survival and 5-year overall survival vs placebo + CRT. A phase 3 confirmatory study (TrilynX NCT04459715) and a phase 3 study to evaluate xevinapant + RT in post-operative, high-risk, cisplatin-ineligible patients (XRay Vision NCT05386550) are ongoing. In these studies, we investigate 3 additional cycles of xevinapant or placebo monotherapy after completing combination treatment. Based on the role of IAPs in apoptosis, immunity, and stromal activation, continual dosing of xevinapant post RT may have additional therapeutic benefits. Methods: MC38 syngeneic tumor-bearing mice were treated with vehicle control, RT alone (3.6Gy once daily [QD], 5 days), or RT + xevinapant (100 mg/kg QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation, immune cells in tumors were evaluated by fluorescence-activated cell sorting (FACS)–based immunophenotyping, and tumor-specific T cell responses were investigated by enzyme-linked immunosorbent spot (ELISpot) assays. To understand the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation, CAF gene expression was profiled on cell lines and patient-derived primary cultures. Results: Xevinapant + RT increased antitumor efficacy compared with vehicle control or RT alone. Moreover, RT in combination with extended dosing of xevinapant for 4 weeks significantly improved therapeutic efficacy and prolonged survival compared with RT + shorter xevinapant dosing durations. FACS analysis demonstrated trends of increased numbers of CD8+ T cells, natural killer cells, and dendritic cells and decreased numbers of regulatory T cells in mice treated with xevinapant + RT. FACS and ELISpot further suggested an increase in antigen-specific T cell counts from baseline with combination therapy arm vs vehicle control or RT alone. Xevinapant may also suppress RT-mediated CAF activation, as indicated by downregulation of activation markers and CAF-derived secretory proteins. Conclusions: In preclinical models, extended dosing of xevinapant for 4 weeks post RT improved antitumor efficacy. Our data suggest that the effect may be, in part, modulated by TME responses, including enhanced antitumor immunity and suppressed pro-tumorigenic phenotype of CAFs. Based on these results, further evaluation is warranted to determine the mechanisms that underscore the therapeutic benefit offered by extended dosing of xevinapant.

Assessment of cytotoxic and apoptotic activities of the Cassia angustifolia aqueous extract against SW480 colon cancer

Abstract The current natural extract modalities for colorectal cancer are limited. This research seeks to assess the process of extracting the plant’s bioactive constituents from Cassia angustifolia and to show the anticancer role played by the plant’s aqueous extract at 0°C by identifying the genes that alter in expression after the Cassia angustifolia treatment in colon cancer cells. The bioactive components of Cassia angustifolia extract were revealed using gas chromatography-mass spectrometry analysis. The colon carcinoma cell lines (SW480) were treated with Cassia angustifolia macrophages at concentrations of 50, 150, and 200 µg/mL for 48 h. Apoptosis was examined by fluorescence-activated cell sorting analysis of Cassia angustifolia-treated and -untreated cells. Microarray analysis was performed by using human microarray chips (HG-U95A) for untreated and treated SW480 cells. Microarray data were confirmed by the reverse transcription polymerase chain reaction. The findings showed that the Cassia angustifolia aqueous extract at 0°C/24 h contained the entire absolute phenolic content of 28.43 mg/g and the entire absolute flavonoid content of 9.16 mg/g. Cassia angustifolia enhanced the hindrance of cell development and apoptosis and decreased glucose uptake. Western blot analysis showed induction in the expression of cleaved caspases 3 and 9 in SW480 cells. Microarray data identified 11 genes and 7 expressed sequence tags (ESTs) markedly altered in treated vs non-treated SW480 cells. Several of these genes have been embroiled in multiple malignancies. In conclusion, the current study provides remarkable new data for Cassia angustifolia extracted at 0°C/24 h. We discovered 7 ESTs and 11 genes that are significantly altered in the progression of SW480 cells treated with Cassia angustifolia therapy versus no treatment, with anticancer and apoptosis inducer effects.

