Cell Ki-67 Research Articles

Expression of Markers Ki-67, Nestin, VEGF, CD34 and Apoptosis in Relatively Healthy Lung Tissue with Non-Changed and Metaplastic Bronchial Epithelium.

Knowledge about the occurrence of processes such as proliferation, apoptosis and angiogenesis in healthy lung tissues with different bronchial epitheliums is limited, and further exploration can contribute to a better understanding of the physiological renewal of lung tissues. The processes mentioned above occur with the help of important tissue factors; therefore, the aim of the study was to determine the expression of markers Ki-67, nestin, CD34 and vascular endothelial growth factor (VEFG) and detect apoptotic cells in relatively healthy lung tissue. Samples of relatively healthy lung tissue were obtained from 19 patients and divided into groups of patients with non-changed and patients with metaplastic bronchial epithelium. Tissue samples were examined by hematoxylin and eosin staining. Ki-67, nestin, VEGF and CD34-positive cells were detected by the immunohistochemistry method. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was carried out to detect apoptotic cells. The number of positive structures was counted semi-quantitatively by microscopy. Ki-67-positive cells were detected in only one case. An occasional to moderate number of nestin-positive structures was found in various tissues of relatively healthy lungs with different bronchial epitheliums. No apoptotic cells were seen in non-changed bronchial epithelium, compared with few apoptotic cells in metaplastic bronchial epithelium. Metaplastic bronchial epithelium contained more VEGF-positive cells than non-changed bronchial epithelium. Samples with non-changed, and metaplastic bronchial epithelium both contained a similar number of CD34-positive structures. Proliferative activity and programmed cell death are not prominent events in normal lung tissue. A moderate number of nestin-positive cells in the alveolar epithelium and cartilage of bronchi with pseudostratified ciliated epithelium suggests a significant role of neuronal origin cells in these structures, to be intensified in metaplastic bronchial epithelium. A practically non-changed number of CD34-positive cells excludes any difference in stimulation of endothelial origin cells between lungs with different types of epithelium, while an increase in VEGF in structures with metaplastic epithelium suggests the presence/influence of tissue ischemia impact on possible development/maintenance of metaplasia.

SCAND1 Reverses Epithelial-to-Mesenchymal Transition (EMT) and Suppresses Prostate Cancer Growth and Migration.

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many carcinomas depend on EMT activation, partial EMT, or hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGFβ signal and EMT-inducing transcription factors, such as ZEB1/2, in tumor cells. However, reverse EMT factors are less studied. We demonstrate that prostate epithelial transcription factor SCAND1 can reverse the cancer cell mesenchymal and hybrid E/M phenotypes to a more epithelial, less invasive status and inhibit their proliferation and migration in DU-145 prostate cancer cells. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and the transcriptional co-repression of target genes. We found that SCAND1 expression correlated with maintaining epithelial features, whereas the loss of SCAND1 was associated with mesenchymal phenotypes of tumor cells. SCAND1 and MZF1 were mutually inducible and coordinately included in chromatin with hetero-chromatin protein HP1γ. The overexpression of SCAND1 reversed hybrid E/M status into an epithelial phenotype with E-cadherin and β-catenin relocation. Consistently, the co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression was negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBRs, in prostate adenocarcinoma specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, in a mouse tumor xenograft model, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer. Kaplan-Meier analysis showed high expression of SCAND1 and MZF1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that SCAND1 induces expression and coordinated heterochromatin-binding of MZF1 to reverse the hybrid E/M status into an epithelial phenotype and, inhibits tumor cell proliferation, migration, and metastasis, potentially by repressing the gene expression of EMT drivers and the MAP3K-MEK-ERK signaling pathway.

High expression of RARG accelerates ovarian cancer progression by regulating cell proliferation.

