Number Of Stromal Cells Research Articles

Abstract A062: Neurotropic fibroblast population increases following exposure to chemotherapy in pancreatic adenocarcinoma

Abstract Introduction Pancreatic adenocarcinoma (PDA) will be the second leading cause of cancer related death by 2030 and patient response to chemotherapy is poor with a strong likelihood of cancer recurrence. While past studies have investigated pre- and post-chemotherapy patient samples, there is a paucity of studies looking at fresh, patient matched samples which allow us to observe interpatient heterogeneity in chemotherapy response. Methods Here, we study the effects of chemotherapy on five different patients using sc-RNA sequencing of matched treatment naïve and post treatment tissue biopsies to compare the effects of chemotherapy, not only on the tumor cells, but also on the tumor microenvironment – a key contributor to tumor survival and proliferation. We utilized the Seurat v4 pipeline and CellChat v2 to observe transcriptomic differences in epithelial cells, myeloid cells, t-Cells, and fibroblasts and their corresponding putative interactions in pre- and post- treatment states. Results Unsurprisingly, we found insufficient tumor epithelial cells post-treatment to perform further analysis on due to the intended cytotoxic effects of chemotherapy. Therefore, we turned our focus to the tumor microenvironment, where we had sufficient numbers of stromal and immune cells, to investigate the effects of chemotherapy. We identified that fibroblasts changed the most consistently across all patients in response to chemotherapy. The fibroblasts clustered into three distinct groups: an immunomodulatory group, a myofibroblastic and neurotropic group, as well as a mixed group expressing genes from all three categories. Interestingly, the neurotropic fibroblast signature was universally enriched after chemotherapy in all five patients, despite having a small and clinically heterogeneous cohort. Conclusions Through an analysis of each fibroblast group in the treatment naïve and post treatment categories, we observed that a specific set of fibroblasts become less myofibroblastic and increasingly neurotropic following treatment, leading us to posit that fibroblasts altered by exposure to chemotherapy may potentially contribute to a ‘pro-tumor’ environment that supports recurrence. To test this in vitro, we treated patient derived fibroblasts with Gemcitabine and used RTPCR to analyze their relative changes in gene expression. We specifically probed for genes that were definitive of myofibroblasts and neurotropic fibroblasts and saw that post-chemotherapy exposure, neurotropic genes such as NEGR1 and NFIA were upregulated. This validated our scRNA conclusions that fibroblasts become increasingly neurotropic following treatment and then differentially interact with their surrounding environment. In future studies, we will further analyze the role these neurotropic fibroblasts play in chemoresponse and tumor recurrence. Matched samples are critical to studies of chemotherapy effects on PDA due to the fact that they allow us to deconvolute one aspect of PDA heterogenity - interpatient heterogeneity - which is a large limitation in studies of this disease. Citation Format: Aylin Z Henstridge, Padma Kadiyala, Ahmed M Elhossiny, Jianhua Liu, Vaibhav Sahai, Nicole Peterson, Jorge Machicado, Richard Kwon, Allison Schulman, Erik Wamsteker, George Philips, Martin Fernandez-Zapico, Mark Truty, Costas Lyssiotis, Timothy Frankel, Filip Bednar, Marina Pasca di Magliano, Eileen S Carpenter. Neurotropic fibroblast population increases following exposure to chemotherapy in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A062.

Unveiling the potential of novel 5α-reductase inhibitors via ligand based drug design, molecular docking and ADME predictions to manage BPH

Benign prostatic hyperplasia (BPH), a prevalent condition among elderly males characterized by the non-cancerous enlargement of the prostate gland is due to the increased stromal and epithelial cell numbers. The enlargement is driven by an active dihydrotestosterone (DHT), synthesized from its prohormone testosterone under the catalytic effect of 5α-reductase Type-II (5-AR2) enzyme. To reduce the increased DHT levels and ultimately to control prostate growth, inhibition of the 5-AR represent an interesting therapeutic target. The 3D crystallography structure 5-AR2, a member of the steroid 5-AR family belonging to the class of oxidoreductase has been resolved recently and opened the door to develop selective novel inhibitors of 5-AR Type-II isoform using molecular modeling techniques. The Force field and Gaussian-field three-dimensional quantitative structure–activity relationship (3D-QSAR) models have been developed using 42 selective 5-AR2 inhibitors belonging to chemical class of steroidal androstene. Partial least squares analysis produced statistically significant model with R2training = 0.9367, 0.9459 and predictability Q2test = 0.7233, 0.8595. Developed 3D-QSAR model include steric, electrostatic, hydrophobic, and hydrogen bond acceptor and donor field indicators, whereas the potential field contributions indicated the importance of steric feature in governing their biological activity. Protein-ligand interaction pattern analysis revealed that amino acid residues GLU57, ARG114, TYR91 present in the active site of 5-AR2 are crucial for lower energy complexes with good binding affinity. Identified hits were finally adjudged for their druggability by determining in-silico pharmacokinetic parameters. Further, RMSD, binding free energy calculations and post MD analysis demonstrated the enhanced binding affinity with better ΔGbind value (-79.6 ± 3.9 kcal/mol) of identified ligand A-10 than the reference molecule finasteride (-69.13 ± 4.69 kcal/mol). Combined approach of ligand-based and structure-based models have provided an improved understanding of the structural properties of androstene class that may be further used to develop novel 5-AR2 inhibitors to be useful in BPH.

