Stromal Tissue Research Articles

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

BackgroundMesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.MethodsA total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.ResultsBy comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.ConclusionsWe established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Immunocompetent PDMS-Free Organ-on-Chip Model of Cervical Cancer Integrating Patient-Specific Cervical Fibroblasts and Neutrophils.

Despite preventive measures and available treatments, cervical cancer still ranks as the fourth most prevalent cancer among women worldwide and remains the leading cause of cancer death in women in many developing countries. To gain further insights into pathogenesis and to develop novel (immuno)therapies, more sophisticated human models recreating patient heterogeneities and including aspects of the tumor microenvironment are urgently required. A novel polydimethylsiloxane-free microfluidic platform, designed specifically for the generation and ccultivation of cervical cancerous tissue, is introduced. The microscale open-top tissue chambers of the cervical cancer-on-chip (CCoC) enable facile generation and long-term cultivation of SiHa spheroids in co-culture with donor-derived cervical fibroblasts. The resulting 3D tissue emulates physiological architecture and allows dissection of distinct effects of the stromal tissue on cancer viability and growth. Treatment with cisplatin at clinically-relevant routes of administration and dosing highlights the platform's applicability for drug testing. Moreover, the model is amenable for integration and recruitment of donor-derived neutrophils from the microvasculature-like channel into the tissue, all while retaining their ability to produce neutrophil extracellular traps. In the future, the immunocompetent CCoC featuring donor-specific primary cells and tumor spheroids has the potential to contribute to the development of new (immuno)therapeutic options.

Donut-shaped Corneal Allogeneic Intrastromal Segment as an Alternative to Deep Anterior Lamellar Keratoplasty in Advanced Keratoconus.

The aim of this study was to describe a new technique of selective corneal stromal transplantation for keratoconus treatment, donut-shaped CAIRS (corneal allogeneic intrastromal ring segment). A donut-shaped corneal graft is obtained using a double-bladed trephine. Descemet membrane, endothelium, and epithelium are all removed from the graft. Only stromal tissue is transplanted. A wide 360-degree intrastromal tunnel is created using the femtosecond laser, with a 30-degree angulation. The diameter is from 5.4 mm to 8 mm optical zone. After dehydration, the corneal graft is inserted into the tunnel. We report the clinical and tomographic outcomes after the procedure in 3 patients. The mean follow-up time after donut-shaped CAIRS was 6.01 ± 1.02 months. In case 1, best spectacle-corrected visual acuity improved from 20/150 to 20/40. In case 2, it improved from 20/400 to 20/40, and in case 3 from 20/200 to 20/40. The mean preoperative K was 57.3 ± 4.5 D and reduced to 44.2 ± 2.5 D after donut-shaped CAIRS. The mean spherical equivalent decreased from -9.8 ± 3.2 preoperatively to -3.2 ± 2.2 postoperatively. No intraoperative or postoperative complications were observed. Anterior segment OCT showed a mid-stroma implant, fusiform in shape, equidistant from the epithelium and endothelium. Donut-shaped CAIRS is a variation of the traditional CAIRS technique and showed to be an alternative option for keratoconus treatment, especially in moderate to advanced cases with a central clear cornea without scars. The technique is minimally invasive, and the visual axis remains untouched.

Ayurvedic Management of Adenomyosis

Adenomyosis is a gynaecologic condition characterized by ectopic endometrial tissue within the uterine myometrium. Adenomyosis, if not treated can lead to endometriosis, and hence can lead to more complications and hysterectomy might be the option. But by using Ayurvedic formulation with properties like Anulomaka, Vatashamaka, Lekhagana, Rakta prasadaka etc properties patient got significant relief from pain and irregular menses along with improvement in quality of life. Uterine adenomyosis is a benign condition in which endometrium like epithelial and stromal tissues appear in myometrium, surrounded by hypertrophic smooth muscle. Endometrial cells from the lining of endometrial cavity, migrate; most commonly into the posterior side or back wall of uterus, as these cells respond to monthly hormonal change. Severity and symptoms associated with adenomyosis, directly proportional to degree of involvement and penetration of uterine muscle. Adenomyosis can cause heavy menstrual bleeding (cyclic or acyclic) or scanty menstruation (oligomennorhoea or hypomenorrhoea) along with dysmenorrhoea. So in this case study patient having adenomyosis presented symptom of dysmenorrhoea with scanty menses and irregular menses. In this study there is Sanga of Doshas predominantly of Vata- kapha, so Lekhana is accepted and Vatavaigunya is present were Mamsa-rakta dusti is present, so Vatanulomana and Rakta prasadan chikitsa is adapted. So adenomyosis can be compared in Ayurveda with Udavartini yonivyapada among 20 Yonivyapada.

