Reduced Tumor Cell Proliferation Research Articles

Inhibiting circ_0000673 blocks the progression of colorectal cancer through downregulating CPSF6 via targeting miR-548b-3p.

Colorectal cancer (CRC) is one of the most common cancers, and its progression is regulated by several factors, including circular RNA (circRNA). The objective of this study was to determine the role, or roles, of circ_0000673 in CRC. We used quantitative real-time polymerase chain reaction (qPCR) to detect the expression of circ_0000673, miR-548b-3p and cleavage and polyadenylation specific factor 6 (CPSF6) in DLD-1 and RKO cells. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to determine circ_0000673 roles in proliferation. Wound healing and transwell assays were used to detect cell migration and invasion abilities. Expression of CPSF6 protein and stem cell-associated proteins were examined using western blot. The putative relationship between miR-548b-3p and circ_0000673 or CPSF6 was verified with dual-luciferase reporter assay. The role of circ_0000673 in CRC was also investigated in a tumor xenograft assay in nude mice. Circ_0000673 expression was increased in CRC tissues and cancer cells. Silencing circ_0000673 reduced tumor cell proliferation, migration and invasion, while also decreasing cell stemness. MiR-548b-3p was found to be a target of circ_0000673, while CPSF6 was a downstream target of miR-548b-3p. The tumor-regulatory effects of si-circ_0000673, anti-miR-548b-3p and oe-CPSF6 were partially reversed by anti-miR-548b-3p, si-CPSF6 and si-circ_0000673, respectively, in rescue assays. Downregulation of circ_0000673 reduced solid tumor growth in vivo. Circ_0000673 inhibition reduced CPSF6 expression by targeting miR-548b-3p, thereby blocking proliferation, migration and invasion of CRC tumor cells.

Disrupting glioblastoma networks with tumor treating fields (TTFields) in in vitro models.

This study investigates the biological effect of Tumor Treating Fields (TTFields) on key drivers of glioblastoma's malignancy-tumor microtube (TM) formation-and on the function and overall integrity of the tumor cell network. Using a two-dimensional monoculture GB cell network model (2DTM) of primary glioblastoma cell (GBC) cultures (S24, BG5 or T269), we evaluated the effects of TTFields on cell density, interconnectivity and structural integrity of the tumor network. We also analyzed calcium (Ca2+) transient dynamics and network morphology, validating findings in patient-derived tumoroids and brain tumor organoids. In the 2DTM assay, TTFields reduced cell density by 85-88% and disrupted network interconnectivity, particularly in cells with multiple TMs. A "crooked TM" phenotype emerged in 5-6% of treated cells, rarely seen in controls. Ca2+ transients were significantly compromised, with global Ca2+ activity reduced by 51-83%, active and periodic cells by over 50%, and intercellular co-activity by 52% in S24, and almost completely in BG5 GBCs. The effects were more pronounced at 200kHz compared to a 50kHz TTFields. Similar reductions in Ca2+ activity were observed in patient-derived tumoroids. In brain tumor organoids, TTFields significantly reduced tumor cell proliferation and infiltration. Our comprehensive study provides new insights into the multiple effects of Inovitro-modeled TTFields on glioma progression, morphology and network dynamics in vitro. Future in vivo studies to verify our in vitro findings may provide the basis for a deeper understanding and optimization of TTFields as a therapeutic modality in the treatment of GB.

Molecular Mechanisms of Anticarcinogenic Potential of Hydrocotyle umbellata and Its Major Components

Despite the development of several anticancer treatments, there remains a need for new drugs that can overcome resistance and reduce side effects. While the medicinal herb Hydrocotyle umbellata (H. umbellata) has been used to relieve pain and inflammation, its antitumor properties have not yet been explored. In this study, we investigated the anticarcinogenic potential of H. umbellata extract (HUE) and its major components, as well as the underlying molecular mechanisms. Our results showed that HUE inhibited the growth of various tumor cell lines, including B16F10, without affecting non-cancer cells. Furthermore, HUE was effective in treating and preventing tumor growth in mice. Our mechanistic studies revealed that HUE inhibited cellular respiration, thereby reducing tumor cell proliferation. When combined with 2-deoxy-D-glucose, HUE demonstrated an enhanced anticancer effect by increasing the rate apoptosis. Analysis of the ethyl acetate and n-butanol fractions of HUE identified 1,3,4-trihydroxy-2-butanyl-α-d-glucopyranoside and caffeoylquinic acid derivatives as the major components responsible for the observed anticancer effects. In conclusion, our findings suggest that HUE and its two major components have the potential to be developed as effective therapeutic agents for a wide range of tumors by targeting cancer cell metabolism.