Croton grewioides essential oil and anethole reduce oxidative stress and improve growth of bovine primordial follicles during culture of ovarian tissue.

This study aims to evaluate the effects of Croton grewioides essential oil (CGEO) and anethole on follicle survival, growth, and oxidative stress in cultured bovine ovarian tissues. Ovarian tissues were cultured for 6 days in a medium supplemented with different concentrations (1, 10, 100, or 1000 µg mL-1) of CGEO or anethole and then, follicular survival and growth, collagen content, and stromal cell density in ovarian tissues cultured in vitro were evaluated by histology. The mRNA levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1), peroxirredoxin 6 (PRDX6), and nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated by real-time PCR. The activity of SOD, CAT, glutathione peroxidase (GPx), and thiol concentrations were investigated. Ovarian tissues cultured with 1 µg mL-1 CGEO or anethole had a higher percentage of healthy follicles than those cultured in a control medium (P < .05). The 1 µg mL-1 CGEO also increased the number of stromal cells, collagen fibers, and thiol levels. Anethole (1 µg mL-1) increased CAT activity and reduced that of GPx. The activity of SOD was reduced by CGEO. In contrast, 1 µg mL-1 anethole reduced mRNA for CAT, PRDX1, and NRF2 (P < .05). In addition, 1 µg mL-1 CGEO reduced mRNA for CAT, PRDX6, and GPx1 (P < .05). The presence of 1 µg mL-1 anethole or CGEO in a culture medium promotes follicle survival and regulates oxidative stress and the expression of mRNA and activity of antioxidant enzymes in cultured bovine ovarian tissues.

Effects of carvedilol on human prostate tissue contractility and stromal cell growth pointing to potential clinical implications

BackgroundApart from antagonizing ß-adrenoceptors, carvedilol antagonizes vascular α1-adrenoceptors and activates G protein-independent signaling. Even though it is a commonly used antihypertensive and α1-adrenoceptors are essential for the treatment of voiding symptoms in benign prostatic hyperplasia, its actions in the human prostate are still unknown. Here, we examined carvedilol effects on contractions of human prostate tissues, and on stromal cell growth.MethodsContractions of prostate tissues from radical prostatectomy were induced by electric field stimulation (EFS) or α1-agonists. Growth-related functions were examined in cultured stromal cells.ResultsConcentration-response curves for phenylephrine, methoxamine and noradrenaline were right shifted by carvedilol (0.1–10 µM), around half a magnitude with 100 nM, half to one magnitude with 1 µM, and two magnitudes with 10 µM. Right shifts were reflected by increased EC50 values for agonists, with unchanged Emax values. EFS-induced contractions were reduced by 21–54% with 0.01–1 µM carvedilol, and by 94% by 10 µM. Colony numbers of stromal cells were increased by 500 nM, but reduced by 1–10 µM carvedilol, while all concentrations reduced colony size. Decreases in viability were time-dependent with 0.1–0.3 µM, but complete with 10 µM. Proliferation was slightly increased by 0.1–0.5 µM, but reduced with 1–10 µM.ConclusionsCarvedilol antagonizes α1-adrenoceptors in the human prostate, starting with concentrations in ranges of known plasma levels. In vitro, effect sizes resemble those of α1-blockers used for the treatment of voiding symptoms, which requires concentrations beyond plasma levels. Bidirectional and dynamic effects on the growth of stromal cells may be attributed to "biased agonism".

Comparative characteristics of the stem cells’ number in the stromal vascular fraction of infrapatellar fat pad and subcutaneous fat tissue

ObjectivesThe use of infrapatellar fat pad adipose stem cells (IPFP-ASCs) shows an age-independent proliferation and differentiation potential. In addition, the pronounced chondrogenic potential of IPFP-ASCs makes them promising candidates for research for use in other methods of regenerative therapy. The purpose of this study was to ascertain the presence and compare the relative abundance of cells exhibiting an immunohistochemical profile characteristic of adipose-derived mesenchymal stem cells in selected samples of the stromal vascular fraction (SVF) obtained from the IPFP and subcutaneous fat tissue. MethodsA direct immunohistochemical study was carried out in serial paraffin sections of the SVF of the infrapatellar fat pad (IPFP) and subcutaneous tissue, using monoclonal antibodies. The minimum criteria were established by the International Society for Cell Therapy to ensure the identity of mesenchymal stem cells use CD73, CD90, and CD105 as positive markers and CD34, CD31, and CD45 as a negative. ResultsAccording to the results of histological, immunohistochemical, morphometric, and statistical studies, it was found that in the SVF of IPFP and subcutaneous adipose tissue, the relative number of cells with the profile CD105+, CD73+, CD34+, CD31−, CD45− in the standard field of view (×200), the SVF of IPFP was 1.58%, whereas the SVF of subcutaneous adipose tissue was 6.92 %, which was statistically significantly greater by 4.38 times (p ​< ​0.05). ConclusionThe presence of a sufficient number of mesenchymal stromal cells in IPFP in combination with their topographic relationship with the structures of the joint determines the use of the SVF of the IPFP for the treatment of diseases of the knee joint. Level of evidenceIII.