To explore the relationship between retinoic acid receptor gamma (RARG) and ovarian cancer (OC) cell proliferation and the prognosis of patients. The transcriptome and clinical information of 379 OC and 88 normal ovarian samples were downloaded from the Cancer Genome Atlas (TCGA) database and the Genotype Tissue Expression (GTEx) database. We compared the mRNA level of RARG between ovrian normal and tumor tissues with the Wilcoxon rank sum test.The R package "limma" was used to analyze the differences in RARG expression between different clinical subgroups. Kaplan-Meier analysis was applied to evaluate the correlation between RARG and prognosis of patients. A nomogram was established to predict the effect of RARG on prognosis of OC patients. Immunohistochemistry and qRT-PCR experiments were conducted to determine the differential expression of RARG between ovarian normal and tumor tissues. Finally, we altered RARG expression using specific siRNA and lentiviral expression vectors to explore the function of RARG by CCK-8, cell cycle, colony formation, and xenograft assays in nude mice. RARG was highly expressed in ovarian tumors and was an independent predictor of poor overall survival outcomes. Subgroup analysis showed the high expression of RARG was related to FIGO stage III-IV (P=0.027), overall survival time <5 years (P=0.013) and dead status (P=0.041). The Kaplan-Meier curve indicated that patients with high RARG expression level had poor prognosis. The area under the curve (AUC) of RAGR expression for predicting patient survival rates at 1, 5 and 9 years were 0.659, 0.616 and 0.627, respectively. The GSEA enrichment analysis revealed that RARG was involved in ovarian cancer progression through multiple pathways. In cellular experiments in vitro, downregulation of RARG expression significantly suppressed the proliferation and colony formation capacity of OC cells. In cellular experiments in vivo, knockdown of RARG significantly reduced tumor growth in nude mice, decreased expression levels of Ki-67 and proliferation cell nuclear antigen (PCNA). High expression of RARG could promote OC cell proliferation and was an independent predictor of poor prognosis. RARG might work as a potential molecular target and biomarker for individualized diagnosis and treatment in OC patients.

492 Altered focal adhesion pattern of G0-positive melanoma cells

Cancer cell drug resistance develops via several mechanisms, including apoptosis evading, drug efflux, cell reprogramming, DNA repair. Recently numerous data pointed out an ability of cancer cells reversibly exit from cell cycle to enter in G0 phase that may favor cancer cell survival. Therefore, the goal of the study was to determine transcriptional phenotype associated with quiescent/senescent state acquisition. Melanoma cells were treated by Dacarbazine to induce their transition in G0 phase of a cell cycle followed by Ki-67 and flow cytometry-based cell cycle analysis.

DDEL-03. ASSESSING THE DEPTH OF DRUG PENETRATION INTO BRAIN PARENCHYMA FROM LOCAL DRUG DELIVERY SYSTEMS IN AN ORTHOTOPIC HIGH GRADE GLIOMA ALLOGRAFT USING ORBITRAP-SECONDARY ION MASS SPECTROMETRY

Abstract Local intracranial drug delivery for high-grade glioma has been developed over the last two decades with chemotherapeutic agents administered immediately adjuvant to neurosurgery to overcome poor blood brain barrier permeability. In addition to this, the incorporation of nanoparticles (NPs) into local drug delivery systems (DDS) can result in increased penetration of chemotherapeutics through brain parenchyma, transporting the drug further from the administration site than by drug molecule diffusion alone.Here we adopted two model local DDS containing NPs to compare the depth of penetration through brain parenchyma. Approach one consisted of a thermosensitive in situ assembling polymer matrix loaded with polymer-doxorubicin conjugated NPs which can line the resection cavity walls, while approach two consisted of drug-loaded (etoposide and olaparib) poly(lactic acid)-poly(ethylene glycol) polymeric NPs held within a bioadhesive pectin hydrogel and delivered via a spray device.We assessed the efficacy of both DDS at preventing recurrence of high-grade glioma in an orthotopic rat 9L gliosarcoma model following surgical resection. In addition to monitoring long-term survival, the effect of each DDS was measured histologically by H&amp;E and by caspase-1 (inflammatory response) and ki67 (cell proliferation) immunostaining staining. Depth of drug penetration was evaluated on post-sacrificial sections using Orbitrap-Secondary Ion Mass Spectroscopy (OrbiSIMS) and fluorescent microscopy. In vivo data suggests that the delivery mechanism of the NPs affects the efficacy of the DDS, whereby long-term survival was observed in rats treated with the sprayed olaparib/etoposide NP formulation, relative to rats where olaparib/etoposide was simply pipetted into the resection cavity. OrbiSIMS confirmed the presence of doxorubicin, olaparib and etoposide in brain tissue. Our work encourages consideration of mass spectrometry modalities to complement in vivo efficacy studies, as an analytical tool to assess brain distribution of systemically administered drugs, or localised brain penetration of drugs released biomaterial-based DDS.