DIPG-35. OVERCOMING THE BLOOD-BRAIN BARRIER CHALLENGE IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are aggressive paediatric brainstem tumours with no known cure. The development of effective treatments is greatly impeded by the failure of most anti-tumour agents to penetrate the blood-brain barrier (BBB). Our research, along with that of others, has revealed that the BBB remains intact in DMG. We employed DMG orthotopic models to explore potential region-specific variations in response to the therapeutic agent temsirolimus as well as confocal microscopy and single-cell RNA sequencing (scRNAseq) to elucidate the impact of DMGs on brain endothelial morphology and pathways governing BBB integrity. We first assessed the effectiveness of the mTOR inhibitor temsirolimus when administered in cortex-injected DMG models compared to brainstem-injected DMG. We found significantly increased survival rates, elevated temsirolimus levels, and reduced tumour cell proliferation in the cortex versus the brainstem. Confocal imaging analysis indicated no structural differences between DMG and Matrigel-injected animals. However, scRNAseq unveiled distinct transcriptomic changes: brainstem-injected DMG exhibited downregulation of genes related to inflammatory and apoptotic pathways, whereas cortex-injected DMG showed downregulation of interferon pathways. To overcome the tightened barrier in the brainstem, we explored three MCL1 inhibitors and SNGR-TNFa (targeting CD13) as potential blood-brain barrier modulators. These inhibitors significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro DMG BBB model, and decreased the protein expression of claudin-5. Moreover, in vivo, we found that administration of a single dose of either SNGR-TNFa or the MCL1 inhibitor S63845 improved penetration of Texas-Red (3KDa) in DMG-engrafted animals, indicating the effective opening of the BBB. In summary, our study revealed that DMG directly affects BBB pathways in endothelial cells, tightening the barrier and reducing treatment efficacy. MCL1 inhibitors and SNGR-TNFa show promise in modulating the BBB and have the potential to enhance therapeutic activity.

Dicerandrol C Suppresses Proliferation and Induces Apoptosis of HepG2 and Hela Cancer Cells by Inhibiting Wnt/β-Catenin Signaling Pathway.

Overwhelming evidence points to an aberrant Wnt/β-catenin signaling as a critical factor in hepatocellular carcinoma (HCC) and cervical cancer (CC) pathogenesis. Dicerandrol C (DD-9), a dimeric tetrahydroxanthenone isolated from the endophytic fungus Phomopsis asparagi DHS-48 obtained from mangrove plant Rhizophora mangle via chemical epigenetic manipulation of the culture, has demonstrated effective anti-tumor properties, with an obscure action mechanism. The objective of the current study was to explore the efficacy of DD-9 on HepG2 and HeLa cancer cells and its functional mechanism amid the Wnt/β catenin signaling cascade. Isolation of DD-9 was carried out using various column chromatographic methods, and its structure was elucidated with 1D NMR. The cytotoxicity of DD-9 on HepG2 and HeLa cells was observed with respect to the proliferation, clonality, migration, invasion, apoptosis, cell cycle, and Wnt/β-catenin signaling cascade. We found that DD-9 treatment significantly reduced tumor cell proliferation in dose- and time-dependent manners in HepG2 and HeLa cells. The subsequent experiments in vitro implied that DD-63 could significantly suppress the tumor clonality, metastases, and induced apoptosis, and that it arrested the cell cycle at the G0/G1 phase of HepG2 and HeLa cells. Dual luciferase assay, Western blot, and immunofluorescence assay showed that DD-9 could dose-dependently attenuate the Wnt/β-catenin signaling by inhibiting β-catenin transcriptional activity and abrogating β-catenin translocated to the nucleus; down-regulating the transcription level of β-catenin-stimulated Wnt target gene and the expression of related proteins including p-GSK3-β, β-catenin, LEF1, Axin1, c-Myc, and CyclinD1; and up-regulating GSK3-β expression, which indicates that DD-9 stabilized the β-catenin degradation complex, thereby inducing β-catenin degradation and inactivation of the Wnt/β-catenin pathway. The possible interaction between DD-9 and β-catenin and GSK3-β protein was further confirmed by molecular docking studies. Collectively, DD-9 may suppress proliferation and induce apoptosis of liver and cervical cancer cells, possibly at least in part via GSK3-β-mediated crosstalk with the Wnt/β-catenin signaling axis, providing insights into the mechanism for the potency of DD-9 on hepatocellular and cervical cancer.

