Low Proliferation Research Articles

The mechanical, wear, antibacterial properties and biocompatibility of injectable restorative materials under wet challenge

ObjectivesTo evaluate the mechanical, wear, antibacterial properties, and biocompatibility of injectable composite materials. MethodsTwo injectable composite resins (GU and BI), one flowable composite resin (FS), and one flowable compomer (DF), in A2 shade, were tested. Mechanical properties were tested via three-point bending test immediately after preparation and after 1-day, 7-day, 14-day, and 30-day water storage. Under water-PMMA slurry immersion, specimens were subjected to a 3-body wear test (10,000 cycles) against stainless steel balls, while the roughness, wear depth, and volume loss were recorded. After 1-day and 3-day MC3T3-E1 cell culture, cell viability was evaluated with CCK-8 test kits, while the cell morphology was observed under CLSM and SEM. Antibacterial properties on S. mutans were assessed via CFU counting, CLSM, and SEM observation. SPSS 26.0 was used for statistical analysis (α = 0.05). ResultsThe mechanical properties were material-dependent and sensitive to water storage. Flexural strength ranked GU > FS > BI > DF at all testing levels. Three nanocomposites had better wear properties than DF. No significant difference on 1-day cell viability was found, but DF showed significantly lower cell proliferation than nanocomposites on 3-day assessment. GU and FS had more favourable cell adhesion and morphology. CFU counting revealed no significant difference, while FS presented a slightly thicker biofilm and BI showed relatively lower bacteria density. ConclusionsInjectable nanocomposites outperformed the compomer regarding mechanical properties, wear resistance, and biocompatibility. The tested materials presented comparable antibacterial behaviours. Flowable resin-based composites’ performances are affected by multiple factors, and their compositions can be attributed. Clinical SignificanceA profound understanding of the mechanical, wear, and biological properties of the restorative material is imperative for the clinical success of dental restorations. The current study demonstrated superior properties of highly filled injectable composite resins, which imply their wider indications and better long-term clinical performances.

Acute Toxicity and Hepatoprotective Influence of Hypericum perforatum Against Thioactamid-Induced Liver Fibrosis in Rats

Background: Hypericum perforatum (MEHP) is a broadly used therapeutic plant exhibiting numerous bioactivities. The present study evaluates acute toxicity and hepatoprotective effects of MEHP against thioacetamide (TAA)-induced liver injury in rats. Methods: The toxicity trial included a single oral administration of 2000 and 5000 mg/kg to rats. In the hepatoprotective experiment, 30 adult rats were arbitrarily clustered into 5 groups: Normal (A) and TAA control rats (B) treated with daily distilled water; reference rats received 3 oral doses/week of 50 mg/kg silymarin; D and E, rats received daily doses of 250 and 500 mg/kg MEHP, respectively. In addition, group B-E received 3 injections of 200 mg/kg TAA in a week for 60 days. Results: The results have shown a lack of any toxic signs in rats following oral administration of up to 5000 mg/kg. The hepatoprotective evaluations revealed a noticeably lower hepatic injury in MEHP-treated rats shown by reduced liver index and hepatocyte proliferation. Histopathological evaluation (H&E and Masson trichrome stains) showed a significant inhibitory potential of MEHP on the incidence rate of hepatic lesions represented by decreased liver necrosis and lower fibrous connective tissue proliferation initiated by TAA in rats. MEHP treatment meaningfully decreased proliferating cell nuclear antigen and α-SMA (myofibroblasts) in liver parenchymal tissues as well as improved redox (up-regulated SOD, CAT, GPx, and down-regulated MDA) and inflammatory state (decreased serum TNF-α and IL-6 cytokines) compared to fibrosis control rats. In addition, MEHP treatment caused significant restoration of serum liver biomarkers (enzymes and proteins) against TAA-induced hepatotoxicity. Conclusion: The present hepatoprotectives of MEHP could be attributed to its chemical contents (hypericin, hyperforin, quercitrin, and p-coumaric acid) that may validate it as a therapeutic additive for liver fibrosis after some pharmacological evaluations.

Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages.