Metformin ameliorates endometrial thickness in a rat model of thin endometrium.

Metformin, a well-established anti-diabetic drug, is also used in managing various other metabolic disorders including polycystic ovarian syndrome (PCOS). There are evidences to show that metformin improves endometrial functions in PCOS women. However, fewer studies have explored the direct effects of metformin on endometrium. Previous in vitro studies have shown that therapeutic serum concentrations of metformin enhance endometrial epithelial cell proliferation. The present study was undertaken to investigate in vivo effects of metformin on endometrial proliferation in a rat model of thin endometrium. Toward this, a rat model of thin endometrium was developed. Metformin (0.1% or 1% w/v) was administrated orally for 15 days in rats with thin endometrium. Oral metformin administration for three consecutive estrous cycles (15 days) in the thin endometrium rat model led to an increase in endometrial thickness compared to sham endometrium. Histological analysis showed a significant increase in the number of endometrial glands (P < 0.05), stromal cells (P < 0.01) and blood vessels (P < 0.01) in metformin-treated (n = 10 in each group) uterine horns compared to sham (saline-treated) uterine horns in rats. The expression of proliferating cell nuclear antigen and vascular epithelial growth factor was found to be upregulated on treatment with 1% metformin-treated group (n = 7). However, pregnancy outcomes in the rats treated with metformin remained unaltered despite the restoration of endometrial thickness. In conclusion, the study demonstrated that metformin ameliorates endometrial thickness in a rat model of thin endometrium by increasing endometrial proliferation and angiogenesis, without restoration of embryo implantation.

Single-cell analysis reveals a unique microenvironment in peri-implantitis.

This study aimed to reveal the unique microenvironment of peri-implantitis through single-cell analysis. Herein, we performed single-cell RNA sequencing (scRNA-seq) of biopsies from patients with peri-implantitis (PI) and compared the results with healthy individuals (H) and patients with periodontitis (PD). Decreased numbers of stromal cells and increased immune cells were found in the PI group, which implies a severe inflammatory infiltration. The fibroblasts were found to be heterogeneous and the specific pro-inflammatory CXCL13+ sub-cluster was more represented in the PI group, in contrast to the PD and H groups. Furthermore, more neutrophil infiltration was detected in the PI group than in the PD group, and cell-cell communication and ligand-receptor pairs revealed most neutrophils were recruited by CXCL13+ fibroblasts through CXCL8/CXCL6-CXCR2/CXCR1. Notably, our study demonstrated that the unique microenvironment of the PI group promoted the differentiation of monocyte/macrophage lineage cells into osteoclasts, which might explain the faster and more severe bone resorption in the progression of PI than PD. Collectively, this study suggests a unique immune microenvironment of PI, which may explain the differences between PI and PD in the clinic. These outcomes will aid in finding new specific and effective treatments for PI.

Melatonin acts through different mechanisms to control oxidative stress and primordial follicle activation and survival during in vitro culture of bovine ovarian tissue

This study aims to evaluate the effects of melatonin and its mechanisms of action on preantral follicle activation and survival, stromal cell density and collagen distribution in extracellular matrix (ECM). The involvement of melatonin receptors and mTORC1 pathway in these procedures were also investigated. To this end, ovarian fragments were cultured for six days in α‐MEM+ alone or supplemented with 1000 pM melatonin, 1000 pM melatonin with 1000 pM luzindole (inhibitor of melatonin receptors), or 1000 pM melatonin with 0.16 µg/ml rapamycin (mTORC1 inhibitor). At the end of culture period, tissues were processed for classical histology, and the follicles were classified as normal or degenerated, as well as in primordial or growing follicles. The ovarian stromal cell density and ECM collagen distribution were also evaluated. Samples of ovarian tissues were also destined to measure the levels of thiol and mRNA for CAT, SOD, GPX1 and PRDX1, as well as the activity of antioxidant enzymes CAT, SOD, and GPX1. The results demonstrated that ovarian tissues cultured with melatonin, melatonin with luzindole or melatonin with rapamycin had significantly higher percentage of morphologically normal follicles than those cultured in control medium (α‐MEM+). However, the presence of either luzindole or rapamycin, did not block the positive effects of melatonin on follicle survival (P > 0.05). Although the presence of melatonin in culture medium reduced the percentage of primordial follicles and increased the percentage of development follicles, these positive effects of melatonin were blocked by either luzindole or rapamycin (P < 0.05). Melatonin, melatonin with luzindole or melatonin with rapamycin did not influence the number of ovarian stromal cells. In contrast, melatonin significantly increased the percentages of collagen in ovarian tissues, but the positive effects of melatonin were blocked by either luzindole or rapamycin. Tissues cultured with melatonin and rapamycin had higher levels of mRNA for CAT and lower GPx activity when compared to those cultured in control medium. In conclusion, melatonin promotes primordial follicle activation, increases collagen fiber in ECM of in vitro cultured bovine ovarian tissue through its membrane-coupled receptors and mTORC1. Oppositely, melatonin increase follicles survival by acting through other pathways, since it can pass through cell membranes and directly regulate oxidative stress.