Therapeutic Potential of Natural Resources Against Endometriosis: Current Advances and Future Perspectives.

Endometriosis (EMS) is defined as the appearance, growth, infiltration, and repeated bleeding of endometrioid tissue (glands and stroma) outside the uterus cavity, which can form nodules and masses. Endometriosis is a chronic inflammatory estrogen-dependent disease and occurs in women of reproductive age. This disorder may significantly affect the quality of life of patients. The pathogenic processes involved in the development and maintenance of endometriosis remain unclear. Current treatment options for endometriosis mainly include drug therapy and surgery. Drug therapy mainly ties to the use of non-steroidal anti-inflammatory drugs (NSAIDs) and hormonal drugs. However, these drugs may produce adverse effects when used for long-term treatment of endometriosis, such as nausea, vomiting gastrointestinal reactions, abnormal liver and kidney function, gastric ulcers, and thrombosis. Although endometriosis lesions can be surgically removed, the disease has a high recurrence rate after surgical resection, with a recurrence rate of 21.5% within 2 years and 40% to 50% within 5 years. Thus, there is an urgent need to develop alternative or additional therapies for the treatment of endometriosis. In this review, we give a systematic summary of therapeutic multiple component prescriptions (including traditional Chinese medicine and so on), bioactive crude extracts of plants/herbs and purified compounds and their newly found mechanisms reported in literature in recent years against endometriosis.

Feasibility of 3D bioprinting of a corneal stroma tissue equivalent using a desktop 3D printer

Aims/Purpose: The aim of this poster is to present a proof of concept for the possibility of three‐dimensional bioprinting of a corneal stroma equivalent, using a modified (open source) three‐dimensional desktop printer.Methods: We present the experimental process designed for the realization of the three‐dimensional bioprinting of a corneal stroma equivalent, utilizing a tissue‐specific deposition nozzle based on commercially available and easily printable parts. The final design considers all the required modifications of the 3D printer and the selection of the appropriate bioink.Results: We designed a prototype proprietary device to be fitted on a MendelMax 3 3D printer. The particular open‐source device works as a syringe extruder, with the syringe containing the bioink. The composition of the bioink is based on a carefully selected blend of collagen, alginate, and keratocytes. The mixture demonstrates suitable structural integrity and transparency after crosslinking.Conclusions: This proof‐of‐concept work demonstrates that, with proper modification, a standard commercial three‐dimensional printer can be modified to use bioinks and bioprint corneal stroma tissue equivalents. More research is required regarding the viability of the tissue equivalents and their clinical applications.

Tumor proliferation and invasion are intrinsically coupled and unraveled through tunable spheroid and physics-based models

Proliferation and invasion are two key drivers of tumor growth that are traditionally considered independent multicellular processes. However, these processes are intrinsically coupled through a maximum carrying capacity, i.e., the maximum spatial cell concentration supported by the tumor volume, total cell count, nutrient access, and mechanical properties of the tissue stroma. We explored this coupling of proliferation and invasion through in vitro and in silico methods where we modulated the mechanical properties of the tumor and the surrounding extracellular matrix. E-cadherin expression and stromal collagen concentration were manipulated in a tunable breast cancer spheroid to determine the overall impacts of these tumor variables on net tumor proliferation and continuum invasion. We integrated these results into a mixed-constitutive formulation to computationally delineate the influences of cellular and extracellular adhesion, stiffness, and mechanical properties of the extracellular matrix on net proliferation and continuum invasion. This framework integrates biological in vitro data into concise computational models of invasion and proliferation to provide more detailed physical insights into the coupling of these key tumor processes and tumor growth. Statement of significanceTumor growth involves expansion into the collagen-rich stroma through intrinsic coupling of proliferation and invasion within the tumor continuum. These processes are regulated by a maximum carrying capacity that is determined by the total cell count, tumor volume, nutrient access, and mechanical properties of the surrounding stroma. The influences of biomechanical parameters (i.e., stiffness, cell elongation, net proliferation rate and cell-ECM friction) on tumor proliferation or invasion cannot be unraveled using experimental methods alone. By pairing a tunable spheroid system with computational modeling, we delineated the interdependencies of each system parameter on tumor proliferation and continuum invasion, and established a concise computational framework for studying tumor mechanobiology.