Mesenchymal stem cells (MSCs) have been extensively studied for therapeutic potential, due to their regenerative and immunomodulatory properties. Serial passage and stress factors may affect the biological characteristics of MSCs, but the details of these effects have not been recognized yet. To investigate the effects of stress factors (high glucose and severe hypoxia) on the biological characteristics of MSCs at different passages, in order to optimize the therapeutic applications of MSCs. In this study, we investigated the impact of two stress conditions; severe hypoxia and high glucose on human adipose-tissue derived MSCs (hAD-MSCs) at passages 6 (P6), P8, and P10. Proliferation, senescence and apoptosis were evaluated measuring WST-1, senescence-associated beta-galactosidase, and annexin V, respectively. Cells at P6 showed decreased proliferation and increased apoptosis under conditions of high glucose and hypoxia compared to control, while the extent of senescence did not change significantly under stress conditions. At P8 hAD-MSCs cultured in stress conditions had a significant decrease in proliferation and apoptosis and a significant increase in senescence compared to counterpart cells at P6. Cells cultured in high glucose at P10 had lower proliferation and higher senescence than their counterparts in the previous passage, while no change in apoptosis was observed. On the other hand, MSCs cultured under hypoxia showed decreased senescence, increased apoptosis and no significant change in proliferation when compared to the same conditions at P8. These results indicate that stress factors had distinct effects on the biological processes of MSCs at different passages, and suggest that senescence may be a protective mechanism for MSCs to survive under stress conditions at higher passage numbers.

ASSESSMENT OF THYROID FUNCTION AND ULTRASONOGRAPHIC FINDINGS IN PATIENTS WITH MUCOPOLYSACCHARIDOSIS

Objective: The thyroid gland, with its high vascularity and low proliferation index of thyrocytes, is highly susceptible to storage diseases, however it has not been evaluated adequately in patients with in mucopolysaccharidosis (MPS). Therefore, the aim of this study is to assess the function, morphology, B- mode, and Doppler ultrasonography features of the thyroid gland in pediatric and adolescent patients with MPS and to evaluate whether the thyroid gland is involved in this disease. Material and Methods: Thyroid hormone functions were measured in all patients, and B-mode ultrasound and color Doppler imaging were performed. Results: Eight boys and 17 girls with MPS were included in the study. Eight patients were diagnosed with MPS I, 2 with MPS II, 3 with MPS III, 3 with MPS IVA, and 9 with MPS VI. Nineteen patients were receiving enzyme replacement therapy, while three patients diagnosed with MPS III remained untreated due to the unavailability of treatment options. Thyroid hormone levels were within normal limits for all patients. B-Mode ultrasound imaging revealed slightly heterogeneous echo texture in only 2 (8%) patients, both with MPS VI. Except for one patient with MPS VI, all color Doppler assessments were within normal limits. Conclusion: The results of our study demonstrate that both thyroid function tests and thyroid gland morphology are normal in MPS through childhood and adolescence. Therefore, we believe that thyroid gland dysfunction does not play a crucial role in the development of symptoms such as growth retardation, dry skin, coarse facial features, and intellectual disability, which could potentially be attributed to thyroid dysfunction. Instead, we think that these findings are more likely attributed to the primary disease involvement process

Rhabdomyomatous Mesenchymal Hamartoma: Report of 4 Cases With Histochemical and Immunohistochemical Findings and Emphasis on Potential Pitfalls.

Rhabdomyomatous mesenchymal hamartoma (RMH) typically presents as a congenital midline head and neck cutaneous polyp in infants. Perianal and mucocutaneous lesions have been reported, and recently, acquired adult-onset variants have been proposed. This makes the true prevalence, etiopathogenesis, and clinicopathologic distribution and classification of RMHs in children compared with those in adults uncertain. We performed a retrospective review to highlight the salient histopathologic, histochemical, and immunohistochemical features in RMHs and to emphasize their specific clinicopathologic criteria to avoid diagnostic pitfalls. We found 4 (0.3%) infants [2 female infants and 2 male infants, average age: 4 months] with mental, nasal, lingual, and perianal midline RMHs (average size: 1.0 cm) of 1303 patients with cutaneous polypoid lesions. Three were isolated, and 1 was associated with Goldenhar syndrome. The cutaneous polyps demonstrated intermixed skeletal muscle, adipose, and fibrocollagenous core stroma that extended into the dermis and around the dermal appendages. The lingual lesion demonstrated skeletal muscle and fibrocollagenous stroma with prominent nerve bundles and little adipose tissue. All showed interstitial loose mesenchyme. Masson trichome demarcated the triphasic stromal components. Alcian blue demonstrated the loose myxoid mesenchyme. Elastic van Gieson did not show elastic fibers. Desmin demonstrated the skeletal muscle bundles, S100 highlighted the adipose tissue lobules and the nerve bundles, and CD34 displayed the mesenchymal stroma. Ki67 showed a low proliferation index in the loose mesenchyme. Smooth muscle actin did not reveal smooth muscle bundles, but with CD31, they highlighted the thick blood vessels. CD117 revealed prominent mast cells. From our retrospective review series, 4 cases that originally diagnosed as RMHs were excluded. Likewise, we found some examples of the reported cases in the English literature that might have been mistaken for RMHs. This is because they did not fulfill the diagnostic clinicopathologic criteria. RMH constitutes a rare entity with specific clinicopathologic features. Most lesions are isolated. Some are associated with congenital anomalies and syndromes. Strict clinicopathologic diagnostic criteria should be applied to avoid mislabeling look-alike lesions for RMHs.