Assessment of ovarian tissue and follicular integrity after cryopreservation via slow freezing or vitrification followed by in vitro culture

To evaluate ovarian tissue and follicle integrity before and after slow freezing or vitrification and postthawing invitro culture. A laboratory study using bovine ovarian cortical tissue. Academic laboratory. Ovaries from healthy cattle. Bovine ovarian cortical tissue was subjected to either slow freezing or vitrification and subsequent invitro culture. Tissue and follicle integrity were assessed before and after cryopreservation and culture. Hematoxylin and eosin staining was used to assess follicle stages, morphology, and stromal cell density. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to examine apoptosis, and Masson's trichrome staining was used to evaluate collagen content in the stromal environment. Immunofluorescent labeling was used to localize and quantify connexin 37 (CX37) and Ki67 expression. Regardless of previous cryopreservation, ovarian tissue culture resulted in a decreased percentage of primordial follicles and an increased percentage of primary follicles compared with fresh tissue, indicating that follicle activation was not negatively affected by cryopreservation. However, both culture and cryopreservation followed by culture decreased the percentage of normal preantral follicles compared with fresh tissue that had not been cultured. Culture and/or cryopreservation did not impact stromal cell number, but there was increased cell apoptosis in tissue that was cultured after vitrification compared with tissue that was not cultured. Tissue culture, regardless of cryopreservation, resulted in decreased collagen deposition. There were fewer follicles expressing CX37 in vitrified and thawed tissue compared with all other treatments. Cryopreservation and/or culture of ovarian tissue did not change the percentage of follicles that contained Ki67-positive granulosa cells or the percentage of Ki67-positive granulosa cells within those follicles. Based on these data, we conclude that tissue cryopreservation followed by culture does not affect follicle activation and growth, but it decreases the proportion of viable follicles within the tissue. Slow freezing was superior to vitrification as indicated by a higher proportion of follicles with normal morphology, lower stromal cell apoptosis, and maintenance of CX37 expression postthawing and after culture.

Impact of Latent Virus Infection in the Cornea on Corneal Healing after Small Incision Lenticule Extraction.

The aim of the present study is to analyze the impact of cornea virus latent infection on corneal healing after small incision lenticule extraction (SMILE) and predict the positive rate of virus latent infection in corneal stroma. A total of 279 patients who underwent SMILE were included in this study. Fluorescence quantitative PCR was used to detect virus infection in the lenticules, which were taken from the corneal stroma during SMILE. Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) were detected. Postoperative visual acuity, spherical equivalent, intraocular pressure, corneal curvature (Kf and Ks), corneal transparency, and corneal staining were compared between the virus-positive group and the virus-negative group. The number of corneal stromal cells and inflammatory cells, corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were evaluated using an in vivo confocal microscope. Out of 240 herpes simplex virus (HSV) tested samples, 11 (4.58%) were positive, among which 5 (2.08%) were HSV-1-positive and 6 (2.50%) were HSV-2-positive. None of the 91 CMV- and EBV-tested samples were positive. There was no statistical significance in the postoperative visual acuity, spherical equivalent, intraocular pressure, Kf and Ks, corneal transparency, corneal staining, the number of corneal stromal cells and inflammatory cells, CNFD, CNBD, CNFL, CTBD, and CNFW between the virus-positive and virus-negative groups (p > 0.05). In conclusion, there is a certain proportion of latent HSV infection in the myopia population. Femtosecond lasers are less likely to activate a latent infection of HSV in the cornea. The latent infection of HSV has no significant impact on corneal healing after SMILE.

Heterotypic Cell Culture from Mouse Bone Marrow under Simulated Microgravity: Lessons for Stromal Lineage Functions.