ODP364 Prolactinomas in men: management challenges

Abstract Introduction Prolactinomas are the most common pituitary adenomas (Pas), with an estimated higher prevalence in women. Over 90% of prolactinomas are small, intrasellar tumors that rarely increase in size, but occasionally, these PAs can be aggressive or locally invasive and cause compression of vital structures. Usually, prolactinomas in male patients are large, invasive, but the reasons for the more aggressive course remain poorly understood. Also, male prolactinomas have a lower expression of estrogen receptor alpha (ERα), which is associated with higher proliferation rates, resistance to dopamine agonists and a poor prognosis (1). Objective To investigate the impact of sex-related differences in prolactinomas. Material and Methods We analyzed 76 patients with confirmed diagnosis of prolactinomas. The patient`s information was collected retrospectively between 2018 and 2021. The patients had a mean age of 40,9 years old (range 18-77) and the majority were males (56,75%). We identified 15 patients (11 males, 4 females) with prolactin (PRL) secreting macroadenomas who underwent pituitary surgery (transsphenoidal, ten patients; transcranial five patients, and three of them had two transsphenoidal interventions and one of them had two transcranial interventions), four had also radiotherapy (three patients had high voltage radiation therapy and one had gamma knife radiosurgery). In the group of patients who underwent pituitary surgery, mean pre-operative maximal diameter was 35.65 ± 14.83 mm (6 giant prolactinomas, all of them in males) with mean PRL levels at presentation of 3640 ng/ml (range 560–16000), with significant higher levels observed in male patients. Eleven patients had visual field defects and the same number had panhypopituitarism with hormonal replacement therapy. Only two patients (females) are now normoprolactinemic without treatment with dopamine agonists. Nine tumors were considered invasive, according to Knosp classification (grade 3-4). Two female patients had remission of the disease following surgery. Nine patients had residual tumors after surgery, with controlled PRL secretion under treatment with dopamine agonists. The nuclear positive cells for Ki-67 were quantified by bright field microscopy (magnification x200) using QuantCenter software, its values ranging from 1% to 20%, with a value of 15-20% in the case of a male patient with giant prolactinoma with cavernous sinus invasion. Conclusion Significantly higher prolactin concentrations are observed in men as compared to women, in PRL secreting pituitary adenomas. The reasons for a more aggressive course of prolactinomas in men remain poorly understood, the higher rates of proliferation markers, such as the Ki-67 labeling index or the mitotic count, the low expression of ERα may explain the more aggressive behavior in men. Reference: Maiter D. Prolactinomas in Men. In: Tritos N., Klibanski A. (eds) Prolactin Disorders. Contemporary Endocrinology. Humana, Cham, 2019. Pp 184-204. https://doi.org/10.1007/978-3-030-11836-5_1 Presentation: No date and time listed

Zinc Oxide Tetrapods Modulate Wound Healing and Cytokine Release In Vitro-A New Antiproliferative Substance in Glaucoma Filtering Surgery.

Glaucoma filtering surgery is applied to reduce intraocular pressure (IOP) in cases of uncontrolled glaucoma. However, postoperative fibrosis reduces the long-term success of both standard trabeculectomy and microstents. The aim of this study was to test the antiproliferative and anti-inflammatory potential of ZnO-tetrapods (ZnO-T) on human Tenon's fibroblasts (HTFs) for glaucoma surgery. The toxicity of ZnO-T on HTFs was determined using an MTT test. For analysis of fibroblast proliferation, migration, and transdifferentiation, cultures were stained for Ki67, alpha-smooth muscle actin (α-SMA), and p-SMAD. A fully quantitative multiplex ELISA was used to determine the concentrations of different cytokines, platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF) in culture supernatants with and without previous ZnO-T treatment. Treatment with higher concentrations (10 and 20 µg/mL) was associated with HTF toxicity, as shown in the wound healing assay. Furthermore, the number of Ki67, α-SMA-positive, and pSMAD-positive cells, as well as IL-6 and HGF in supernatants, were significantly reduced following incubation with ZnO-T. In conclusion, we were able to show the antiproliferative and anti-inflammatory potentials of ZnO-T. Therefore, the use of ZnO-T may provide a new approach to reducing postoperative fibrosis in glaucoma filtering surgery.

Survival and prognostic factors in patients undergoing the resection of solitary brain metastasis from non-small cell lung cancer: a retrospective cohort study.

Neurosurgery is the standard of care for resectable solitary brain metastasis (BM) from non-small cell lung cancer (NSCLC), but still with a poor outcome. Postoperative whole-brain radiotherapy (WBRT) was reported to reduce local recurrence, whether it could prolong survival was uncertain. In this study, we attempted to evaluate WBRT and other prognostic for overall survival (OS) in these patients. In this retrospective study, NSCLC patients with a solitary BM and controlled primary tumor who underwent neurosurgical resection were selected from the medical records database between January 2014 and December 2018. Clinical data, disease control/progression results and survival outcomes were obtained from the medical records, regular outpatient follow-up and telephone interviews. Univariable and multivariable Cox analyses of potential prognostic factors including patients' characteristics, BM features, tissue-based parameters and postoperative treatments were conducted. OS was illustrated using Kaplan-Meier curves, and group differences were assessed using the log-rank test. The subgroup analysis compared each variable between the WBRT group and the untreated control by the hazard ratio and its 95% confidence interval (CI). A total of 94 patients were included, with a median OS of 812 days. Univariable analysis showed that postoperative WBRT and targeted therapy were associated with OS. Multivariable analysis demonstrated that postoperative WBRT [P<0.001, hazard ratio (HR) 0.357], chemotherapy (P=0.008, HR 0.512), targeted therapy (P<0.001, HR 0.265), and smaller tumor size (P=0.018, HR 0.553) were independent prognostic factors for prolonged OS. However, tissue-based parameters (Ki67 tumor cell proliferation index, epidermal growth factor receptor, and checkpoint levels) were identified as statistically insignificant factors. In the subgroup analysis, the beneficial effect of WBRT was only observed in patients that did not receive systematic treatments. Postoperative WBRT and systematic treatments after solitary BM resection improve the prognosis of NSCLC patients with a controlled primary tumor. Postoperative WBRT could be considered, especially for those who not receive systematic chemotherapy or targeted therapy treatments, as they might be more likely to benefit from it.