Cellular and Transcriptional Response of Human Astrocytes to Hybrid Protein Materials.

Collagen is a major component of the tissue matrix, and soybean can regulate the tissue immune response. Both materials have been used to fabricate biomaterials for tissue repair. In this study, adult and fetal human astrocytes were grown in a soy protein isolate (SPI)-collagen hybrid gel or on the surface of a cross-linked SPI-collagen membrane. Hybrid materials reduced the cell proliferation rate compared to materials generated by collagen alone. However, the hybrid materials did not significantly change the cell motility compared to the control collagen material. RNA-sequencing (RNA-Seq) analysis showed downregulated genes in the cell cycle pathway, including CCNA2, CCNB1, CCNB2, CCND1, CCND2, and CDK1, which may explain lower cell proliferation in the hybrid material. This study also revealed the downregulation of genes encoding extracellular matrix (ECM) components, including HSPG2, LUM, SDC2, COL4A1, COL4A5, COL4A6, and FN1, as well as genes encoding chemokines, including CCL2, CXCL1, CXCL2, CX3CL1, CXCL3, and LIF, for adult human astrocytes grown on the hybrid membrane compared with those grown on the control collagen membrane. The study explored the cellular and transcriptional responses of human astrocytes to the hybrid material and indicated a potential beneficial function of the material in the application of neural repair.

Fat fraction quantification with MRI estimates tumor proliferation of hepatocellular carcinoma.

To assess the utility of fat fraction quantification using quantitative multi-echo Dixon for evaluating tumor proliferation and microvascular invasion (MVI) in hepatocellular carcinoma (HCC). A total of 66 patients with resection and histopathologic confirmed HCC were enrolled. Preoperative MRI with proton density fat fraction and R2* mapping was analyzed. Intratumoral and peritumoral regions were delineated with manually placed regions of interest at the maximum level of intratumoral fat. Correlation analysis explored the relationship between fat fraction and Ki67. The fat fraction and R2* were compared between high Ki67(>30%) and low Ki67 nodules, and between MVI negative and positive groups. Receiver operating characteristic (ROC) analysis was used for further analysis if statistically different. The median fat fraction of tumor (tFF) was higher than peritumor liver (5.24% vs 3.51%, P=0.012). The tFF was negatively correlated with Ki67 (r=-0.306, P=0.012), and tFF of high Ki67 nodules was lower than that of low Ki67 nodules (2.10% vs 4.90%, P=0.001). The tFF was a good estimator for low proliferation nodules (AUC 0.747, cut-off 3.39%, sensitivity 0.778, specificity 0.692). There was no significant difference in tFF and R2* between MVI positive and negative nodules (3.00% vs 2.90%, P=0.784; 55.80s-1 vs 49.15s-1, P=0.227). We infer that intratumor fat can be identified in HCC and fat fraction quantification using quantitative multi-echo Dixon can distinguish low proliferative HCCs.

Clinicopathologic and Molecular Characterization of Xanthomatous Giant Cell Renal Cell Carcinomas: Further Support for a Close Morphologic Spectrum to Eosinophilic Solid and Cystic Renal Cell Carcinomas.