Automated Analysis of Extracellular Matrix Invasion of Cancer Cells from Tumor Spheroids.

The main cause of mortality among cancer patients is metastatic disease. Metastasis develops from cancer cells that invade the stromal tissue and intravasate the circulatory or lymphatic systems to eventually form new tumors in other organs. Blocking cancer cell invasion can potentially prevent or reduce the metastatic progression of cancers. Testing different chemical compounds against cell invasion in three-dimensional cultures is a common laboratory technique. The efficacy of the treatments is often evaluated from confocal microscopic images of the cells using image processing. However, the analysis approaches are often subject to variations and inconsistencies due to user decisions that must be made while processing each image. To overcome this limitation, we developed a fully automated method to quantify the invasion of cancer cells from a 3D tumor spheroid into the surrounding extracellular matrix. We demonstrated that this method resolves cell invasion from spheroids of different shapes and sizes and from cells that invade as a cluster or individually. We also showed that this approach can help quantify the dose-dependent anti-invasive effects of a commonly used chemotherapy drug. Our automated method significantly reduces the time and increases the consistency and accuracy of cancer cell invasion analysis in three-dimensional cultures.

Hypomethylation of the ENPP3 promoter region contributes to the occurrence and development of ovarian endometriosis via the AKT/mTOR/4EBP1 signaling pathway.

Growing evidence indicates that aberrant methylation is pivotal in the development and progression of endometriosis (EMs). This study explores the relationship between abnormal methylation of the ENPP3 promoter and the pathogenesis of ovarian EMs, focusing on its regulatory effect on ENPP3 expression. We analyzed the methylation levels of ENPP3 in ectopic endometrial tissues from ovarian EMs patients and in normal endometrial tissues from women without EMs. The expression and distribution of ENPP3 were evaluated using RT-qPCR and immunohistochemistry. Transwell assays were conducted to examine the impact of ENPP3 overexpression on the migratory and invasive capabilities of endometrial stromal cells. Our results demonstrated significantly reduced methylation levels at the CpG sites of the ENPP3 promoter region in ectopic endometrial tissues compared to normal endometrial tissues. RT-qPCR findings revealed a marked increase in ENPP3 expression in ovarian EMs tissues relative to endometrial tissues from patients without EMs, and this upregulation was negatively correlated with the methylation levels of the ENPP3 promoter region. Immunohistochemical analyses confirmed elevated ENPP3 expression in the glandular epithelial cells and stroma of ovarian EMs tissues. Furthermore, in vitro experiments showed that overexpressed ENPP3 notably intensified the invasion and migration of endometrial stromal cells. Transcriptome sequencing and functional analyses indicated that the increased ENPP3 expression activated the AKT/mTOR/4EBP1 signaling pathway. In summary, the study suggests that hypomethylation in the ENPP3 promoter region may contribute to the initiation and advancement of ovarian EMs by activating the AKT/mTOR/4EBP1 pathway, supporting the theory that EMs might be an epigenetically regulated disorder.

Rutin-loaded chitosan nanoparticles alleviated Freund's adjuvant induced rheumatoid arthritis via modulating oxidative stress and inflammatory parameters in Wistar rats.

Rheumatoid arthritis (RA) is the most common chronic inflammatory disease, primarily affecting the joints and with stromal tissue dysregulation causing chronic inflammation and joint destruction. Rutin is a natural flavonoid with potential therapeutic properties in chronic destructive conditions including rheumatoid diseases. In this study, the protective effects of rutin nanoformulation in an animal model of rheumatoid arthritis caused by Freund's complete adjuvant (FCA) were investigated. Sixty male rats were randomly divided into ten groups including normal, negative control, prednisolone 10 mg/kg (positive control), 3 doses of rutin (15, 30, 45mg/kg), rutin nanoparticles (15, 30, 45 mg/kg), and nanoparticle without rutin, for 28 days. Different behavioral parameters including the open field test, acetone drop test, hot plate test, Von Frey test, and inclined plane test were evaluated. Serum levels of glutathione (GSH), catalase, and nitric oxide as well as histopathological analyses were measured in different groups. Also, matrix metalloproteinase (MMP)-2 and MMP-9 activity were appraised by gelatin zymography. The injection of FCA prolonged the rats' immobility duration in comparison to the control group. Rheumatoid arthritis induction also increased nitric oxide and decreased GSH and catalase levels, while these effects were reversed in the groups that received nanoparticles containing rutin and prednisolone. Rutin nanoparticles suppressed MMP-9 and activated MMP-2. Also, this rutin drug delivery system plays a significant role in the improvement of histopathological symptoms. Considering the improvement of behavioral and tissue symptoms and the modulation of the level of inflammatory cytokines, nanoparticles containing rutin can be proposed as a suitable approach in the management of patients with rheumatoid arthritis.