A self-healable and injectable hydrogel for pH-responsive doxorubicin drug delivery in vitro and in vivo for colon cancer treatment

Hydrogels are three-dimensional solid-like structures with a wide range of applications in drug transport, tissue engineering, wound dressing, etc. We developed herein a new hydrogel by mixing 5′-guanosine monophosphate (5′-GMP) disodium salt (16 mM) and 1,4,5,8-naphthalene tetracarboxylic acid tetra potassium salt (NAP) (4 mM) in 4:1 (GMP:NAP) stoichiometry in aqueous media (pH ∼4 at 25 °C). The mechanical properties of the resulting hydrogel were investigated by rheological studies, which showed that the hydrogel formed at pH 4 is the most stable [storage modulus (Gꞌ) of ∼4000 Pa] and has higher endurance for the oscillatory strain (20% strain). Spectroscopic investigations provided insights into the unique self-assembly of 5′-GMP and NAP. Further, the composite exhibited pH-responsive hydrogelation over the pH range of 3–6, and the gel was transformed into a sol form at pH > 5. This stimuli-sensitive nature of the hydrogel was then exploited for the encapsulation and efficacious delivery of an anticancer drug, doxorubicin (DOX), specifically toward the colorectal cancer. Microscopic studies revealed the presence of a regular entangled fibrillar network for the hydrogels, which allowed for its exceptionally high drug loading capacity. The controlled release of DOX from the hydrogel was then studied at the physiological pH of the gastrointestinal tract, i.e. pH ∼7.4. The DOX-loaded hydrogel (DOX-Gel) exhibited a ∼82% release of DOX over 100 h at pH ∼7.4. The enhanced cellular toxicity of DOX was observed in CT-26 murine colorectal carcinoma cell lines treated with the DOX-loaded hydrogel compared to DOX only as manifested from the fluorescence-activated cell sortinganalyses and fluorescence microscopic studies. The fluorescence-activated cell sorting analysis showed a ∼4.5-fold increase in the drug internalization for the DOX-Gel as compared to DOX only formulations. The co-localization of DOX into the cell nucleus was confirmed from confocal microscopy. The in vivo drug delivery efficacy of this pH-responsive hydrogel was further assessed on an animal tumor model, Balb/c mice (n = 9). A significant regression of ∼87.5% in the tumor volume was observed after 21 days of DOX-gel administration, in contrast to DOX only formulation, which showed only a ∼46.0% regression in the tumor volume. The blood reports of the non-treated control mice and the mice treated with DOX only indicated a metastatic behavior in comparison with the mice treated with the DOX-Gel. This, the present system, is a self-healable, injectable hydrogel, which makes it an ideal drug delivery vehicle with stimuli-responsive controlled drug release capabilities even in vivo.

Genome Engineering of Human Urine-Derived Stem Cells to Express Lactoferrin and Deoxyribonuclease.

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on piggyBac transposon modification. USCs from a healthy donor sample were isolated according to established protocols. Within 2 months, 10 clones had been expanded, analyzed, and frozen. Fluorescence-activated cell sorting analysis of individual clones revealed that all 10 clones expressed characteristic USC markers (97-99% positive for CD44, CD73, CD90, and CD146; negative for CD31, CD34, and CD45). The isolated USCs were successfully differentiated along the osteogenic, adipogenic, and chondrogenic lineages, suggesting multipotent differentiation capacity. Additionally, the USCs were differentiated into podocytes positive for NEPHRIN (NPHS1), podocalyxin, and Wilms tumor 1 (WT1). Transfection of USCs with a strongly expressing Green fluorescent protein plasmid was optimized to achieve 61% efficiency in live cells using several commercially available lipophilic reagents. Transgene promoters were compared in five luciferase-expressing piggyBac transposons by live animal imaging. The CMV promoter produced the highest luciferase signal, followed by EF1-α. Finally, HEK-293 and USCs were transfected with piggyBac transposons expressing lactoferrin and DNase1 for treatment of acute kidney injury associated with rhabdomyolysis. We found that both proteins were expressed in USCs and that lactoferrin was successfully secreted into the cell culture media. In conclusion, USCs represent a clinically relevant cell type that can express nonviral transgenes. Impact statement Acute kidney injury (AKI) affects over 13 million people worldwide each year, with hospitalization rates on the rise. There are no therapies that directly regenerate the kidney after AKI. Each human kidney contains approximately one million nephrons that process ∼100 L of urinary filtrate each day. Thousands of kidney cells become detached and are excreted in the urine. A small percentage of these cells can be clonally derived into urine-derived stem cells. We have optimized methods for genome engineering of adult human urine-derived stem cells for future applications in regenerative approaches to treat kidney injury.