A recent study described a rare subtype of tuberous sclerosis complex ( TSC )-mutated renal cell carcinoma primarily characterized by Xanthomatous giant cell morphology. Only 2 cases in young individuals have been reported so far, making the correct diagnosis challenging from a pathological perspective. It remains unknown whether this tumor represents an independent subtype or belongs to other TSC -mutated tumors. We conducted a clinicopathologic evaluation and immunohistochemical profiling of 5 cases of Xanthomatous Giant Cell Renal Cell Carcinoma (XGC RCC) with confirmed TSC2 mutations through targeted DNA sequencing. In addition, we analyzed transcriptomic profiles using RNA-seq for the following samples: XGC RCC, Low-grade Oncocytic tumors (LOT), High-grade Oncocytic tumors/Eosinophilic Vacuolar Tumors (HOT/EVT), Eosinophilic Solid and Cystic Renal Cell Carcinomas (ESC RCC), Chromophobe cell Renal Cell Carcinomas (ChRCC), Renal Oncocytomas (RO), clear cell Renal Cell Carcinomas (ccRCC), and normal renal tissues. There were 2 female and 3 male patients, aged 22 to 58 years, who underwent radical nephrectomy for tumor removal. The tumor sizes ranged from 4.7 to 9.5cm in diameter. These tumors exhibited ill-defined boundaries, showed an expansive growth pattern, and featured distinctive tumor giant cells with abundant eosinophilic to Xanthomatous cytoplasm and prominent nucleoli. All tumors had low Ki-67 proliferation indices (<1%) and demonstrated immune reactivity for CD10, PAX8, CK20, CathepsinK, and GPNMB. Next-generation sequencing confirmed TSC2 mutations in all cases. RNA sequencing-based clustering indicated a close similarity between the tumor and ESC RCC. One patient (1/5) died of an accident 63 months later, while the remaining patients (4/5) were alive without tumor recurrences or metastases at the time of analysis, with a mean follow-up duration of 43.4 months. Our research supports the concept that Xanthomatous giant cell renal cell carcinoma (XGC RCC) shares clinicopathological and molecular characteristics with ESC RCC and shows a relatively positive prognosis, providing further support for a close morphologic spectrum between the two. We propose considering XGC RCC as a distinct subtype of ESC RCC.

DataXflow: Synergizing data-driven modeling with best parameter fit and optimal control – An efficient data analysis for cancer research

Building data-driven models is an effective strategy for information extraction from empirical data. Adapting model parameters specifically to data with a best fitting approach encodes the relevant information into a mathematical model. Subsequently, an optimal control framework extracts the most efficient targets to steer the model into desired changes via external stimuli. The DataXflow software framework integrates three software pipelines, D2D for model fitting, a framework solving optimal control problems including external stimuli and JimenaE providing graphical user interfaces to employ the other frameworks lowering the barriers for the need of programming skills, and simultaneously automating reoccurring modeling tasks. Such tasks include equation generation from a graph and script generation allowing also to approach systems with many agents, like complex gene regulatory networks. A desired state of the model is defined, and therapeutic interventions are modeled as external stimuli. The optimal control framework purposefully exploits the model-encoded information by providing those external stimuli that effect the desired changes most efficiently. The implementation of DataXflow is available under https://github.com/MarvelousHopefull/DataXflow.We showcase its application by detecting specific drug targets for a therapy of lung cancer from measurement data to lower proliferation and increase apoptosis. By an iterative modeling process refining the topology of the model, the regulatory network of the tumor is generated from the data. An application of the optimal control framework in our example reveals the inhibition of AURKA and the activation of CDH1 as the most efficient drug target combination. DataXflow paves the way to an agile interplay between data generation and its analysis potentially accelerating cancer research by an efficient drug target identification, even in complex networks.

Abstract CT235: RNA-seq molecular subtypes and gene expression signatures in the phase 3 IMpower151 study of 1L bevacizumab + pemetrexed/paclitaxel ± atezolizumab in metastatic NSCLC