Transcriptomics Analysis of Stromal Breast Cancer Tissues Reveals Up-Regulation of GABA-A Receptor Activity

Breast cancer is the second most prevalent cancer overall among newly diagnosed cases and the most common cancer among women. 90% of cancer deaths are caused by metastasis, which is a factor that precedes local invasion, but very little is understood about the molecular mechanisms of invasion and metastasis. Thus, uncovering the root causes of this illness at the Transcriptomics level may result in a cutting-edge method of treating breast cancer. The total RNA microarray processed data from GEO for breast cancer patients was analyzed to discover hidden differences between epithelial breast cancer tissues (ET), stromal breast cancer tissues (ST), normal control epithelial breast cancer tissue samples (EC), and normal control stromal breast cancer tissue samples (SC) at the Transcriptomics level. Therefore, multiple bioinformatics analyses of the transcriptional profiles of 64 samples—including 28 TEC, 28 SCC, 5 EN, and 5 SN controls—received from the NCBI-Bio project were performed in the current study (PRJNA107497). First, ET versus EC and ST vs SC samples showed significant patterns in exploratory data analysis based on gene expression data using principal component analysis (PCA). Following this, 13512 substantially differentially expressed genes (Fold change (&gt;= 1.5), p.adj value 0.1) between these conditions were found by Welch's T-test differential gene expression analysis. This study identifies the main element that may significantly contribute to the spread of breast cancer from epithelial cells to stromal cells in the mammary glands as the gene like the GBRP. This study was also able to identify the affected biological pathways for both the ST vs. SC samples and the ET vs. EC samples as a result of the up-regulated and down-regulated genes. This most surely provides a crucial hint about the cause of the deadly metastatic cancer problem. Finally, the results presented here provide novel insights on breast cancer metastasis.

P53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.

Exploration of cancer associated fibroblasts phenotypes in the tumor microenvironment of classical and pleomorphic Invasive Lobular Carcinoma.

Multiplex immunofluorescence were performed on formalin fixed paraffin embedded tissues using Opal-7 color kit. The antibodies used for phenotyping CAFs were Pan CK (AE1/AE3), CD45, A-SMA, FAP, S100, Thy-1 with optimized dilutions. The images were acquired and analyzed using Vectra 3.0 imaging system and InForm software respectively. We studied 19 different CAFs colocalized phenotypes in the tumor, stroma and overall tissue compartments between classic and pleomorphic ILC. Total A-SMA+, A-SMA+FAP+S100+ and A-SMA+S100+ CAFs demonstrated higher densities in classic ILC cases while FAP+S100+ and S-100+ CAFs were increased in the pleomorphic subtype samples. Our study explores multiple CAFs phenotypes between classical and pleomorphic ILC. We showed that CAFs subset differ between Classic ILC and Pleomorphic ILC. A-SMA CAFs are more prevalent in the TME of classic ILCs whereas Pleomorphic ILCs are dominated by CAFs without A-SMA expression. This also iterates the importance of exploring this particular type of breast carcinoma in more detail, paving the way for meaningful translational research.

BHLHE40 Inhibits Ferroptosis in Pancreatic Cancer Cells via Upregulating SREBF1.