Muscle and skeleton structures are considered most susceptible to negative factors of spaceflights, namely microgravity. Three-dimensional clinorotation is a ground-based simulation of microgravity. It provides an opportunity to elucidate the effects of microgravity at the cellular level. The extracellular matrix (ECM) content, transcriptional profiles of genes encoding ECM and remodelling molecules, and secretory profiles were investigated in a heterotypic primary culture of bone marrow cells after 14 days of 3D clinorotation. Simulated microgravity negatively affected stromal lineage cells, responsible for bone tissue formation. This was evidenced by the reduced ECM volume and stromal cell numbers, including multipotent mesenchymal stromal cells (MSCs). ECM genes encoding proteins responsible for matrix stiffness and cell-ECM contacts were downregulated. In a heterotypic population of bone marrow cells, the upregulation of genes encoding ECM degrading molecules and the formation of a paracrine profile that can stimulate ECM degradation, may be mechanisms of osteodegenerative events that develop in real spaceflight.

Prognostic value of CD4&lt;sup&gt;+&lt;/sup&gt; in patients with Т1–2N0M0 breast cancer

Aim. To evaluate the prognostic value of CD4+ in patients with T1–2N0M0 breast cancer.Materials and methods. We performed a quantitative assessment of CD4+ T-lymphocytes in tumor stromal cells and determined the prognostic parameter using the method developed by us (patent No. RU2697709C1 dated 19.08.2019) in 394 patients diagnosed with infiltrative breast cancer. Survival of patients in the analyzed group was assessed over a period of 10 years.Archival material from paraffin blocks of the «target group» was used in the work for molecular genetic profiling techniques (T1–2N0M0, 1118 patients, 2000–2009). A histological preparation stained with hematoxylin and eosin was scanned, and the central and peripheral zones of the tumor were isolated. To identify CD4+ T-lymphocytes, the immunohistochemical study of preparations obtained with rabbit anti-CD4 monoclonal antibodies was performed. After rescanning histological and immunohistochemical preparations, combining different immunohistochemical stains, the cell density in the tumor and stromal components was calculated in preparations stained with hematoxylin and eosin and CD4. For further calculations, we used the data obtained as a result of the number of stromal cells reduced to 1 mm2 (according to the preparation stained with hematoxylin and eosin), the so-called cell density per calculated number of CD4+-stained cells (cell area × proportion of CD4+ cells in percentages). The resulting ratio is used to predict the outcome of breast cancer: if its value is ≤ 50 %, the prognosis is assessed as favorable (in which the patient’s survival is 10 years or more).Results. In 296 (75 %) of 394 observed patients, the proportion of CD4+ T-lymphocytes in tumor stromal cells was ≤ 50 %. Over 10 years, the overall survival of patients in this group was 93 %, which is considered a statistically favorable group for breast cancer (stages T1–2, N0). In 98 (25 %) of 394 observed patients, the proportion of CD4+ T-lymphocytes in tumor stromal cells was ˃50 %. Over 10 years, the overall survival of patients in this group was 82 %, which is considered a statistically unfavorable group for breast cancer (stages T1–2, N0).Conclusion. Our findings suggest high accuracy of our method of breast cancer prognosis based on the quantitative assessment of CD4+ T-lymphocytes in tumor stromal cells. It can be used for breast cancer prognosis in early stages (T1–2, N0).

P-677 Letrozole inhibits ovulation via reducing responsiveness to luteinizing hormone in in vitro-grown mouse follicles