Abstract 351: Developing A Human Bone Marrow Organoid Culture System To Study The Diabetic Immune System

Objectives: There is limited understanding on how diabetes negatively impacts wound healing after ischemic tissue damage. Murine models for limb ischemia and myocardial infarction have demonstrated that recruitment of bone marrow hematopoietic stem cell (HSC)-derived immune cells at all stages of ischemic tissue remodeling and healing. By leveraging our lab’s three-dimensional (3D) bone marrow organoid culture system used to study human HSC, we aim to study the effect of diabetes on immune cell development. Methods: Surgically discarded bone marrow tissue samples from patients undergoing lower extremity amputation for non-healing ulcer or tissue loss was used to develop our bone marrow 3D organoid culture system. Human HSCs are identified using fluorescence-tagged antibodies against cell surface markers. Hematopoietic potential of bone marrow-derived HSCs was tested using colony forming unit assay. In vitro immune cell differentiation into monocyte and macrophage was performed. Bulk RNA libraries were sequenced on the NextSeq 500 system (Illumina) and analysis done on DeBrowser. Results: Our novel human bone marrow co-culture system provides a method for bone marrow cell expansion from primary human bone marrow tissue explants. De novo sprouting cells can be observed growing from bone marrow tissue explants harvested from non-diabetic and diabetic donors. Using fluorescence-activated cell sorting analysis, we identified a trend towards reduced HSC numbers in diabetic when compared to non-diabetic donors. HSC derived from both diabetic and non-diabetic bone marrow tissue explants were able to undergo hematopoiesis and immune cell differentiation. Bulk RNA sequencing demonstrates distinct gene expression profiles of the cells expanded from non-diabetic and diabetic bone marrow cells. Conclusions: Our 3D bone marrow organoid culture system will allow for future comparative studies between the non-diabetic and diabetic immune system. The preliminary data suggest that there are gene expression profile differences between bone marrow progenitor cells from diabetic and non-diabetic donors. These gene expression differences may attribute to alterations in the HSC number and immune cell development observed in diabetes.

Perivascular localized cells commit erythropoiesis in PDGF-B-expressing solid tumors.

Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. To test if vascular cells can differentiate into RBCs, genome-wide expression profiling was performed using mouse-derived pericytes. Genetic tracing of perivascular localized cells employing NG2-CreERT2:R26R-tdTomato mouse strain was used to validate the findings in vivo. Fluorescence-activated cell sorting (FACS), single-cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation-specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA, magnetic-activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Genome-wide expression profiling showed that in response to platelet-derived growth factor subunit B (PDGF-B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor-like features and underwent differentiation towards the erythroid lineage. PDGF-B simultaneously targeted cancer-associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell-derived subpopulation of hematopoietic cells. Single-cell sequencing and colony formation assays validated the fact that, upon PDGF-B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell-derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.

Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing.

The mechanistic target of rapamycin complex 1 (mTORC1) is an essential hub that integrates nutrient signals and coordinates metabolism to control cell growth. Amino acid signals are detected by sensor proteins and relayed to the GATOR2 and GATOR1 complexes to control mTORC1 activity. Here we perform genome-wide CRISPR/Cas9 screens, coupled with an assay for mTORC1 activity based on fluorescence-activated cell sorting analysis of pS6, to identify potential regulators of mTORC1-dependent amino acid sensing. We then focus on interleukin enhancer binding factor 3 (ILF3), one of the candidate genes from the screen. ILF3 tethers the GATOR complexes to lysosomes to control mTORC1. Adding a lysosome-targeting sequence to the GATOR2 component WDR24 bypasses the requirement for ILF3 to modulate amino-acid-dependent mTORC1 signalling. ILF3 plays an evolutionarily conserved role in human and mouse cells, and in worms to regulate the mTORC1 pathway, control autophagy activity and modulate the ageing process.