PD-L1 regulates tumor proliferation and T-cell function in NF2-associated meningiomas.

Programmed death-ligand 1 (PD-L1) expression is an immune evasion mechanism that has been demonstrated in many tumors and is commonly associated with a poor prognosis. Over the years, anti-PD-L1 agents have gained attention as novel anticancer therapeutics that induce durable tumor regression in numerous malignancies. They may be a new treatment choice for neurofibromatosis type 2 (NF2) patients. The aims of this study were to detect the expression of PD-L1 in NF2-associated meningiomas, explore the effect of PD-L1 downregulation on tumor cell characteristics and T-cell functions, and investigate the possible pathways that regulate PD-L1 expression to further dissect the possible mechanism of immune suppression in NF2 tumors and to provide new treatment options for NF2 patients. PD-L1 is heterogeneously expressed in NF2-associated meningiomas. After PD-L1 knockdown in NF2-associated meningioma cells, tumor cell proliferation was significantly inhibited, and the apoptosis rate was elevated. When T cells were cocultured with siPD-L1-transfected NF2-associated meningioma cells, the expression of CD69 on both CD4+ and CD8+ T cells was partly reversed, and the capacity of CD8+ T cells to kill siPD-L1-transfected tumor cells was partly restored. Results also showed that the PI3K-AKT-mTOR pathway regulates PD-L1 expression, and the mTOR inhibitor rapamycin rapidly and persistently suppresses PD-L1 expression. Invivo experimental results suggested that anti-PD-L1 antibody may have a synergetic effect with the mTOR inhibitor in reducing tumor cell proliferation and that reduced PD-L1 expression could contribute to antitumor efficacy. Targeting PD-L1 could be helpful for restoring the function of tumor-infiltrating lymphocytes and inducing apoptosis to inhibit tumor proliferation in NF2-associated meningiomas. Dissecting the mechanisms of the PD-L1-driven tumorigenesis of NF2-associated meningioma will help to improve our understanding of the mechanisms underlying tumor progression and could facilitate further refinement of current therapies to improve the treatment of NF2 patients.

Exploring the multi-targeting phytoestrogen potential of Calycosin for cancer treatment: A review.

Cancer remains a significant challenge in the field of oncology, with the search for novel and effective treatments ongoing. Calycosin (CA), a phytoestrogen derived from traditional Chinese medicine, has garnered attention as a promising candidate. With its high targeting and low toxicity profile, CA has demonstrated medicinal potential across various diseases, including cancers, inflammation, and cardiovascular disease. Studies have revealed that CA possesses inhibitory effects against a diverse array of cancers. The underlying mechanism of action involves a reduction in tumor cell proliferation, induction of tumor cell apoptosis, and suppression of tumor cell migration and invasion. Furthermore, CA has been shown to enhance the efficacy of certain chemotherapeutic drugs, making it a potential component in treating malignant tumors. Given its high efficacy, low toxicity, and multi-targeting characteristics, CA holds considerable promise as a therapeutic agent for cancer treatment. The objective of this review is to present a synthesis of the current understanding of the antitumor mechanism of CA and its research progress.

Abstract PO5-18-11: Use of DiviTum®TKa as a biomarker assay for CDK4/6 inhibitor medication compliance and drug-drug interaction assessment in ER/PR positive metastatic breast cancer