HOXB13 facilitates hepatocellular carcinoma progression by activating AKT/mTOR signaling pathway

Introduction and ObjectivesHepatocellular carcinoma (HCC) is one of the sixth most common malignancies worldwide and is accompanied by high mortality. Homeobox B13 (HOXB13) has been shown to be involved in the development of various cancers. This study aimed to investigate the role of HOXB13 in HCC progression. Materials and MethodsThe expression of HOXB13 in HCC tumor tissues was analyzed using qRT-PCR and immunohistochemical staining . After overexpression or downregulation of HOXB13 in HCC cell lines, cell proliferation was detected by CCK8 assay and Ki67 staining and cell invasion ability were tested by transwell assay. Western blot assay was applied to analyze the effect of HOXB13 on related signaling pathways. In addition, the role of HOXB13 on HCC in vivo was explored using a HCC mouse model. IF and WB were performed to detect cell proliferation, apoptosis and related protein expression in mice tumor tissues. ResultsThe results showed that the expression of HOXB13 was significantly increased in HCC tissues compared with adjacent tissues and positively correlated with the tumor stage and survival of HCC patients. Overexpression of HOXB13 promoted the proliferation and invasion of HCC cells and up-regulated the protein expression of AKT, mTOR and MMP2. In contrast, the downregulation of HOXB13 resulted in the opposite results. In vivo experiments, HOXB13 significantly promoted tumor growth in mice bearing HCC by promoting cell proliferation and inhibiting cell apoptosis. ConclusionsThis study suggested that HOXB13 can facilitate HCC progression by activation of the AKT/mTOR signaling pathway. HOXB13 may be a novel target for HCC therapy.

Identification of alanine aminotransferase 1 interaction network via iTRAQ-based proteomics in alternating migration, invasion, proliferation and apoptosis of HepG2 cells.

Objective: To investigate the mechanism of alanine aminotransferase 1 (ALT1) in the progression of HCC, the differentially expressed proteins (DEPs) in the ALT1 interaction network were identified by targeted proteomic analysis.Methods: Wound healing and transwell assays were conducted to assess the effect of ALT1 on cellular migration and invasion. Cell Counting Kit-8 (CCK-8), colony formation, and flow cytometry assays were performed to identify alterations in proliferation and apoptosis. After coimmunoprecipitation processing, mass spectrometry with iso-baric tags for relative and absolute quantitation was utilized to explore the protein interactions in ALT1 knockdown HepG2 cells.Results: The results showed that ALT1 knockdown inhibits the migration, invasion, proliferation of HepG2 cells, and promotes apoptosis. A total of 116 DEPs were identified and the bioinformatics analysis suggested that the ALT1-interacting proteins were primarily associated with cellular and metabolic processes. Knockdown of ALT1 in HepG2 cells reduced the expression of Ki67 and epithelial cell adhesion molecule (EP-CAM), while the expression of apoptosis-stimulating protein 2 of p53 (ASPP2) was increased significantly. Suppression of the ALT1 and EP-CAM expression contributed to alterations in epithelial–mesenchymal transition (EMT) -associated markers and matrix metalloproteinases (MMPs). Additionally, inhibition of ALT1 and Ki67 also decreased the expression of apoptosis and proliferation factors. Furthermore, inhibition of ALT1 and ASPP2 also changed the expression of P53, which may be the signaling pathway by which ALT regulates these biological behaviors.Conclusions: This study indicated that the ALT1 protein interaction network is associated with the biological behaviors of HepG2 cells via the p53 signaling pathway.

Impact of MyD88, Microbiota, and Location on Type 1 and Type 3 Innate Lymphoid Cells during Toxoplasma gondii Infection.