Abstract Introduction IMpower151 (NCT04194203) was a phase 3 study to evaluate the efficacy and safety of atezolizumab, bevacizumab, carboplatin, plus paclitaxel or pemetrexed (ABCP) vs BCP, as 1L treatment for metastatic nsqNSCLC. IMpower151 did not meet its primary endpoint of INV-PFS. The objectives of this biomarker analysis were to characterize the molecular subtypes of metastatic nsqNSCLC and to explore the relationship between molecular subtype, predefined gene expression signatures, PD-L1 status and clinical outcomes with ABCP and BCP. Methods RNA sequencing of 250 nsqNSCLC tumor samples from IMpower151 was analyzed in the ACBP (n=123) and BCP (n=127) treatment arms. Non-negative matrix factorization (NMF) was applied to variably expressed genes to define molecular subtypes. Gene expression signatures were defined based on previously published associations with their respective biology. Tumor PD-L1 levels were determined by SP263 immunohistochemistry (Ventana). Results No PD-L1 dependent difference of survival benefit of ABCP compared to BCP was observed at the cutoff of TC 1% or TC 50%. Four molecular subtypes (NMF1-NMF4) were identified based on RNA sequencing. NMF1 (n=78) were immune-depleted and had low tumor proliferation. NMF2 (n=73) were characterized by low level of anti-tumor immune cells, increased expression of stromal signatures and high tumor proliferation. NMF3 (n=65) had the most inflamed transcriptomic profile with high enrichment of immune cells, and increased expression of stromal signatures as well. NMF4 (n=34) had the enrichment of granulocytes, a less inflamed immunophenotype and decreased expression of stromal signatures. The positive rate of PD-L1 expression with TC&amp;gt;=1% was significantly lower in NMF1 compared to other subtypes. None of patients with the four NMF subtypes showed significantly longer PFS or OS with ABCP than BCP. Overall, patients with NMF2 showed numerically shorter PFS and patients with NMF3 showed numerically longer PFS than other subtypes. Gene expression signatures of antitumor cytokines are correlated with survival benefit of ABCP compared to BCP. Patients with high expression level of antitumor cytokines showed substantially improved PFS and OS benefit from ABCP than BCP in both ITT population and EGFR mutation population. Conclusion Four molecular subtypes of nsqNSCLC with different immune-cell and tumor microenvironmental features were characterized. NMF2 may identify a fibrotic and immune-depleted subgroup correlated with worse prognosis. NMF3 may identify an immune-inflamed subgroup correlated with better prognosis. Neither NMF subtypes nor PD-L1 IHC level showed a correlation with survival benefit of ABCP controlled by BCP. The gene expression signature of antitumor cytokines could serve as a potential predictive biomarker for survival benefit of ABCP in metastatic nsqNSCLC including EGFR mutant patients. This finding is based on the exploratory analysis of a single clinical trial with limited sample size. It needs further investigation in other cohorts of tumor tissues and with larger clinical potential in blood samples as well. Citation Format: Caicun Zhou, Charlie Sun, Stacey Huang, Xiaorong Dong, Gongyan Chen, Zhehai Wang, Xianghua Wu, Yu Yao, Yiping Zhang, Ying Cheng, Hongming Pan, Xiaodong Zhang, Jiuwei Cui, Lifeng Wang, Xi Chen, Xiaoling Li, Ziping Wang, Qiming Wang, Jianxing He, Mengzhao Wang, Li Qian, Habib Hamidi, Shanshan Li, Marcus Ballinger, David S. Shames, Minu Srivastava. RNA-seq molecular subtypes and gene expression signatures in the phase 3 IMpower151 study of 1L bevacizumab + pemetrexed/paclitaxel ± atezolizumab in metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT235.

Clinical and Analytical Validation of Two Methods for Ki-67 Scoring in Formalin Fixed and Paraffin Embedded Tissue Sections of Early Breast Cancer.

Proliferation determined by Ki-67 immunohistochemistry has been proposed as a useful prognostic and predictive marker in breast cancer. However, the clinical validity of Ki-67 is questionable. In this study, Ki-67 was retrospectively evaluated by three pathologists using two methods: a visual assessment of the entire slide and a quantitative assessment of the tumour margin in 411 early-stage breast cancer patients with a median follow-up of 26.8 years. We found excellent agreement between the three pathologists for both methods. The risk of recurrence for Ki-67 was time-dependent, as the high proliferation group (Ki-67 ≥ 30%) had a higher risk of recurrence initially, but after 4.5 years the risk was higher in the low proliferation group. In estrogen receptor (ER)-positive patients, the intermediate Ki-67 group initially followed the high Ki-67 group, but eventually followed the low Ki-67 group. ER-positive pN0-1 patients with intermediate Ki-67 treated with endocrine therapy alone had a similar outcome to patients treated with chemotherapy. A cut-off value of 20% appeared to be most appropriate for distinguishing between the high and low Ki-67 groups. To summarize, a simple visual whole slide Ki-67 assessment turned out to be a reliable method for clinical decision-making in early breast cancer patients. We confirmed Ki-67 as an important prognostic and predictive biomarker.

Huge mesenchymal hamartoma in a young adult: a case report

Abstract Mesenchymal hamartoma of the liver (MHL) is rare. Less than 50 adult cases have been described. Due to their potential degeneration or recurrence, a complete surgical resection must be performed. We describe a case of a 26-year-old with a palpable solid lesion, which displaced abdominal organs. Percutaneous needle biopsies suggested the diagnosis of MHL. A right hemi-hepatectomy without segment 1 was performed; the post-operative course was uneventful. The mesenchymal component of the tumour was reactive to desmin and smooth muscle actin. Low proliferation index was confirmed (MIB1). Genetic counselling: the sequencing analysis of DICER1 and CDKN1C gene was negative, DNA methylation analysis on the chromosome 11p15 region was normal. After 42 months, there was no recurrence. In conclusion, clinicians should consider MHL in the differential diagnosis. The dimension and the need of radicality impose major liver resections or liver transplantations, which should be performed in referral centres.