Abstract Background: CDK4/6 inhibitor (CDK4/6i) with endocrine therapy (ET) is standard first line (1L) treatment for metastatic hormone positive (ER/PR+) HER2- negative breast cancer (mBC). Some patients have progression free survival (PFS) of 2 years or more whereas some patients have very short PFS on these medications. Thymidine kinase is a biomarker that reflects cell proliferation. DiviTum®TKa is an assay that was cleared by the FDA in July 2022 based on data that showed that thymidine kinase activity (TKa) value of < 250 DiviTum units (DuA) is associated with the decreased likelihood of disease progression within 30 days or 60 days post testing. The purpose of this study is to assess the use of TKa as a biomarker for identifying potential medical compliance issues and drug-drug interactions (DDI) in patients on CDK4/6i and to evaluate whether counseling and optimization of concurrent medications will lead to lower TKa levels in subsequent treatment cycles and possibly longer PFS. Trial design: This is a pilot study to assess whether TKa can serve as a biomarker to identify patients with a suboptimal reduction in tumor cell proliferation caused by a potential CDK4/6i compliance or DDI issue. We will also assess whether correction of the issue can improve suppression of TKa levels in ER/PR+ mBC patients on 1L CDK4/6i and ET. For screening, we are utilizing a novel automated real-time data driven cohort identification system designed by Dr. Guannan Gong at Yale. Patients will be treated and monitored as per standard of care. An aliquot of serum obtained during routine blood draws will be sent to a CLIA-certified Biovica lab for TKa testing. TKa results will be returned in real time. Patients with a detectable TKa baseline level (above 145 DuA), followed by a sustained TKa level (>145 DuA) at cycle 1 D15 and D28 will be assessed for medication compliance and DDI. TKa levels will be repeated at subsequent standard of care blood draws to assess if the intervention affected TKa levels. Patients will be followed for 2 years. Eligibility criteria: Participants must have ER+ mBC and starting 1L CDK4/6i + ET and be previously CDK4/6i -naïve. Specific aims: Primary objective: to estimate the rate of improvement in CDK4/6i response (change in TKa levels) after counseling for medication compliance and adjustment of potential deleterious DDIs. Secondary objectives: 1) estimate the rate of sub-optimal CDK 4/6i response in cycle 1 of treatment. 2) Compare clinical benefit rate (CBR) in patients with sub-optimal and optimal CDK4/6i response after both cycles 1 and cycle 3. 3) Compare PFS in patients with sub-optimal and optimal CDK 4/6i response after both cycles 1 and cycle 3. 4) Assess CDK4/6i response via TKa levels upon CDK4/6i dose reductions or changes in CDK4/6i regimens. 5) Compare CDK4/6i response profiles across the three CDK 4/6i among different patients and within the same patients if CDK4/6i is changed throughout treatment course. 6) Correlate TKa levels with tumor marker levels. 7) Assess plasma concentrations of CDK4/6i in patients with suboptimal TKa levels. Statistical methods: For binary endpoints measured at a given time point, we will estimate the two-sided 95% exact CI using Clopper-Pearson method. With a sample size of 120 patients, assuming 20% drop out rate, we predict that about 15% of patients will have suboptimal CDK4/6i response, estimating a 95% CI of 0.086-0.235 in the first cycle. Comparisons between paired outcomes will be completed using the paired t-test or Wilcoxon signed-rank test for continuous parameters of interest, or McNemar’s Chi-square test for categorical parameters of interest. Time to event endpoints will be estimated using Kaplan-Meier method. Present accrual and target accrual: This trial has just been activated. Target accrual is 120 patients. Contact information for people with a specific interest in the trial: Mariya.Rozenblit@yale.edu Citation Format: Mariya Rozenblit, Adriana Kahn, Guannan Gong, Amy Williams, Lajos Pusztai. Use of DiviTum®TKa as a biomarker assay for CDK4/6 inhibitor medication compliance and drug-drug interaction assessment in ER/PR positive metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-11.

Abstract 4181: AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia

Abstract Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold prediction software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients. Citation Format: Niels Vandewalle, Hatice Satilmis, Emma M. Verheye, Rong Fan, Yanmeng Wang, Timo W. De Groof, Tessa Kerre, Nathan De Beule, Ann De Becker, Elke De Bruyne, Eline Menu, Karin Vanderkerken, Karine Breckpot, Nick Devoogdt, Kim De Veirman. AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia [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 4181.

Abstract 3802: Integrating multiplex-staining and multiplex-DIA: Profiling the tumor microenvironment spatial proteome for precision cancer research