Abstract 2457: Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer

Abstract Dysregulated WNT signaling caused by mutation in the APC gene occurs in 80-90% of all colorectal cancer (CRC) cases. Progressive overpopulation of ALDH+ stem cells occurs during development of CRC in familial adenomatous polyposis (FAP) patients. Given that ALDH is a key member of the retinoic acid signaling (RA) pathway, we hypothesize that human CRC is caused by an imbalance between dysregulated WNT and RA signaling. Herein, we aim to restore proper WNT and RA signaling and identify a common link or target to induce differentiation of CSCs along the neuroendocrine cell (NEC) lineage. Accordingly, to determine the effects of restoring wt-APC in APC-mutant HT29 CRC cells, we conducted in vitro experiments to examine Wnt/β-catenin signaling, sensitivity to retinoids, cell proliferation, expression of stem cell markers, quantification of ALDH+ stem cells, and NEC maturation. We utilized Nanostring profiling, TCF reporter assay, western blot, and fluorescence activated cell sorting analyses to ascertain these effects. We found that induction of wt-APC expression decreased WNT/β-catenin signaling and reduced protein expression of WNT-target genes. Notably, inducing wt-APC decreased ATRA-induced expression of the WNT target gene CYP26A1 (by 50%), which is predicted to, in turn, increase RA signaling by lowering degradation of RA. Indeed, inducing wt-APC increased sensitivity of CRC cells to ATRA-induced apoptosis and inhibition of cell proliferation in a dose-dependent manner. Expression of ALDH1A1 decreased (3-fold) with ATRA treatment and inducing wt-APC extended the decrease by an additional 2-fold. Furthermore, upon inducing wt-APC, we found an increase in the protein expression of several NEC markers including CHGA, GLP2R, NSE (ENO2), and SSTR1, and an increased number of GLP2R+ NECs. We also discovered a very highly regulated protein network of 248 proteins and identified a cell signaling mechanism that links the WNT and retinoic acid pathways via CYP26A1. Thus, by inducing wt-APC expression and exogenously activating RA signaling, we found a way to increase differentiation of SCs along the NEC (and other) lineage(s). Translating this strategy in vivo might lead to new, more effective treatments involving retinoids for CRC patients. Citation Format: Caroline O. B. Facey, Victoria O. Hunsu, Brian T. Osmond, Chi Zhang, Lynn M. Opdenaker, Bruce M. Boman. Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2457.

Fluorescence-Based Detection of Fatty Acid β-Oxidation in Cells and Tissues Using Quinone Methide-Releasing Probes.

Detection of metabolic activity enables us to reveal the inherent metabolic state of cells and elucidate mechanisms underlying cellular homeostasis and growth. However, a fluorescence approach for the study of metabolic pathways is still largely unexplored. Herein, we have developed a new chemical probe for the fluorescence-based detection of fatty acid β-oxidation (FAO), a key process in lipid catabolism, in cells and tissues. This probe serves as a substrate of FAO and forms a reactive quinone methide (QM) as a result of metabolic reactions. The liberated QM is covalently captured by intracellular proteins, and subsequent bio-orthogonal ligation with a fluorophore enables fluorescence analysis. This reaction-based sensing allowed us to detect FAO activity in cells at a desired emission wavelength using diverse analytical techniques including fluorescence imaging, in-gel fluorescence activity-based protein profiling (ABPP), and fluorescence-activated cell sorting (FACS). The probe was able to detect changes in FAO activity induced by chemical modulators in cultured cells. The probe was further employed for fluorescence imaging of FAO in mouse liver tissues and revealed the metabolic heterogeneity of FAO activity in hepatocytes by the combination of FACS and gene expression analysis, highlighting the utility of our probe as a chemical tool for fatty acid metabolism research.

Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 μM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-β-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

An anti-leishmanial compound 4',7-dihydroxyflavone elicits ROS-mediated apoptosis-like death in Leishmania parasite.

The treatment for leishmaniasis is currently plagued by side effects such as toxicity, and the emergence of drug resistance to the available repertoire of drugs, as well as the expense of these drugs. Considering the rising concerns, we report here , the anti-leishmanial activity and mechanism of a flavone compound 4',7-dihydroxyflavone (TI 4). Four flavanoids were initially screened for anti-leishmanial activity and cytotoxicity. The results showed that the compound TI 4 exhibited higher activity and selectivity index while maintaining low cytotoxicity. Preliminary microscopic studies and FACS analysis reported that the parasite underwent apoptosis on TI 4 treatment. Further in-depth studies revealed high ROS production and thiol levels in the parasites suggesting ROS mediated apoptosis in the parasites upon TI 4 treatment. Other apoptotic indicators like intracellular Ca2+ and mitochondrial membrane potential also indicated the onset of apoptosis in the treated parasites. The mRNA expression levels signified that the redox metabolism genes were upregulated by 2-fold along with the apoptotic genes. In summary, the use of TI 4 on Leishmania parasites induces ROS-mediated apoptosis, therefore the compound has immense potential to be an anti-leishmanial drug. However, in vivo studies would be required to ascertain its safety and efficacy before we can exploit the compound against the growing leishmaniasis crisis.