270 Inducing Neural Maturation and Increasing Survival in SHH Medulloblastoma Using EZH2 Inhibition in a Mouse Model

INTRODUCTION: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Genetically, MB can be divided into four subgroups of which the SHH subtype histologically shows nodular architecture. Within this nodular architecture, there are islands of mature cells, with more abundant neuropil and a low proliferation rate, scattered among sheets of primitive cells. METHODS: We performed laser capture microdissection followed by whole transcriptome analysis, spatial transcriptomics using Digital Spatial Profiling, whole genome DNA methylation and ChIP-Seq analysis of mature and primitive areas from 8 medulloblastomas. We developed a genetically-engineered mouse model of SHH MB showing spontaneous maturation and lack of maturation with a conditional EZH2 genetic ablation or EZH2 overactivation respectively. Finally, we developed a fucoidan-based nanoparticle drug delivery across the blood brain barrier (BBB) for targeted molecular inhibition. RESULTS: Using whole transcriptome and DNA methylation analysis, we identified ∼120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3. ChIP-Seq analysis showed striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Medulloblastoma specific EZH2 genetic ablation resulted in diffuse tumor cell differentiation and prolonged survival in mice (n = 10 per group, log-rank p = 0.01). Conversely, conditional EZH2 (Y641F) activation prevented medulloblastoma differentiation. A fucoidan-based nanoparticle successfully delivered the EZH2 inhibitor (EPZ-6438) across the murine BBB to achieve significant extension of mouse survival (median 70 days compared to 21 days in control mice; *p = 0.01, Mantel-Cox). CONCLUSIONS: Spontaneous maturation of medulloblastoma cells can be induced by inhibition of EZH2. Fucoidan-based nanoparticle delivery systems allows tumor specific targeted delivery across the BBB extending survival in mice.

Cancer Stem Cells from Definition to Detection and Targeted Drugs.

Cancers remain the second leading cause of mortality in the world. Preclinical and clinical studies point an important role of cancer/leukaemia stem cells (CSCs/LSCs) in the colonisation at secondary organ sites upon metastatic spreading, although the precise mechanisms for specific actions are still not fully understood. Reviewing the present knowledge on the crucial role of CSCs/LSCs, their plasticity, and population heterogeneity in treatment failures in cancer patients is timely. Standard chemotherapy, which acts mainly on rapidly dividing cells, is unable to adequately affect CSCs with a low proliferation rate. One of the proposed mechanisms of CSC resistance to anticancer agents is the fact that these cells can easily shift between different phases of the cell cycle in response to typical cell stimuli induced by anticancer drugs. In this work, we reviewed the recent studies on CSC/LSC alterations associated with disease recurrence, and we systematised the functional assays, markers, and novel methods for CSCs screening. This review emphasises CSCs' involvement in cancer progression and metastasis, as well as CSC/LSC targeting by synthetic and natural compounds aiming at their elimination or modulation of stemness properties.

Patients with recurrent vulvovaginal candidiasis exhibit a decrease in both the fungicidal activity of neutrophils and the proliferation of peripheral blood mononuclear cells.

Recurrent vulvovaginal candidiasis (RVVC) is an important and underestimated fungal infection. We aimed to determine the fungicidal and proliferative capacities of neutrophils and peripheral blood mononuclear cells (PBMCs), respectively and the clinical and microbiological characteristics of a cohort of Colombian patients diagnosed with RVVC. A cross-sectional study was conducted. A total of 66 women were included (40 diagnosed with RVVC and 26 healthy women [HW]). Demographic and clinical data were recorded. Vaginal fluid samples were obtained for isolation, identification and antifungal susceptibility testing of Candida species using selective culture media and the Vitek 2.0® system. Blood samples were also obtained to evaluate cell subpopulations; furthermore, neutrophils and PBMCs were isolated to determine their fungicidal and proliferative capacities, respectively. The median age was 29 (IQR: 34-23) for RVVC and 24 (IQR: 30-23) for HW. Only two species of the genus Candida were identified: Candida albicans (92.5%) and Candida lusitaniae (7.5%). Resistance to fluconazole, voriconazole, flucytosine and amphotericin B was observed on six C. albicans isolates and one C. lusitaniae isolate. Only the family history of vulvovaginal candidiasis was associated with RVVC occurrence. The RVVC group exhibited a significantly higher number of neutrophils but with lower fungicidal activity in comparison to HW; likewise, PBMCs from RVVC patients presented a lower proliferation index when stimulated with C. albicans. Contrary to what has been reported worldwide, in Colombian patients with RVVC, C. albicans was the main isolated species without increased antifungal resistance. The diminished fungicidal and proliferative capacities of neutrophils and PBMCs, respectively, could suggest a possible alteration in the innate and adaptive immune responses.