Abstract Our innovative dual-dimensional multiplexing approach ushers in a new era of spatial proteomic exploration. Multiplex-staining deepens understanding of tumor-stroma interactions. Our robust dimethyl labeling-based multiplex-DIA workflow enhances throughput and proteome depth in the Deep Visual Proteomics (DVP) technology. Now, our audacious integration of multiplex-staining with multiplex-DIA holds the promise of pushing the boundaries of spatial proteome profiling. This innovation combines the power of mass spectrometry (MS) with clinical viability in 1) requiring just one FFPE section for spatial proteome analysis at the resolution of individual cell types; 2) ingeniously transmuting MS revelations into a clinically feasible staining strategy. A force in personalized oncology, our innovation unravels the intricate dynamics of the cancer ecosystem while ensuring direct clinical relevance. We conducted multiplex immunofluorescent staining on human cancers using the MACSima platform, followed by cellular phenotyping and automated single-cell laser microdissection. Subsequently, we loaded dimethyl-labelled 3-plex tryptic peptides onto Evotips and performed LC-MS analysis using the Evosep One coupled to timsTOF SCP. Pre- and post-22-plex staining proteome analysis confirmed no significant protein loss, validating microdissection compatibility and excellent recovery. The key findings include: 1) profiling of 21 distinct cell types/subtypes in human tonsil cancer tissues, resulting in the identification of 4941 protein groups from a cellular volume of 110,859 µm3, equivalent to approximately 55 cells; 2) observation of a loss of CD45RA/RO expression in tumor-infiltrating cytotoxic T cells, contributing to a reduction in MET and PDGF signaling activity within tumor cells; 3) the presence of abundant cytotoxic T cells, B cells, and plasma cells, coupled with a reduction in tumor cell proliferation, indicative of a hot tumor microenvironment. Proteome analysis revealed enhanced immune cell mobility and interactions with tumor cells. Moreover, our workflow facilitated the identification of potential diagnostic markers for mycosis fungoides, a marker-lacking skin lymphoma, where we achieved the identification of 2964 significant proteins out of a total of 6009. In addition, a rare lymphoma case study not only guided ABVD chemotherapy and suggested IL4 inhibition to address chemoresistance but also identified CDK, MCM, and PSMA/B/C/D/E inhibitors as promising candidates for targeted therapy. The integration of multiplex-staining and multiplex-DIA represents a significant stride in advancing personalized diagnostics and therapeutic strategies. Citation Format: Xiang Zheng, Andreas Mund, Matthias Mann. Integrating multiplex-staining and multiplex-DIA: Profiling the tumor microenvironment spatial proteome for precision cancer research [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 3802.

Proteomic study on nintedanib in gastric cancer cells.

Gastric cancer is a very common gastrointestinal tumor with a high mortality rate. Nintedanib has been shown to significantly reduce tumor cell proliferation and increase apoptosis in gastric cancer cells in vitro. However, its systemic action mechanism on gastric cancer cells remains unclear. A high-throughput proteomic approach should help identify the potential mechanisms and targets of nintedanib on gastric cancer cells. The effects of nintedanib on the biological behavior of gastric cancer cells were evaluated. A cytotoxic proliferation assay was performed to estimate the half maximal inhibitory concentration (IC50). AGS cells were divided into control, and nintedanib-treated groups (5 µM, 48 h), and differential protein expression was investigated using tandem mass tags (TMT) proteomics. The molecular mechanisms of these differentially expressed proteins and their network interactions were then analyzed using bioinformatics, and potential nintedanib targets were identified. This study identified 845 differentially expressed proteins in the nintedanib-treated group (compared to the control group), comprising 526 up-regulated and 319 down-regulated proteins. Bioinformatics analysis revealed that the differentially expressed proteins were primarily enriched in biological pathways for branched-chain amino acid metabolism, steroid biosynthesis, propionate metabolism, fatty acid metabolism, lysosome, peroxisome, and ferroptosis. Key driver analysis revealed that proteins, such as enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH), isocitrate dehydrogenase 1 (IDH1), acyl-CoA oxidase 1 (ACOX1), acyl-CoA oxidase 2 (ACOX2), acyl-CoA oxidase 3 (ACOX3), and acetyl-CoA acyltransferase 1 (ACAA1) could be linked with nintedanib action. Nintedanib inhibits the proliferation, invasion, and metastasis of gastric cancer cells. The crossover pathways and protein networks predicted by proteomics should provide more detailed molecular information enabling the use of nintedanib against gastric cancer.

Danggui Buxue Tang improves therapeutic efficacy of doxorubicin in triple negative breast cancer via ferroptosis