Pancreatic cancer (PCa) is one of the most fatal human malignancies. The enhanced infiltration of stromal tissue into the PCa tumor microenvironment limits the identification of key tumor-specific transcription factors and epigenomic abnormalities in malignant epithelial cells. Integrated transcriptome and epigenetic multiomics analyses of the paired PCa organoids indicate that the basic helix-loop-helix transcription factor 40 (BHLHE40) is significantly upregulated in tumor samples. Increased chromatin accessibility at the promoter region and enhanced mTOR pathway activity contribute to the elevated expression of BHLHE40. Integrated analysis of chromatin immunoprecipitation-seq, RNA-seq, and high-throughput chromosome conformation capture data, together with chromosome conformation capture assays, indicate that BHLHE40 not only regulates sterol regulatory element-binding factor 1 (SREBF1) transcription as a classic transcription factor but also links the enhancer and promoter regions of SREBF1. It is found that the BHLHE40-SREBF1-stearoyl-CoA desaturase axis protects PCa cells from ferroptosis, resulting in the reduced accumulation of lipid peroxidation. Moreover, fatostatin, an SREBF1 inhibitor, significantly suppresses the growth of PCa tumors with high expressions of BHLHE40. This study highlights the important roles of BHLHE40-mediated lipid peroxidation in inducing ferroptosis in PCa cells and provides a novel mechanism underlying SREBF1 overexpression in PCa.

Investigation of MT1-MMP Activity in Cancer Cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP, also called MMP14) is one of the significant cell invasion drivers. MT1-MMP has been shown to play a crucial role in cancer invasion, cartilage degradation in rheumatoid arthritis, angiogenesis, and collagen homeostasis in different stromal tissues. Thus, investigating MT1-MMP activities in different cell types is of interest to investigators in different research fields. Several methods are available to assess the unique biological activity of MT1-MMP in the cells. This chapter describes various cell-based assays to evaluate unique MT1-MMP activity.

Abstract C168: Prognostic value of tumor microenvironment (TME) features in advanced, EGFR-mutant non-small cell lung cancer (NSCLC)

Abstract Background: Prognostic role of tumor microenvironment (TME) features, although well-studied in the context of immunotherapy responses, remains unclear for oncogene-driven non-small cell lung cancer (NSCLC). Here, we interrogate the impact of TME features on survival outcomes of patients with advanced EGFR-mutant NSCLC. Methods: A single-center, retrospective study was carried out on patients with advanced EGFR-mutant (exon 19 deletion/L858R) NSCLC who underwent first-line targeted therapy or chemotherapy. Clinical, radiological, molecular, pathological data were collected. Hematoxylin-eosin diagnostic tissue slides, digitalized at 20x magnification, were analyzed by an expert pathologist and a medical oncologist, blinded to clinical data, to describe TME. Tumor-infiltrating lymphocytes (TILs), excluding polymorphonucleates, were assessed in the stromal compartment as positive (+) or negative (-), with positivity threshold of 10% of stromal tissue area. Fibrosis, necrosis, tertiary lymphoid structures (TLSs) and lymphangitis were assessed with a positivity threshold of 1%. Progression free survival (PFS) and overall survival (OS) were calculated from the start of first-line therapy and compared by log-rank test. Multivariable analysis by Cox regression model for OS included age, gender, PS-ECOG, number (n) of metastatic sites, presence of brain metastases and TME features. Results: A total of 118 patients were included: 52 treated with first-line osimertinib, 47 with gefitinib/erlotinib, 19 with platinum-based chemotherapy. The patient population included 84 (71%) females, 77 (65%) never smokers. Median age was 64 years (range 34-91), median n of metastatic sites was 2, PS ECOG was &amp;lt;2 in 91 (77%) and brain metastases were present in 48 (41%) cases. Most common biopsy site was lung (70%). Fibrosis, TILs, lymphangitis, necrosis, TLSs were found in 76/95 (80%), 34/93 (37%), 35/95 (37%), 28/94 (30%), and 12/92 (13%) of evaluable samples, respectively. After a median follow-up of 50.3 months (m), median OS was 35.2 (95%CI 27.8-41.6) m in the overall population; 23.1 (95%CI 18.2-39.3) vs 36.3 (95%CI 32.1-51.3) m (p=0.007) in necrosis+ vs -; 27.7 (95%CI 26.6- not reached, NR) vs 35.5 (95% CI 27.8-48.5) m (p=0.85) in TILs+ vs TILs-; 38.1 (95%CI 36.6-NR) vs 32.1 (95%CI 27-44.7) m (p=0.94) in TLS+ vs TLS-. Nine (26%) TILs+ tumors were also necrosis+. Necrosis (HR 2.05, p=0.026) and PS&amp;gt;1 (HR 2.52, p&amp;lt;0.01) were independently associated with worse OS in the multivariable analysis. In necrosis+ vs -, median PFS to first-line was 11.4 (95%CI 3,6-NR) vs 11.2 (95%CI 9.3-NR) m in osimertinib (p=0.67) and 7.8 (95%CI 5.4-NR) vs 13 (95%CI 8.7-20.1) in erlotinib/gefitinib group (p=0.84). Conclusions: Necrosis emerges as a negative prognostic factor for EGFR-mutant NSCLC. The role of osimertinib in counteracting the impact of necrosis should be elucidated on a larger scale to yield more substantial data. Further analyses will be performed on patients treated with anti-angiogenic agents. Citation Format: Lodovica Zullo, Maria Rosa Ghigna, Wael Salem Zrafi, Arianna Marinello, Damien Vasseur, Maxime Frelaut, Pamela Abdayem, Marco Tagliamento, Pernelle Lavaud, Anas Gazzah, Jean-Yves Scoazec, Jordi Remon, Fabrice Barlesi, Benjamin Besse, David Planchard, Mihaela Aldea. Prognostic value of tumor microenvironment (TME) features in advanced, EGFR-mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C168.