Toxoplasma gondii induces strong IFN-γ-based immunity. Innate lymphoid cells (ILC), in particular ILC1, are an important innate source of this protective cytokine during infection. Our objective was to determine how MyD88-dependent signaling influences ILC function during peroral compared with i.p. infection with T. gondii. MyD88 +/+ and MyD88 -/- mice were orally inoculated with ME49 cysts, and small intestinal lamina propria ILC were assessed using flow cytometry. We observed T-bet+ ILC1, retinoic acid-related orphan receptor γt+ ILC3, and a population of T-bet+retinoic acid-related orphan receptor γt+ double-positive ILC. In MyD88 -/- mice, IFN-γ-producing T-bet+ ILC1 frequencies were reduced compared with wild-type. Treatment of MyD88 -/- mice with an antibiotic mixture to deplete microflora reduced IFN-γ+ ILC1 frequencies. To examine ILC responses outside of the mucosal immune system, peritoneal exudate cells were collected from wild-type and knockout mice after i.p. inoculation with ME49 cysts. In this compartment, ILC were highly polarized to the ILC1 subset that increased significantly and became highly positive for IFN-γ over the course of infection. Increased ILC1 was associated with expression of the Ki67 cell proliferation marker, and the response was driven by IL-12p40. In the absence of MyD88, IFN-γ expression by ILC1 was not maintained, but proliferation remained normal. Collectively, these data reveal new aspects of ILC function that are influenced by location of infection and shaped further by MyD88-dependent signaling.

Comparative Study on Clinicopathological Characteristics of Breast Cancer in Vietnam and Italy

Background: In Vietnam and Italy, breast cancer (BC) occurs in women more frequently than any other cancer. Vietnam has a substantially lower incidence of breast cancer than Italy, but a higher mortality rate. Most Vietnamese patients present to the hospital with severe tumors at the late stages of diagnosis. The purpose of this study was to compare clinicopathologic features, biomarkers, and subtypes of BC between Vietnamese women and Italian ones. Methods: The sample was collected from all Vietnamese patients undergoing surgery with the diagnosis of primary invasive breast carcinoma in Hue Central Hospital as well as Hue University Hospital in Vietnam during 1 year from April 2016 to April 2017, and from the same category of women in Sassari University Hospital, Italy during the year 2016. The study parameters in both groups consisted of age at diagnosis, tumor size, histologic grade, histologic type, axillary node status, stages of diagnosis, biomarkers (Estrogen Receptor - ER, Progesterone Receptor - PR, Ki-67 cell proliferation marker - Ki-67, Human epidermal growth factor receptor 2 - HER2), and molecular subtypes of BC. Results: 323 patients were collected in total (235 from Sassari, Italy, and 88 from Thua Thien - Hue province, Vietnam). Vietnamese patients were diagnosed at a younger age than Italian patients, at just 52.5 on average, while the figure for Italian was 62. The Vietnamese BC patients also had a bigger tumor in size, higher grade, more axillary node positivity as well as a later stage of disease in comparison with Italian ones. The proportion of ER-positive was higher in the Italian group than that in Vietnam (88.1 vs 55.7%). The high Ki-67 expression prevalence was significantly higher in Vietnamese patients compared with Italian (81.8 vs 45.1%). The HER2-positive rate in Vietnam was 33%, higher than that in Italy (8.1%). Histologic grade and cell proliferation index Ki-67 were significantly correlated with HER2 positivity in both groups. Conclusions: Vietnamese patients demonstrated more aggressive tumor features and worse prognostic biomarkers than Italian patients. The prevalence of HER2 positive and high Ki-67 expression in Vietnamese patients was considerably higher than in Italian patients. The remarkable differences in clinicopathological characteristics between the two populations suggest the diversity of biological tumor, ethnicity, and environment as well as the effectiveness of the screening program.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

HIF-1α Stabilization Boosts Pulp Regeneration by Modulating Cell Metabolism

Stem cell–based therapeutics is a promising strategy in dental pulp regeneration. However, low cell viability after transplantation in vivo due to the ischemic microenvironment is still a critical challenge for future clinical application. With the aim of improving postimplantation cell survival and pulp tissue regeneration, stem cells from human exfoliated deciduous teeth (SHED) were preconditioned to a hypoxic condition by hypoxia-inducible factor 1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using lentiviral short hairpin RNA. HIF-1α–stabilized SHED were encapsulated in PuraMatrix hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28 d, enhanced dental pulp–like tissue formation was observed with a significantly higher level of vascularization, which could be attributed to both endothelial differentiation of SHED and recruitment of host blood vessels. Furthermore, dentin-like tissue formation in vivo and accelerated odontogenic/osteogenic differentiation both in vivo and in vitro were observed. At 7 d postimplantation, significantly less DNA damage and higher Ki67 expression were detected in the HIF-1α–stabilized SHED group compared with the control SHED. Accordingly, cell viability assay and staining for Ki67 and apoptotic cells in vitro showed that HIF-1α stabilization could decrease cell apoptosis and enhance cell survival significantly. We demonstrated that PI3K/AKT pathway activation had resulted in low caspase 3 expression in HIF-1α–stabilized SHED in hypoxic conditions. Furthermore, we found that HIF-1α–induced cell survival could also be attributed to the upregulated expression of PDK1, HK2, and Glut1, which contributes to the maintenance of reactive oxygen species homeostasis and metabolic adaptation in hypoxia. In addition, we identified Smad7 as 1 of the top 3 upregulated genes through RNA sequencing in HIF-1α–stabilized SHED and demonstrated its essential role in HK2 and Glut1 upregulation. Taken together, HIF-1α stabilization enhances cell survival of SHED through modulating various target genes and potential signaling pathways, as well as odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell–based therapy in general.