Ependymomas of the spinal region in adults: Clinical and pathological features and MYCN expression levels in spinal ependymomas and myxopapillary ependymomas

BackgroundEpendymomas (EPNs) of the spinal region are a heterogeneous group of tumors that account for 17.6 % in adults. Four types have been recognized: subependymoma, spinal ependymoma (Sp-EPN), myxopapillary ependymoma (MPE), and Sp-EPN-MYCN amplified, each with distinct histopathological and molecular features. MethodsThis study investigated the clinical and pathological characteristics and MYCN expression levels of 35 Sp-EPN and MPE cases diagnosed at a tertiary university hospital over a decade-long period. ResultsTwenty-five cases were Sp-EPN and 10 cases were MPE, and were graded as WHO grade 2, except for 1 Sp-EPN case with grade 3 features. The most common symptoms were lower back pain and difficulty in walking. Radiology showed different tumor sizes and locations along the spinal cord, with MPEs exclusively in the lumbosacral region. Surgery was the main treatment, and gross total resection was achieved in all cases except for one. Immunohistochemistry showed low Ki-67 proliferation indices in all cases, and no MYCN expression. During follow-up, 3 (8.6 %) cases recurred and/or metastasized and 5 cases (14.3 %) died. No significant difference was found in disease-free survival or overall survival between Sp-EPN and MPE cases. However, 3 cases with grade 2 histology demonstrated recurrence and/or metastasis, despite the lack of MYCN expression. ConclusionOur results underscore the multifactorial nature of tumor aggressiveness in EPNs of the spinal region. This study enhances our knowledge of the clinical and pathological features of Sp-EPNs and MPEs and highlights the need for better diagnostic and prognostic markers in these rare tumors.

Abstract 2568: High infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to treatments or survival in estrogen receptor-positive breast cancer

Abstract Introduction: Psychological stress aggravates cancer cell biology which leads to cancer progression and metastasis through its influence on the sympathetic nervous system. Recently, infiltration of a local anesthetic lidocaine around a tumor prior to its removal was reported to lead to better survival in early-stage breast cancer (BC). Therefore, we hypothesized that neuron infiltration within the bulk tumor of BC could be associated with cancer aggressiveness and worse survival. Methods: A total of 5755 BC patients from five independent cohorts, including METABRIC (n=1903), SCAN-B (n=3273), GSE25066 (n=508), and ISPY2 (GSE173839; n=105), which have both clinical and transcriptomic data, were included in the study. The fraction of neuron infiltration was calculated using the xCell algorithm. Results: High neuron infiltration in BC was not associated with survival in either the METABRIC or SCAN-B cohorts. BC with high neuron infiltration had high expressions of neurotransmitter receptors: adrenergic receptor α1B and 2A, dopamine receptor D2, muscarinic acetylcholine receptor M3 and M4, and TAC1, which encodes TACR1, the receptor for substance P, but had lower expression of adrenergic receptor β2 and cholinergic acetylcholine receptor 1 and 10. Further, levels of neuron infiltration were highest in estrogen receptor-positive/HER2-negative (ER+/HER2-) among the subtypes and lower in tumors with lymph node metastasis, but was not associated with tumor size. Neuron infiltration was associated with higher AJCC staging in METABRIC, which was not validated in SCAN-B cohort. High neuron infiltration in BC was associated with reduced homologous recombination defects, mutation rate, and SNV neoantigen, and with less cell proliferation, evidenced by lower Nottingham histological grade and Ki67 gene expression, as well as less enrichment of Hallmark cell proliferation-related gene sets: E2F targets, G2M checkpoint, and Mitotic spindle. Despite its association with less cell proliferation, BC with high neuron infiltration was not associated with pathological complete response (pCR) in the GSE25066 cohort. High neuron infiltration in BC was associated with higher infiltration of lymph endovascular cells as well as less immune cell infiltration; CD8, CD4 memory, T-helper 1 and 2, dendritic cells, M1 and M2 macrophages, regulatory T cells, and B cells, lower cytolytic activity, and immune response; interferon-alpha and interferon-gamma and IL2 stat signaling. Despite this association, BC with high neuron infiltration was not associated with pCR after immunotherapy in the ISPY2 clinical trial cohort. Conclusion: BC with high infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to systemic therapies or patient survival in ER+/HER2- subtype. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Jun Arima, Pia Sharma, Itaru Endo, Kenichi Hakamada, Kazuaki Takabe. High infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to treatments or survival in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2568.