Abstract Study question Does letrozole reduce responsiveness to luteinizing hormone (LH) in in vitro-grown mouse follicles? Summary answer We found that letrozole reduced responsiveness to LH via reducing of luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) transcription in in vitro. What is known already The continuous dosing of letrozole, the third-generation aromatase inhibitor, is used for the controlled ovarian stimulation (COS) of fertility preservation cycles in women with hormone-sensitive breast cancer to prevent the transient rise of serum estrogen levels. Recently, lower oocyte maturation rates and higher abnormal fertilization rates in COS with letrozole compared to standard COS have been reported. The studies using aromatase knockout mice and estrogen receptor beta knockout mice showed that intra-follicular estrogen is essential for responsiveness to LH of granulosa cells. However, whether letrozole reduces responsiveness to LH of follicles remains unknown. Study design, size, duration We evaluated the effect of letrozole on responsiveness to LH using in vitro mouse follicle culture system. Preantral follicles (150-200 µm) with a small number of stromal cells were isolated from 3 weeks old C57BL/6jjcl mouse ovaries and cultured in the presence of letrozole. We analyzed in vitro ovulation ability, and expression levels of ovulation-related genes to clarify the effect of letrozole on responsiveness to LH of follicles. Participants/materials, setting, methods The follicles were cultured with letrozole (0.01µM, 0.1µM, or 1µM) or vehicle (0.1% DMSO) for 5 days followed by 5 IU/ml hCG and 5 ng/ml hEGF stimulation as ovulation induction. The diameter and survival rate of follicles were assessed. After16 hours after ovulation induction, we assessed the ovulation and measured the transcription of Lhcgr, Ptgs2, Runx1 and Actb (internal control) in the follicles by RT-qPCR. We conducted a collateral experiment in the presence of estradiol. Main results and the role of chance The follicle growth and survival rate did not change by letrozole in vitro. However, in vitro ovulation was reduced by the addition of letrozole in a dose-dependent manner (Cochran-Armitage trend test, P &amp;lt; 0.0001): 41.2% in the 0.01µM letrozole treated group (n = 23), 10.5% in the 0.1µM letrozole treated-group (n = 17), and no ovulation in the 1µM letrozole treated-group (n = 19) compared with 69.6% in the vehicle-group (n = 18). Exogenous estrogen addition restored the ovulation ability of 1µM letrozole-treated follicle to the same levels as the vehicle group. RT-qPCR revealed that 0.1 µM letrozole significantly reduce the transcription levels of Lhcgr (P = 0.0027), Runx1 (P = 0.0124), and Ptgs2 (P = 0.0067) compared with vehicle-treated follicles. Exogenous estrogen addition restored the transcription of these genes to levels observed in vehicle-treated follicles. We also observed a positive correlation between the transcription of Lhcgr and Ptgs2 (R = 0.55, P = 0.0003), Lhcgr and Runx1 (R = 0.71, P &amp;lt; 0.0001), and Ptgs2 and Runx1 (R = 0.77, P &amp;lt; 0.0001). Our results suggested that letrozole-treated follicles impaired ovulation due to dysregulation of Lhcgr transcription and its downstream cascade caused by estrogen deficiency. Limitations, reasons for caution This is an in vitro experiment using a mouse model. Further research is needed to determine whether our findings are applicable to humans. Wider implications of the findings Our findings imply that letrozole may have disadvantages in not only ovulation, but also subsequent reproductive processes affected by the effectiveness of the LH surge, such as oocyte maturation, fertilization, and preimplantation embryo development. When evaluating oocyte cryopreservation for fertility preservation, it may be necessary to consider the COS protocol. Trial registration number Not applicable

Pancreatic Tissue Dissection to Isolate Viable Single Cells.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Abstract A27: Cellular barcoding of the leukemic niche reveals an apelin-mediated clonal expansion of niche endothelial and mesenchymal stromal cell clones

Abstract Hematopoietic stem and progenitor cells (HSPCs) reside in niches that provide regulatory signals for their function. Perturbations such as acute leukemia induce cellular and molecular insults to the niche to support disease progression. Mutation of the MYC oncogene family is a frequent event leading to leukemogenesis. We developed a new zebrafish model of acute erythroid leukemia (AEL) by overexpression of human CMYC under the blood specific promotor draculin (drl). Analyses of drl:CMYC marrows demonstrated a significant expansion of progenitors and a decrease of erythroid, lymphoid and myeloid mature cells (fc=4.8, -4.5, -3.3, -6.5; p&amp;lt;0.000001). RNA-Sequencing of drl:CMYC marrows revealed an upregulation of the erythroid master regulator gata1a (fc=1.4, p=0.01) and fetal hemoglobins hbbe1.1/2 (fc=4.7, 2.9; p=0.0004). Primary and secondary transplantation of drl:CMYC marrows resulted in engraftment and disease propagation (7/7; 17/18). We used the cellular barcoding GESTALT technique to uniquely barcode single cells using CRISPR-CAS9 during embryonic development. We induced a round of barcoding at the one-cell stage and another one at 28 hours post-fertilization, the time of HSC birth. We injected these GESTALT embryos with drl:CMYC to induce AEL, barcode HSPCs and their niche to perform clone tracing. The number of HSPC clones was decreased by half compared to controls (p=0.008) indicative of a clonal expansion of the disease. We performed barcode and single-cell transcriptome profiling of flk1:GFP+ niche endothelial cells and found a significant decrease in the number of endothelial cells clones (fc= -3.5, p&amp;lt;0.05) paired with the identification of a novel AEL venous endothelial population upregulating 99 genes (fc&amp;gt;1; p&amp;lt;0.05) suggestive of angiogenesis likely supporting leukemogenesis. We sorted cxcl12a:dsRed+ niche stromal cells and found that AEL marrows have significantly less stromal clones (fc= -2.1, p&amp;lt;0.01) that are selectively amplified (&amp;gt;20% of the stromal compartment). We hypothesized that AEL expands a subset of stromal cells to promote disease progression and scRNA-Seq of 3,263 cxcl12a:dsRed+ stromal cells revealed an increased fraction of lepr+ mesenchymal stromal cells (MSCs, 66 vs 24% in controls). We hypothesized that AEL progenitors secrete a signal that can specifically remodel the marrow niche and we mined our scRNA-Sequencing data for a candidate ligand significantly upregulated in AEL cells compared to control HSPCs (fc&amp;gt;1; p&amp;lt;0.05) and paired receptors expressed on more than 20% of AEL associated endothelial and/or stromal cells. We identified the peptide hormone apelin expressed by the leukemia and tested if apelin alone could remodel the normal marrow niche endothelial and stromal cells by overexpressing apelin under the drl promotor. Adult marrow analyses revealed a decrease in the absolute number of stromal cells per marrow (fc= -2.2, p&amp;lt;0.001). Together our data support a model in which leukemia induces a clonal expansion of HSPC niche clones via apelin signaling to promote disease progression. Citation Format: Chloé S Baron, Serine Avagyan, Song Yang, Aaron McKenna, Leonard Zon. Cellular barcoding of the leukemic niche reveals an apelin-mediated clonal expansion of niche endothelial and mesenchymal stromal cell clones [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A27.