A Computer-Aided Diagnosis (CAD) System for Automatic Counting of Ki67 Cells in Meningioma

Meningioma is a type of primary brain tumour where this tumour arises in the three thin layers of tissues, called meninges. Tumour grading is usually used to describe tumour cells' characteristics and behaviours and how they look under a microscope. There were many techniques used for determining the grade of the tumour. Ki67 was the most common proliferation marker used to measure cell proliferation activity. Currently, pathologists used the manual counting technique to count the Ki67 cells before determining tumour grading. However, this technique was time-consuming, tiring and the counting results are often not accurate. Besides that, manual counting has poor reproducibility and discordant between counting values’ among the pathologist. Therefore, this study aimed to develop a Computer-Aided Design (CAD) software that automatically counts the Ki67 cells for determining tumour grading. The purpose of developing this software is to alleviate pathologists’ workload associated with counting Ki67 cells and scoring the Ki67 index. The CAD software was developed through seven stages. Based on Pearson Correlation Coefficient results, there was a good positive correlation between the proposed technique with the manual counting technique in counting positive and negative Ki67 cells with a correlation of 0.99 and 0.72 respectively. The proposed CAD system also showed promising results in computing the Ki67 labeling index with a low percentage absolute error of 1.85%.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2 Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co‐administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle‐treated mice. Compared to vehicle‐treated mice, ERKi‐treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho‐Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi‐treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co‐administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

P-804 Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal mammalian testicular tissue: a comparative study

Abstract Study question What is the impact of different static and dynamic organotypic culture systems on the outcome of prepubertal porcine immature testicular tissue (ITT) culture? Summary answer Using a hybrid silicone-agarose static culture system improves germ cell survival and differentiation in ITT ex-vivo. What is known already In mice, organotypic culture of ITT led to the birth of healthy fertile pups. In other larger mammals, including humans, it remains challenging to efficiently reproduce spermatogenesis ex-vivo. With the advent of tissue engineering, culture systems are becoming more complex to better mimic the in vivo environment by modifying and/or improving physical culture conditions (i.e. tissue perfusion, oxygen diffusion, direct/indirect contact with medium). Recent studies in rodents showed that by using hybrid static and dynamic silicone-based organotypic culture systems, the efficiency of spermatogenesis can be significantly improved. Such culture systems have not been reported in large mammals yet. Study design, size, duration Testes were collected from three 5-7 days old piglets as a by-product of castration for a Belgian farm. The fresh ITT fragments were immediately used for culture experiments. Fragments were cultured for a 30 days duration in the different static i.e. polytetrafluoroethylene membrane inserts (CT), agarose gel (AG) and agarose gel with polydimethylsiloxane cover (AGPC) and dynamic i.e. microfluidic (MF) systems using a single medium. The CT system served as control. Participants/materials, setting, methods Cultured ITT fragments were monitored every 4-5 days by serial imaging. Analyses were performed at days 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity and diameters were evaluated on Periodic Acid Schiff-stained slides. Immunohistochemistry was used to identify: germ (PGP9.5, VASA, SYCP3, CREM), somatic (SOX9, INSL3) and proliferating (Ki67) cells and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured using ELISA. A two-way ANOVA was used for statistical analyses. Main results and the role of chance All systems supported the survival, growth and maturation of ITT fragments in vitro. Between days 0 and 30, ST diameters increased in all systems (p &amp;lt; 0.000) (day 0: 61.2±6.5μm; day 30: CT: 75.3±18.1μm, AG: 77.7±15.7μm, AGPC: 84.9±13.6μm, 66.9±12.9μm). Sertoli cell (SOX9) numbers also increased while spermatogonia (PGP9) numbers per ST decreased (day 0: 6.94±0.31, day 30: CT: 1.84±0.64, AG: 2.65±0.88, AGPC: 3.47±0.7, MF: 2.11±0.84). Meiotic germ cells (SYCP3, CREM) were absent at day 0 and were generated starting day 5 (SYCP3) and 10 (CREM) in the different systems and maintained till day 30 (SYCP3 per ST; CT: 0.31±0.21, AG: 0.5±0.31, AGPC: 0.69±0.20, MF: 0.38±0.20 and CREM per ST: CT: 0.18±0.18, AG: 0.25±0.13, AGPC: 0.34±0.32, MF: 0.14±0.14). When compared to control (CT), significantly larger STs (days 10 to 30), a higher number of spermatogonia (PGP9) (day 30; AGPC: 3.72±0.70, CT: 1.84±0.64, p = 0.04), meiotic SYCP3 + (day 20, AGPC: 0.95±0.49, CT: 0.46±0.39, p = 0.01) and CREM+ cells (day 20, AGPC: 0.49±0.21, CT: 0.23±0.64, p = 0.02) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cells (Ki67/VASA), Leydig cells (INSL3) and testosterone levels were not significantly different between the different systems over the culture period. Limitations, reasons for caution The small sample size and inter-tissue variability could have limited the results of this study. Also it is not excluded that further differences, in terms of tissue growth, germ cell survival and differentiation, could be observed between the different systems if a more complex culture medium is used. Wider implications of the findings In contrast to rodents, modifying physical culture conditions minimally impacts the outcome of porcine ITT maturation ex-vivo. Due to anatomo-physiological similarities between pigs and humans, it is likely for similar results to be observed if applied to human ITT. Studies aiming at determining optimal culture medium components are rather needed. Trial registration number not applicable