Abstract 4070: CIS-demasking cytokine linker technology allows a selective cytokine delivery to activated immune cells while sparing others and peripheral toxicity

Abstract Immunocytokines are promising new therapeutic approaches to enhance T-cell response by delivering interleukins into the tumoral site. Fusion of wild-type or attenuated interleukins to anti-PD-1 antibody has shown some efficacy to preferentially cis-activate PD1-expressing T-cells but on-target/off-tumor activity on PD-1-negative cells is still observed due to the high affinity of the cytokine to its receptor leading a broad systemic effect, toxicity, high clearance and low tumor biodistribution limiting the potential of some immunocytokines (e.g. anti-PD-1/IL-2/IL-15/IL-21). Various strategies have emerged to achieve localized cytokine activation using conditional strategies (e.g. allosteric modulators, MMP-cleavable linkers or pH-dependent binding) that remain challenging due to high dependency to specific TME composition (MMP, acidosis...). We developed the OSE-Cytomask® Platform, a universal and innovative linker technology allowing exclusive cytokine CIS-demasking upon binding of the fused antibody to its target without TRANS-activation associated with undesired effects (e.g. Toxicity). A series of different linkers were screened for optimal cis-activation of cytokine on PD1-expressing vs PD1-negative T-cells using different cytokines fused to a high-affinity anti-PD1. OSE-Cytomask® linker technology decreases IL-2 or IL-15 cytokine activity (pSTAT5 signaling) on PD1-negative cells while maintaining high activation of PD1-transduced T-cells even in co-culture. We confirmed activity of OSE-Cytomask® Immunocytokine on (PD1+) human T-cells while very low activation has been observed in naïve T-cells. Importantly, Cytomask® linker technology does not induce TRANS-activation on PD1-negative T-cells illustrating an innovative and strict CIS-demasking mechanism of action. In vivo, Anti-PD-1/IL-15 and/or anti-PD1/IL-2 OSE-Cytomask® molecules illustrated reduced toxicity after single or multiple injections (2mg/kg). High T-cell proliferation has been observed in tumor-micro-environment where PD1-expressing T cells are located. Low T-cell proliferation in periphery was seen, illustrating the potential of CIS-demasking technology to target the right T-cells at the right place. Altogether, OSE-Cytomask® linker technology illustrates specific intrinsic property to mask cytokine on naïve peripheral immune cells not expressing the target of the antibody while allowing selective CIS-demasking of the cytokine and CIS-activation of activated immune cells expressing (e.g. PD-1). This linker technology could be used with a broad range of cytokine to abrogate OFF-tumor cytokine activity associated with toxicity while selectively CIS-activating activated immune cells in the TME. Citation Format: Aurore Morello, Caroline Mary, Virginie Thepenier, Margaux Seité, Justine Durand, Cécile Batty, Kévin Biteau, Julien Taurelle, Géraldine Teppaz, Amandine Georges, Nicolas Poirier. CIS-demasking cytokine linker technology allows a selective cytokine delivery to activated immune cells while sparing others and peripheral toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4070.

Abstract 1835: Discovery of HSN003839, a highly potent inhibitor of ubiquitin-specific protease USP21 for cancer therapy

Abstract USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs), which catalyze the removal of ubiquitin chain from targeted proteins. Numerous efforts over the last decade have identified a wide range of USP21 substrates and revealed the momentous and multifaceted role of USP21 in physiological and pathological states, especially in tumorigenesis, indicating that USP21 is an appealing target for the therapy of many correlative diseases. Nevertheless, progress in the development of potent USP21 inhibitors remains limited. Herein, we present the discovery of HSN003839, a highly potent and selective USP21 inhibitor with excellent anticancer activity and drug-like properties. Following screening tool compounds to identify hits and subsequent optimization, we discovered a series of compounds with strong enzymatic inhibition for USP21 with IC50 &amp;lt; 100 nM. Among them, HSN003839 displayed low nanomolar cancer cells proliferation inhibitions and excellent ADMET properties. The mouse oral bioavailability (F) is 76.5% at 10 mg/kg. In a preliminary mouse CDX model, monotherapy of HSN003839 showed 105% tumor growth inhibition (TGI) at 30 mg/kg with no obvious body weight reduction. Further biological characterization, efficacy and toxicity studies are ongoing. The structure of HSN003839 was not presented and will not be disclosed at the time of presentation at AACR meeting. Citation Format: Xiaohui Liu, Ying Wang, Dongliang Xia, Wei Zhang, Yan He, Shijie Tian, Xuerong Feng, Xia Yang, Qi Zhang, Fei Liu, Shiyi Jiang, Ran Hu, Ming Tang, Xuelin Tang, Yuqing Liu, Weijiao Yuan, Xuedan You. Discovery of HSN003839, a highly potent inhibitor of ubiquitin-specific protease USP21 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1835.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve invivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.