Involvement of a novel cAMP signaling mediator for beige adipogenesis

BackgroundExposure to cold temperature stimulates the sympathetic nervous system that activates β-adrenergic receptor signals in brown and beige adipocytes, leading to the induction of adaptive thermogenesis in mammals. Prominin-1 (PROM1) is a pentaspan transmembrane protein that is widely identified as a marker for stem cells, although the role of this protein as a regulator of many intracellular signaling cascades has been recently delineated. The main focus of the current study is to identify the previously unknown role of PROM1 in beige adipogenesis and adaptive thermogenesis. MethodsProm1 whole body knockout (Prom1 KO) mice, Prom1 adipogenic progenitor (AP) cell-specific knockout (Prom1 APKO) mice and Prom1 adipocyte-specific knockout (Prom1 AKO) mice were constructed and were subject for the induction of adaptive thermogenesis. The effect of systemic Prom1 depletion was evaluated by hematoxylin and eosin staining, immunostaining, and biochemical analysis in vivo. Flow cytometric analysis was performed to determine the identity of PROM1-expressing cell types, and the resultant cells were subject to beige adipogenesis in vitro. The potential role of PROM1 and ERM in cAMP signaling was also assessed in undifferentiated AP cells in vitro. Finally, the specific effect of Prom1 depletion on AP cell or mature adipocytes on adaptive thermogenesis was evaluated by hematoxylin and eosin staining, immunostaining, and biochemical analysis in vivo. ResultsProm1 KO mice displayed an impairment in cold- or β3-adrenergic agonist–induced adaptive thermogenesis in subcutaneous adipose tissues (SAT) but not in brown adipose tissues (BAT). By fluorescence-activated cell sorting (FACS) analysis, we identified that PROM1 positive cells are enriched in PDGFRα+Sca1+ AP cells from SAT. Interestingly, Prom1 knockout stromal vascular fractions showed reduced PDGFRα expression, suggesting a role of PROM1 in beige adipogenic potential. Indeed, we found that Prom1-deficient AP cells from SAT showed reduced potential for beige adipogenesis. Furthermore, AP cell-specific depletion of Prom1, but not adipocyte-specific depletion of Prom1, displayed defects in adaptive thermogenesis as evidenced by resistance to cold-induced browning of SAT and dampened energy expenditure in mice. ConclusionWe found that PROM1 positive AP cells are essential for the adaptive thermogenesis by ensuing stress-induced beige adipogenesis. Identification of PROM1 ligand might be useful in the activation of thermogenesis that could be potentially beneficial in combating obesity.

Abstract P4-08-15: Identification of target kinases whose inhibition enhances antitumor efficacy of sacituzumab-govitecan in metastatic HER2-negative breast cancer

Abstract BACKGROUND HER2-negative (HER2−) breast cancer (BC) subtypes, such as luminal A and triple-negative breast cancer (TNBC), express human trophoblast cell-surface antigen 2 (Trop-2). A high level of Trop-2 expression is associated with a poor prognosis in solid tumors. Sacituzumab-govitecan (SG), a Trop-2–directed antibody conjugated with SN-38 (topoisomerase Ⅰ inhibitor) significantly improves survival in patients with metastatic TNBC. Also, the IMMU-132-01 phase Ⅰ/Ⅱ study revealed that SG has antitumor activity in previously treated hormone receptor (HR)+/HER2− metastatic BC. In a phase Ⅲ trial (TROPiCS-02), SG demonstrated statistically significant progression-free survival in patients with heavily pre-treated HR+/HER2− endocrine-resistant metastatic BC. To augment the efficacy of SG, we sought to identify potential kinase targets whose inhibition enhances the antitumor efficacy of SG to formulate an SG-based combination therapy. MATERIALS AND METHODS Fluorescence-activated cell sorting analysis and Western blotting were used to evaluate the expression of Trop-2 in 24 TNBC and 8 HR+ BC cell lines. Non-biased high-throughput kinome-wide RNAi screening was performed with BT-20 (TNBC) and T47D tamoxifen/abemaciclib double-resistant (HR+) cell lines to identify target kinases whose inhibition produces synergistic antitumor efficacy of SG. To validate the combination antitumor effect of SG and selected kinase target inhibitors, sulforhodamine B staining proliferation assays, cell cycle analysis, and caspase 3/7 activity assays were performed in Trop-2+ TNBC and HR+ BC cell lines, including cell lines resistant to tamoxifen and double-resistant to tamoxifen and CDK4/6 inhibitor (palbociclib or abemaciclib). RESULTS In vitro proliferation assays revealed that the half-maximal inhibitory concentration (IC50) of SG in tested TNBC cell lines ranged from 10 nM to 84 nM, and that of HR+ cell lines ranged from 12.5 nM to >250 nM. We identified 69 kinase targets in TNBC cell line BT-20 and 44 kinase targets in HR+ BC cell line T47D (tamoxifen/abemaciclib double-resistant) by the sensitivity index analysis (>0.15) of RNAi screening results, and further pathway analysis revealed DNA damage response (DDR), (drug: BAY1895344), PI3k/Akt/mTOR (drug: copanlisib), and MAPK (drug: trametinib) as potential target canonical pathways whose targeting enhanced the cytotoxic effect of SG in both TNBC and HR+ BC. Among these strategies, inhibition of the DDR pathway with ATR inhibitor (BAY1895344) yielded a synergistic antiproliferative effect in combination with SG compared to SG or BAY1895344 alone in all tested TNBC and HR+ BC cell lines. Growth inhibition by SG combined with BAY1895344 averaged 97.1% in TNBC cell lines and ranged from 30.7% to 81.3% in HR+ BC cell lines, with P< 0.001. Other combinations with SG showed partially synergistic antiproliferative effects (copanlisib: effective in 3 of 5 cell lines, trametinib: effective in 2 of 5 cell lines). CONCLUSIONS RNAi screening uncovered DDR, PI3k/Akt/mTOR, and MAPK pathways as potential targets for combination with SG. Among inhibitors of these targets, DDR pathway inhibitor BAY1895344 had the most robust synergy with SG, compared to two other target inhibitors, against Trop-2+ TNBC and HR+ BC cell lines. These in vitro data warrant future in vivo validation studies of the synergistic antitumor effect of SG and BAY1895344 in metastatic HER2– breast cancer. Citation Format: Nakyung Oh, Jon A. Fuson, Huey Liu, Debu Tripathy, Naoto T. Ueno, Jangsoon Lee. Identification of target kinases whose inhibition enhances antitumor efficacy of sacituzumab-govitecan in metastatic HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-15.