Abstract 2: Flavokawain A targets cancer stem cells for inhibiting the growth of prostate cancer

Abstract Prostate cancer stem cells (PCSC) are small tumor subpopulations capable of self-renewal and driving prostate cancer heterogeneity, which is critical for cancer initiation, recurrence, and development of therapeutic resistance. Thus, there’s an urgent need to explore and develop new CSC targeting agents. Flavokawain A (FKA), a novel chalcone from kava plant, can selectively inhibit the growth of pRb deficient cell lines and result in a proteasome-dependent and ubiquitination-mediated Skp2 (S-phase kinase-associated protein 2) degradation, while the potential of FKA to target PCSC is unknown. Therefore, we evaluated the effects of FKA on PCSC self-renewal both in vitro and in vivo. FKA treatment decreased both size and numbers of the tumor spheroids growing from CD44+/CD133+ tumor stem cells, which were sorted out from the prostate tumor cell line DU145 and 22RV1. Expression of the stem cell markers Nanog, Oct4, and Sox2 were all downregulated in the cancer spheroids treated with FKA. Growth of the 22RV1CD44+/CD133+ xenograft tumors was significantly inhibited in FKA diet-fed mice compared to the vehicle control diet-fed mice. Immunohistochemistry analysis revealed less positive Ki67, Nanog, and Oct4 cells in tumor tissues from the FKA dietary group than those from the control dietary group. These results demonstrated that FKA can inhibit prostate cancer stemness and suggested a potential role of FKA for preventing prostate cancer recurrence by targeting PCSC. Citation Format: Liankun Song, Merci Mino, Jana Yamak, Vyvyan Nguyen, Derron Lopez, Victor Pham, Ali Fazelpour, Vinh Le, Dongjun Fu, Matthew Tippin, Xiaolin Zi. Flavokawain A targets cancer stem cells for inhibiting the growth of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2.

Abstract 3484: Development of a custom high-plex GeoMx digital spatial profiler breast cancer protein biomarker assay