Concentrated ultrasound-processed fat (CUPF): More than a mechanically emulsified graft

Autologous fat grafting is still an evolving technique. Researchers have attempted to increase the survival rate of grafts by concentrating adipose-derived stem cells (ASCs). In this study, we investigate a novel method that combines ultrasonic processing and centrifugation to generate small fat particles termed concentrated ultrasound-processed fat (CUPF) for grafting. The standard approach for obtaining CUPF is described. The properties of processed fat, including CUPF, microfat, centrifuged fat, and nanofat, were investigated using histological observation. Comparative analyses were conducted on the cell number, viability, and immunophenotypic profile of stromal vascular fraction cells (SVFs). Cultured ASCs were evaluated for cell proliferation and adipogenic, osteogenic, and chondrogenic potential. The processed fats were transplanted and evaluated using in vivo and histological studies. Compared with microfat, centrifuged fat, and nanofat, CUPF had a condensed tissue content and higher concentration of viable cells in a small tissue structure and could smoothly pass through a 27-gauge cannula. In the CUPF group, SVFs were isolated in great numbers, with high viability and a high proportion of CD29- and CD105-positive cells. ASCs from the CUPF group exhibited high proliferation and multilineage differentiation potential. The grafts from the CUPF group were well preserved, and histological quantification revealed an increase in the abundance of Ki67- and CD31-positive cells in the tissue. Our study established a new fat processing strategy that combines ultrasonic processing and centrifugation to harvest small particle grafts named CUPF. CUPF concentrated a large number of ASCs and has great potential for regenerative therapy.

Patient Demographic Factors Are Not Associated With Mesenchymal Stromal Cell Concentration in Bone Marrow Aspirate Concentrate

To describe the capacity for concentration of a single processing machine for bone marrow aspirate concentrate (BMAC) production and investigate the effects of demographic factors on the number of mesenchymal stromal cells (MSCs) in BMAC. Patients enrolled in our institution's randomized control trials involving BMAC who had complete BMAC flow cytometry data were included. Multipotent MSC phenotype, defined as cell-surface coexpression of specific-identifying antigens (≥95% positive) and the absence of hematopoietic lineage markers (≤2% positive), was determined for both patient bone marrow aspirate (BMA) and BMAC samples. The ratio of cells in BMA:BMAC samples was calculated and Spearman correlations (i.e., body mass index [BMI]) and Kruskall-Wallis (i.e., age: <40, 40-60, >60 years) or Mann-Whitney (i.e., sex) tests were used to determine the relationship of cell concentration to demographic factors. Eighty patients were included in analysis (49% male, mean age: 49.9 ± 12.2 years). Mean concentration of BMA and BMAC was 2,048.13 ± 2,004.14 MSCs/mL and 5,618.87 ± 7,568.54 MSC/mL, respectively, with a mean BMAC:BMA ratio of 4.35 ± 2.09. A significantly greater MSC concentration was observed in the BMAC samples when compared with BMA (P= .005). No patient demographic factors (age, sex, height, weight, BMI) were found to predict MSC concentration in the BMAC samples (P ≥ .01). Demographic factors, including age, sex, and BMI do not impact the final concentration of MSCs in BMAC when using a single harvest technique (anterior iliac crest) and a single processing system. As the role of BMAC therapy expands in clinical application, it becomes increasingly important to understand the determinants of BMAC composition and how it is affected by different harvesting techniques, concentrating processes, and patient demographics.