Premature T cell aging in major depression: A double hit by the state of disease and cytomegalovirus infection

IntroductionPrevious research indicates that premature T cell senescence is a characteristic of major depressive disorder (MDD). However, known senescence inducing factors like cytomegalovirus (CMV) infection or, probably, childhood adversity (CA) have not been taken into consideration so far. ObjectiveDifferentiation and senescent characteristics of T cells of MDD patients were investigated in relation to healthy controls (HC), taking the CMV seropositivity and CA into account. Methods127 MDD and 113 HC of the EU-MOODSTRATIFICATION cohort were analyzed. Fluorescence activated cell sorting (FACS) analysis was performed to determine B, NK, and T cell frequencies. In a second FACS analysis, naïve, effector memory (Tem), central memory (Tcm), effector memory cells re-expressing RA (TEMRA), as well as CD28+ and CD27+ memory populations, were determined of the CD4+ and CD8+ T cell populations in a subsample (N = 35 MDD and N = 36 HC). CMV-antibody state was measured by IgG ELISA and CA by the Childhood Trauma Questionnaire. ResultsWe detected a CMV-antibody positivity in 40% of MDD patients (35% HC, n. s.) with seropositive MDD cases showing a higher total childhood trauma score. Second, a higher inflation of memory CD4+ T helper cells in CMV seronegative patients as compared to seronegative HC and reduced numbers of naïve CD4+ T helper cells in CMV seropositive patients (not in CMV seropositive HC) were found. Third, a higher inflation of memory CD8+ T cytotoxic cells in CMV seropositive cases as compared to CMV seropositive HC, particularly of the TEMRA cells, became apparent. Higher percentages of CD4+ TEMRA and late stage CD27−CD28− TEMRA cells were similar in both HC and MDD with CMV seropositivity. Overall, apportioning of T cell subpopulations did not differ between CA positive vs negative cases. ConclusionsMDD patients show several signs of a CMV independent “MDD specific” premature T cell aging, such as a CMV independent increase in CD4+ T memory cells and a latent naïve CD4 T-cell reduction and a latent CD8+ T-cell increase. However, these two latent T cell senescence abnormalities only become evident with CMV infection (double hit).

Chlorogenic acid improves intestinal morphology by enhancing intestinal stem-cell activity.

Chlorogenic acid (CGA), as one of the most abundant naturally occurring phenolic acids, has been documented to be beneficial for intestinal health. However, the underlying mechanism is still not fully understood. The adult intestinal stem cell is the critical driver of epithelial homeostasis and regeneration. This study hypothesized that CGA exerted intestinal health effects by modulating intestinal stem-cell functions. Lgr5-EGFP mice were treated for 14 days, and intestinal organoids derived from these mice were treated for 3 days, using CGA solution. In comparison with the control group, CGA treatment increased intestinal villous height and crypt depth in mice and augmented the area expansion and the number of budding intestinal organoids. Quantitative polymerase chain reaction (qPCR) analysis revealed that CGA treatment significantly increased the expression of genes coding intestinal stem-cell markers in intestinal tissue and organoids, and upregulated the expression of genes coding secretory cell lineages and enterocytes, although not statistically significantly. Fluorescence-activated cell-sorting analysis further confirmed that CGA augmented the number of stem cells. 5-Ethynyl-2'-deoxyuridine (EdU) incorporation and Ki67 immunostaining results also demonstrated that CGA treatment enhanced intestinal stem-cell proliferation. Altogether, our findings indicate that CGA could activate intestinal stem-cell and epithelial regeneration, which could contribute to the improvement of intestinal morphology or organoid growth of mice. This highlights a promising mechanism for CGA as an excellent candidate for the formulation of dietary supplements and functional foods for intestinal protection. © 2023 Society of Chemical Industry.