Comparison of structural, mechanical, and histochemical characteristics of L-PRF and T-PRF membranes using different centrifugation protocols – An ex-vivo observational study

ObjectivesMany investigators are looking into the possibility of using platelet-rich. hemoderivatives as a source of the protein structures and cytokines necessary for. periodontal healing of wounds. The objective of the study was to evaluate and. compare the structural, mechanical, and histochemical characteristics of L-PRF and TPRF. membranes prepared by using three different centrifugation protocols. MethodsThirty participants around the ages of 20 and 35 who were in good overall. health had their antecubital veins pricked for ten milliliters of the blood. Venous blood. was centrifuged immediately after collection in silica-coated glass tubes and titaniumcoated. tubes utilizing the APRF machine. The mechanical properties of PRF. membranes (n = 30) for L-PRF and T-PRF each protocol) were assessed using a. desktop universal testing machine. ResultsThe forces around 700 g (3200 rpm, 15 min) yielded a PRF membrane with. desirable mechano-degradation characteristics for its clinical application. Histochemical examination revealed mixed inflammatory infiltrate with predominantly. platelets followed by lymphocytes, neutrophils& monocytes in T-PRF. Whereas, in. LPRF thin strands of mixed inflammatory infiltrate predominantly neutrophils &. monocytes are seen in the connective tissue stroma. ConclusionThis study demonstrated that centrifugation speed, force, and time. influenced the structural, and mechanical properties of PRF membranes prepared with. various protocols. This is the first study to reveal the marked impact of glass and. titanium-reinforced centrifugation tubes on the final production of PRF. It was further. found that a significant difference was observed between L-PRF and T-PRF at. 3200 rpm and 2700 rpm protocols.

Adenoid Ameloblastoma- A very rare, odontogenic tumor variant with less than 20 reported cases in the literature: Report of two cases and literature review

Abstract Introduction/Objective We describe two cases of Adenoid Ameloblastoma (AA), a very rare, hybrid variant, of two of the most common odontogenic neoplasms (Ameloblastoma and adenomatoid odontogenic tumors). AA is a benign epithelial odontogenic tumor characterized microscopically by epithelium resembling conventional ameloblastoma, with additional duct-like structures, epithelial whorls, and cribriform architecture. Dentinoid deposits, clusters of clear cells, and ghost-cell keratinization may also be present. These tumors do not harbor BRAF or KRAS mutations and their molecular basis appears distinct from conventional ameloblastoma but remains unknown. The lesion tends to recur, and sometimes, only correctly diagnosed after recurrence. Methods/Case Report Adenoid Ameloblastomas were identified after a review of the Temple University, Department of Pathology archive from 2012-2022. A total of four specimens (two patients) were identified. Results (if a Case Study enter NA) Case# 1 22-years-old female Biopsy Location/ size: Mandible, left, 2.1 cm. Microscopic: Neoplastic proliferation composed of odontogenic epithelium in a plexiform pattern with the formation of pseudo glandular structures of variably sized cystic spaces, the peripheral epithelial cells are palisaded with reversed polarity. The central cells have clear cytoplasm and resemble stellate reticulum with occasional tight whorls of cells noted. Hemorrhage and giant cells are present in granulation tissue. The lesion was incompletely excised. Diagnosis: Benign odontogenic tumor. Case#2 (1st occurrence): -49-years-old male -Biopsy -Location and size of lesion: Maxilla-left anterior, 0.9 cm. -Microscopic: Cyst lined by loosely arranged epithelial cells with a basal cell layer having hyperchromatic and palisaded nuclei. Knots of squamous cells are present within the cyst lining. The luminal layer is flattened to cuboidal and nonkeratinized. Duct-like openings are present within the epithelium and contain myxoid stromal tissue. Focally the epithelium extends into the connective tissue as thin anastomosing strands surround the myxoid tissue. -Diagnosis: Benign odontogenic tumor. Case#2 (2nd occurrence): -Biopsy -Diagnosis: Benign odontogenic tumor Case#2 (3rd occurrence): -Excisional biopsy -Diagnosis: AA Conclusion AA is a rare, aggressive odontogenic tumor variant with morphologic overlap with other odontogenic lesions; therefore, oral pathologists and clinicians should be familiar with this entity to achieve an accurate diagnosis and prompt management.