Ubiquitin-Specific Protease 43 Impacts Pancreatic Ductal Adenocarcinoma Prognosis by Altering Its Proliferation and Infiltration of Surrounding Immune Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer, and the therapy options for PDAC remain restricted. The distinctive tumor immunological microenvironment (TIME) of PDAC, comprising a high number of stromal cells and a limited infiltration of cytotoxic T lymphocytes (CTLs), rendered immunotherapy ineffective. The protein level of ubiquitin-specific protease 43 (USP43) was a prognostic predictor in numerous cancers; however, its function in PDAC is limited. This article focuses on the influence of USP43 expression on PDAC prognosis and TIME alteration. Based on TCGA database and tissue microarray staining, the expression of USP43 in PDAC was evaluated. The association between USP43 and prognosis was then investigated using tissue samples and online databases. In PDAC tumor tissues, the correlation between USP43 expression and clinicopathological characteristics, immune cell infiltration, and prognosis was investigated. The expression of USP43 in PDAC cell lines was evaluated using quantitative polymerase chain reaction. Using a cell counting kit-8 (CCK-8) and a cell colony formation test, the viability of the cells was determined. On the basis of online databases and tissue samples, the link between USP43 and immune cell infiltration around PDAC was also examined. For statistical analyses, the software GraphPad, R, and SPSS 26.0 were utilized. The expression of USP43 was considerably higher in PDAC compared to normal pancreatic tissue in both the TCGA database and the tissue microarrays of PDAC patients (P < 0.001). High USP43 expression was associated with poor overall survival in both the TCGA database and the tissue microarray of PDAC patients (P = 0.046 and 0.021, respectively). USP43 overexpression promoted PANC-1 cell proliferation (P = 0.0018), but USP43 knockdown decreased PANC02 cell proliferation (P < 0.001). According to the TCGA database, USP43 is associated with T cell activation and inhibits CD8+ T cell activation in PDAC, as proven by a study of cell lines. Moreover, in both TCGA and PDAC cell lines, USP43 expression was negatively linked with the chemokine signaling pathway. Overexpression of USP43 is a potential prognostic indicator for PDAC patients. USP43 is a potential biomarker associated with T cell activation, suppression of CD8+ T cell enrichment, and the cytokine signal pathway. Future multicenter studies are needed to confirm our findings and their potential application in the treatment of PDAC patients.

A STUDY ON TRANS URETHRAL RESECTION OF PROSTATE FOR SYMPTOMATIC BPH PRESENTING IN A TERTIARY CARE HOSPITAL

Introduction: Benign prostatic hyperplasia (BPH) is a common chronic progressive disease resulting in urinary obstruction in aging men. It is a pathologic process that consists of androgen-dependent increases in the number of epithelial and stromal cells in the periurethral area of the prostate. BPH contributes to, but is not the sole cause of, lower urinary tract symptoms in aging men. Many patients require surgical treatment and presently TURP (Trans Urethral Resection of Prostate) is considered the gold standard in surgical management of BPH. Aim: To observe and assess the outcome of TURP being done in Narayan Medical College and Hospital Sasaram, Bihar. Materials and Methods: The records of patients who presented to the surgical OPD with LUTS and were assessed to undergo TURP for prostatic hyperplasia. We present our experience with series of 47 patients and can say that TURP can be used for most patients presenting with BPH in peripheral areas. Result: In the study period 47 TURP procedures were performed for BPH. The mean age of the cohort was 67.9 years;( ranging from 56 to 84 years). The mean prostatic volume was 44.06 grams (range 32 to 76 grams) and mean operating time was 48 minutes (range 29 to 85 minutes). Blood transfusion was required in 3 patients (6.3%). No patients required open intervention and there were no mortalities. The common complications were bleeding (12%), UTI and clot retention. Conclusions: TURPis not available to majority of rural population having symptomatic benign prostatic hyperplasia (BPH) due to lack of facility but remains the treatment of choice provided trained surgeons and facilities are there. The overall cost of TURP is marginally higher for an average rural patient as compared to open surgery but is worthwhile in view of its inherent minimal trauma, short hospital stays and early recovery and huge cost advantage over newer therapies like HoLEPor PVP

Changes in Lower Urinary Tract Symptoms in Benign Prostatic Hyperplasia Patients Affected by Covid-19

Introduction: Benign prostatic hyperplasia (BPH) is a histological diagnosis characterized by an increase in the number of epithelial and stromal cells in the transitional zone of the prostate gland. The clinical manifestation of BPH is associated with the appearance of so-called lower urinary tract symptoms (LUTS) which can also be a consequence of other conditions not related to the prostate. Covid-19, also known as severe acute respiratory syndrome coronavirus 2 was discovered as a disease in late 2019 in the city of Wuhan, China. Materials and Methods: A case-control study was conducted between September 2021 and May 2022. Information was initially collected on 60 patients followed up and treated conservatively for BPH in two outpatient practices and recovered from Covid-19. After processing the received information, 27 patients were excluded from the study and 33 patients remained for observation The assessment of patients is carried out through an internationally validated questionnaire – international prostate symptom score (IPPS), prostate specific antigen, digital rectal examination (DRЕ), and ultrasound diagnostics of the prostate gland with consideration of its volume and the amount of residual urine. Results: Our data show a strong correlation between the changes in LUTS in patients with BPH and Covid-19 infection. Conclusion: Although almost 3 years have passed since the beginning of this pandemic, there are still many unanswered questions surrounding this disease. We believe that with our experience we will enrich the information about the relationship between Covid-19 and LUTS, and the results obtained by us can serve as a basis for future large-scale and more in-depth studies on the subject.