Effect of isolated grandivittin from Ferulago trifida Boiss. (Apiaceae) on the proliferation and apoptosis of human lung cancer A549 cells.

Lung cancer is one of the deadliest cancers in the world. Introducing new promising agents can help the chemotherapeutic management of cancer. In the knowledge of oncology, plants are of special interest as a rich source of new antineoplastic and chemotherapeutic agents. Grandivittin (GRA) is one of the main constituents of Ferulago trifida Boiss. with established medicinal, phytochemical, and pharmacological properties. This study aimed to isolate and evaluate the antineoplastic potential of grandivittin and its underlying mechanisms in human lung cancer A549 cells. The viability of the A549 cells after being treated with 0.1, 0.4, 0.7, 1, and 1.3mM of GRA for three following days was measured using the MTT method. The early apoptosis and late apoptosis were assessed by fluorescence-activated cell sorter analysis through annexin V/PI staining. The expression of apoptotic agents' genes (caspase 3, caspase 9, Bcl2, Bax, and P53) was evaluated by the RT-PCR method. GRA increased apoptotic cells and decreased cell viability in a dose- and time-dependent manner, in which only 50% of cells survived at a dose of 0.7mM. The expression of Bax, P53, caspase 3, and caspase 9 genes in the A549 cells was significantly upregulated after GRA treatment compared to control cells (P < 0.05). On the other hand, Bcl2 was significantly downregulated after GRA treatment (P < 0.05). The results indicated that GRA can activate cell death in A549 lung carcinoma cells by inducing both DNA toxicity p53 and cascade-dependent pathways. Therefore, GRA may be a potential new therapeutic agent for the treatment of lung cancer.

Metformin regulates myoblast differentiation through an AMPK-dependent mechanism.

This study aims to investigate how metformin (Met) affects muscle tissue by evaluating the drug effects on proliferating, differentiating, and differentiated C2C12 cells. Moreover, we also investigated the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) in the mechanism of action of Met. C2C12 myoblasts were cultured in growth medium with or without Met (250μM, 1mM and 10mM) for different times. Cell proliferation was evaluated by MTT assay, while cell toxicity was assessed by Trypan Blue exclusion test and Lactate Dehydrogenase release. Fluorescence Activated Cell Sorting analysis was performed to study cell cycle. Differentiating myoblasts were incubated in differentiation medium (DM) with or without 10mM Met. For experiments on myotubes, C2C12 were induced to differentiate in DM, and then treated with Met at scalar concentrations and for different times. Western blotting was performed to evaluate the expression of proteins involved in myoblast differentiation, muscle function and metabolism. In differentiating C2C12, Met inhibited cell differentiation, arrested cell cycle progression in G2/M phase and reduced the expression of cyclin-dependent kinase inhibitor 1. These effects were accompanied by activation of AMPK and modulation of the myogenic regulatory factors. Comparable results were obtained in myotubes. The use of Compound C, a specific inhibitor of AMPK, counteracted the above-mentioned Met effects. We reported that Met inhibits C2C12 differentiation probably by blocking cell-cycle progression and preventing cells permanent exit from cell-cycle. Moreover, our study provides solid evidence that most of the effects of Met on myoblasts and myotubes are mediated by AMPK.

Platelet-Rich stroma from Crohn’s disease patients for treatment of perianal fistula shows a higher myeloid cell profile compared to non-IBD controls