Mucocele of the Anorectal Pouch: Mucinous Cystic Neoplasm with Low-Grade Dysplasia Simulating Low- Grade Appendiceal Mucinous Neoplasm (LAMN)

Abstract Introduction/Objective We present the second case of colonic mucocele with dysplasia. To date, only five cases of colonic mucocele have been reported, all in the setting of a pouch or bypass, with only one of them showing dysplasia. The exceptional rarity of the lesion provides unique insight into etiology of mucocele. Methods/Case Report A 44-year-old male with complex surgical history including pull-through procedure for imperforate anus at birth, and later proctectomy with end colostomy due to poor anorectal function, presented with worsening low back pain radiating down to his left leg. An abdominal CT scan revealed a large pelvic cyst, causing compressive neuropathy. Multilocular cystic mass (17.5 cm) was resected, which was partially filled with mucinous material. Mucin was not seen on the cyst surface. On microscopic examination, the cyst was largely denuded however was partially lined by markedly attenuated ano-rectal type mucosa and focally by dysplastic glandular epithelium. Similar to LAMN, pools of acellular mucin dissecting stromal tissues were noted, associated with degenerative changes, calcifications and fibrosis. No invasive carcinoma or high-grade dysplasia was seen. Given the lack of extra- cystic mucin or evidence of overt malignancy, it was reported as mucinous cystic neoplasm with uncertain malignant potential. The patient has been followed for 16 months without evidence of recurrence or peritoneal disease. Results (if a Case Study enter NA) NA Conclusion Extended stasis within a bowel segment appear to contribute to the development of mucoceles. The blind-ended anatomy of the appendix renders it highly susceptible to this phenomenon, explaining the relatively high incidence of LAMN. Conversely, such prolonged stasis rarely occurs elsewhere in colon unless a pouch or bypassed segment is surgically created, particularly placing patients with a complex surgical history at the highest risk. Therefore, close follow-up is indicated in this patient group to screen for mucocele that may progress to develop dysplasia and mucinous neoplasm.

Listen to Your Gut: Key Concepts for Bioengineering Advanced Models of the Intestine.

The intestine performs functions central to human health by breaking down food and absorbing nutrients while maintaining a selective barrier against the intestinal microbiome. Key to this barrier function are the combined efforts of lumen-lining specialized intestinal epithelial cells, and the supportive underlying immune cell-rich stromal tissue. The discovery that the intestinal epithelium can be reproduced in vitro as intestinal organoids introduced a new way to understand intestinal development, homeostasis, and disease. However, organoids reflect the intestinal epithelium in isolation whereas the underlying tissue also contains myriad cell types and impressive chemical and structural complexity. This review dissects the cellular and matrix components of the intestine and discusses strategies to replicate them in vitro using principles drawing from bottom-up biological self-organization and top-down bioengineering. It also covers the cellular, biochemical and biophysical features of the intestinal microenvironment and how these can be replicated in vitro by combining strategies from organoid biology with materials science. Particularly accessible chemistries that mimic the native extracellular matrix are discussed, and bioengineering approaches that aim to overcome limitations in modelling the intestine are critically evaluated. Finally, the review considers how further advances may extend the applications of intestinal models and their suitability for clinical therapies.