Abstract The goal of this study was to develop a high-plex assay to simultaneously quantitate 27 established and novel breast cancer (BC)-related, immune protein and phosphoprotein biomarkers using the GeoMx® Digital Spatial Profiler (DSP). The custom assay performance was compared to standard clinical BC biomarker assays (e.g.ER, PR, HER2, Ki67) across the spectrum of BC subtypes and in multiple laboratories. Commercially available antibodies to 27 BC-related protein biomarkers, including ER, PR, HER2, Ki-67, AR, immune-related (e.g. PD-L1) and several cell cycle/proliferation markers were oligonucleotide-tagged, and verified by immunohistochemistry for performance against untagged antibodies. The tagged antibodies were combined with 3 isotype controls and 2 housekeeping proteins into a custom BC high-plex assay for DSP. Confirmation of target specificity was done on a custom tissue microarray (TMA) (Run control) composed of (un)treated cancer cell lines and normal tissues. For clinical BC samples, each biopsy had 4x600 µm regions of interest segmented into pan-Cytokeratin+ tumoral epithelium and pan-Cytokeratin- adjacent stroma segments. With targeted UV light, oligonucleotides were collected from each segment sequentially and quantitated with nCounter. Raw counts were geomean normalized for analysis. Intra-site and inter-site assay reproducibility was assessed with the TMA and serial FFPE sections from BC biopsies. Quantitation of proteins showed high reproducibility within sites (e.g. Run 1 v. Run 2, p&amp;gt;0.05 for all protein targets) and the high-plex custom assay showed good concordance between 3 participating laboratories. GeoMx DSP-derived protein data correlated well with orthogonal methodologies performed on a sample subset including immunohistochemistry, reverse phase protein array, DNA and RNA-sequencing. For example, DSP protein counts for p53 protein were higher in tumors with TP53 point mutation than those with truncating TP53 alterations. Expanded sample testing is underway with 120 well-characterized breast cancer biopsies of various histologies, immune subtypes (e.g. TIL scores) and clinical biomarker phenotypes (e.g. ER+/HER2-/Ki-67 high BC; ER+/HER2-/Ki-67 low BC; HER2+ BC; ER-/PR-/HER2- BC).Our preliminary data demonstrate that this custom high-plex BC assay can quantitate protein biomarkers across a wide dynamic range with high intra-lab and inter-lab reproducibility. The assay requirement of a single 5-µm tissue section facilitates complex biomarker profiling in biopsies with limited material. The custom assay alone or in combination with other targeted DSP protein modules can simultaneously interrogate the biomarker profile, immune-based and other drug target profile, and immune microenvironment of BC specimens, providing a novel approach for actionable tumor subtyping. For research use only. Not for use in diagnostic procedures. Citation Format: Christopher Corless, Amber Bridgeman, Jinho Lee, Gary Geiss, Sarah Warren, Sarah Church, Joseph M. Beechem, E Aubrey Thompson, Jodi Carter. Development of a custom high-plex GeoMx digital spatial profiler breast cancer protein biomarker assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3484.

Neoadjuvant survivin-targeted immunotherapy maveropepimut-S (MVP-S) to increase Th1 immune response in Ki67-high hormone receptor-positive (HR+) early-stage breast cancer (ESBC).

TPS1119 Background: HR+ ESBC is associated with suboptimal pathologic complete response rate (pCR, ̃10%) following neoadjuvant cytotoxic chemotherapy. Neoadjuvant anti-endocrine therapy with aromatase inhibitors (AI) may serve as an effective alternative. Efficacy can be gauged using the surrogate Ki67 cell proliferation histologic marker. Patients with poor Ki67 response (defined as Ki67 &gt; 10%) following neoadjuvant AI exhibit poor prognosis and therapeutic resistance to both anti-endocrine therapy and chemotherapy. In a genomic analysis among Ki67-high HR+ tumors, we identified 8-fold upregulation of BIRC5 (survivin), a gene that regulates apoptosis and the cell cycle and that is associated with poor clinical outcome. Maveropepimut-S (MVP-S, previously named DPX-Survivac) leverages the non-aqueous, lipid-based DPX delivery platform to educate a specific and persistent T cell-based immune response to 5 HLA-restricted peptides from Survivin, a cancer-associated protein commonly upregulated in several cancers. Treatment with MVP-S and intermittent, low-dose cyclophosphamide (CPA) has shown tumor infiltration of survivin-specific T cells. Previous clinical trials have shown that MVP-S is well-tolerated, immunogenic, and could lead to clinical response in several cancer indications. Further exploration of the regimen in breast cancer could extend the application of this immunotherapy for this unmet medical need. Methods: NCT04895761 is phase I trial evaluating the safety and immunologic effects of neoadjuvant MVP-S plus letrozole (arm A, n = 6), with/without tumor-directed MR-guided radiotherapy (arm B, n = 6), or intermittent low-dose cyclophosphamide or CPA (arm C, n = 6). Postmenopausal patients with T1c+ HR+HER2- breast cancer with Ki67 &gt; 10% will receive two doses of MVP-S and 7 weeks of neoadjuvant letrozole prior to surgery (all arms), whereas arm B will be treated additionally with concurrent 10Gy x 2 tumor boost radiation to facilitate immunogenic cell death, and arm C (n = 6) will be treated additionally with intermittent low-dose CPA (50mg BID) to facilitate regulatory T cell depletion. The primary objective is safety. Biomarker objectives are to evaluate for each treatment arm: 1) systemic type I survivin-specific immune response, as measured by IFN-γ ELISPOT; 2) changes in immune environment by GeoMx digital spatial genomic profiling; 3) and changes in tumor infiltrating lymphocytes (TILs) and Ki67. These data will be used to identify the most immunogenic MVP-S combination therapy for study in phase II trial powered to assess clinical outcome (pCR). Clinical trial information: NCT04895761.