BackgroundNew cell-based therapies are under investigation to improve perianal fistulizing Crohn’s disease (pCD) healing. Autologous stromal vascular fraction combined with platelet-rich plasma (referred to as platelet-rich stroma [PRS]) is a new adipose-derived stromal therapy. The effect of Crohn’s disease (CD) on adipose tissue, and adipose-derived therapies, is largely unknown. We characterized the cellular composition of subcutaneous lipoaspirate and PRS of pCD patients and non-Inflammatory Bowel Disease (IBD) controls. MethodsConsecutive pCD patients (≥18 years) and non-IBD controls, who underwent liposuction for the purpose of autologous PRS therapy, were included (October 2020 and March 2021). Mechanically fractionated lipoaspirate and the combined PRS product were analyzed for cell surface marker expression using fluorescence-activated cell sorting analysis. ResultsTwenty-three patients (37.8 [IQR 30.7–45.0] years; 9 [39.1 %] male; 11CD patients) were included. Similar total number of cells were found in CD and non-IBD lipoaspirate (CD 8.23 ± 1.62*105 cells/mL versus non-IBD 12.20 ± 3.39*105). Presence of stromal cells, endothelial like cells, immune cells, T-cells, myeloid cells and M2/M1 macrophage ratio were similar in CD and non-IBD lipoaspirate. In PRS samples, more cells/mL were seen in CD patients (P = 0.030). Myeloid cells were more abundant in CD PRS samples (P = 0.007), and appeared to have a higher regulatory M2/M1 ratio. Interdonor variation was observed between lipoaspirate and PRS samples. ConclusionsThe composition of CD and non-IBD lipoaspirate were found to be similar and interdonor variation was observed. However, PRS from CD patients showed more myeloid cells with a regulatory phenotype. Crohn’s disease does not appear to alter the immunological composition of adipose-derived products.

Interstitial Macrophages Lead Early Stages of Bleomycin-Induced Lung Fibrosis and Induce Fibroblasts Activation.

A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed pulmonary macrophages' role as modulators of the fibrotic processes. The proportion, phenotype, and function of alveolar (AM) and interstitial macrophages (IM) at the early stages of bleomycin-induced pulmonary fibrosis have not been clearly described. In this way, our study aimed to characterize these macrophage populations and investigate the effect on fibroblast activation. C57BL/6 mice were intratracheally injected with bleomycin and were sacrificed at day 3, 5, and 7 for the performance of flow cytometry and fluorescent-activated cell sorting analysis for protein and gene expression quantification. After bleomycin administration, the proportion of IM was significantly higher than that of AM, which showed a decay during the inflammatory phase, and peaked at day 7. At day 7 of the inflammatory phase, AM started shifting their phenotype from M1-like towards M2, while IM showed a M2-like phenotype. Conditioned medium derived from IM sorted at day 7 induced fibroblast activation and differentiation in myofibroblasts in vitro. Our findings indicate that IM are the largest macrophage population at the early stages of experimental pulmonary fibrosis and are secreted mediators able to activate fibroblasts, pointing to macrophage modulation as a potential therapeutic strategy to restrain progressive fibrosing lung disorders.

Different Contacted Cell Types Contribute to Acquiring Different Properties in Brain Microglial Cells upon Intercellular Interaction

Microglial cells (MGs), originally derived from progenitor cells in a yolk sac during early development, are glial cells located in a physiological and pathological brain. Since the brain contains various cell types, MGs could frequently interact with different cells, such as astrocytes (ACs), pericytes (PCs), and endothelial cells (ECs). However, how microglial traits are regulated via cell-cell interactions by ACs, PCs, or ECs and how they are different depending on the contacted cell types is unclear. This study aimed to clarify these questions by coculturing MGs with ACs, PCs, or ECs using mouse brain-derived cells, and microglial phenotypic changes were investigated under culture conditions that enabled direct cell-cell contact. Our results showed that ACs or PCs dose-dependently increased the number of MG, while ECs decreased it. Microarray and gene ontology analysis showed that cell fate-related genes (e.g., cell cycle, proliferation, growth, death, and apoptosis) of MGs were altered after a cell-cell contact with ACs, PCs, and ECs. Notably, microarray analysis showed that several genes, such as gap junction protein alpha 1 (Gja1), were prominently upregulated in MGs after coincubation with ACs, PCs, or ECs, regardless of cell types. Similarly, immunohistochemistry showed that an increased Gja1 expression was observed in MGs after coincubation with ACs, PCs, or ECs. Immunofluorescent and fluorescence-activated cell sorting analysis also showed that calcein-AM was transferred into MGs after coincubation with ACs, PCs, or ECs, confirming that intercellular interactions occurred between these cells. However, while Gja1 inhibition reduced the number of MGs after coincubation with ACs and PCs, this was increased after coincubation with ECs; this indicates that ACs and PCs positively regulate microglial numbers via Gja1, while ECs decrease it. Results show that ACs, PCs, or ECs exert both common and specific cell type-dependent effects on MGs through intercellular interactions. These findings also suggest that brain microglial phenotypes are different depending on their surrounding cell types, such as ACs, PCs, or ECs.