Ethnopharmacological relevanceDanggui Buxue Tang (DBT) has been used for over 800 years to enhance Qi and nourish Blood, and it is particularly beneficial for cancer patients. Recent research has shown that combining DBT with chemotherapy agents leads to superior anti-cancer effects, thereby enhancing therapeutic efficacy. Aim of the studyThe aim of this study was to evaluate the effectiveness of a combination therapy involving doxorubicin (DOX) and Danggui Buxue Tang (DBT) in the treatment of triple-negative breast cancer (TNBC) and to elucidate the underlying mechanisms of action. Materials and methodsIn vitro experiments were performed using MDA-MB-231 and 4T1 cells, while in vivo experiments were carried out using MDA-MB-231 xenograft mice. The therapeutic effects of the combination therapy were evaluated using various techniques, including MTT assay, colony formation assay, flow cytometry, transwell assay, immunofluorescence, transmission electron microscopy (TEM), histological analysis, western blotting, and bioluminescence assay. ResultsDBT was found to enhance DOX's anti-TNBC activity in vitro by promoting ferroptosis, as evidenced by the observed mitochondrial morphological changes using TEM. The combination therapy was also found to reduce the expression of Nrf2, HO-1, and GPX4, which are all targets for ferroptosis induction, while simultaneously increasing ROS production. Additionally, the combination therapy reduced nuclear accumulation and constitutive activation of Nrf2, which is a significant cause of chemotherapy resistance and promotes cancer growth. In vivo experiments using an MDA-MB-231 xenograft animal model revealed that the combination therapy significantly reduced tumor cell proliferation and accelerated TNBC deaths by modulating the Nrf2/HO-1/GPX4 axis, with no evidence of tissue abnormalities. Moreover, the combination therapy exhibited a liver protective effect, and administration of Fer-1 was able to reduce the ROS formation produced by the DBT + DOX combination therapy. ConclusionThis study provides evidence that the combination therapy of DOX and DBT has the potential to treat TNBC by promoting ferroptosis through the Nrf2/HO-1/GPX4 axis.

177Lu]Lu-labeled anti-claudin-18.2 antibody demonstrated radioimmunotherapy potential in gastric cancer mouse xenograft models.

Gastric cancer (GC), one of the most prevalent and deadliest tumors worldwide, is often diagnosed at an advanced stage with limited treatment options and poor prognosis. The development of a CLDN18.2-targeted radioimmunotherapy probe is a potential treatment option for GC. The CLDN18.2 antibody TST001 (provided by Transcenta) was conjugated with DOTA and radiolabeled with the radioactive nuclide 177Lu. The specificity and targeting ability were evaluated by cell uptake, imaging and biodistribution experiments. In BGC823CLDN18.2/AGSCLDN18.2 mouse models, the efficacy of [177Lu]Lu-TST001 against CLDN18.2-expressing tumors was demonstrated, and toxicity was evaluated by H&E staining and blood sample testing. [177Lu]Lu-TST001 was labeled with an 99.17%±0.32 radiochemical purity, an 18.50 ± 1.27 MBq/nmol specific activity and a stability of ≥ 94% after 7 days. It exhibited specific and high tumor uptake in CLDN18.2-positive xenografts of GC mouse models. Survival studies in BGC823CLDN18.2 and AGSCLDN18.2 tumor-bearing mouse models indicated that a low dose of 5.55 MBq and a high dose of 11.10 MBq [177Lu]Lu-TST001 significantly inhibited tumor growth compared to the saline control group, with the 11.1 MBq group showing better therapeutic efficacy. Histological staining with hematoxylin and eosin (H&E) and Ki67 immunohistochemistry of residual tissues confirmed tumor tissue destruction and reduced tumor cell proliferation following treatment. H&E showed that there was no significant short-term toxicity observed in the heart, spleen, stomach or other important organs when treated with a high dose of [177Lu]Lu-TST001, and no apparent hematotoxicity or liver toxicity was observed. In preclinical studies, [177Lu]Lu-TST001 demonstrated significant antitumor efficacy with acceptable toxicity. It exhibits strong potential for clinical translation, providing a new promising treatment option for CLDN18.2-overexpressing tumors, including GC.

IMMU-41. IMMUNOLOGICAL TARGETING OF SLOW-CYCLING CELLS TO PREVENT RECURRENCE IN GBM

Abstract Glioblastoma (GBM) is the most common primary malignancy of the central nervous system (CNS) and is typically fatal due to recurrence despite aggressive treatment. Targeting residual cells after surgical tumor removal is challenging due to the infiltrative nature of GBM. It is crucial to develop effective adjuvant therapies that can combat the drivers of disease recurrence. Traditional anticancer treatments tend to eliminate rapidly dividing cells while sparing dormant or infrequently dividing populations. Numerous studies have identified slow-cycling cells (SCCs) in various cancers, including high-grade glioma, which are resistant to therapies. Our laboratory has discovered SCCs in high-grade glioma, which are highly infiltrative and treatment-resistant cells responsible for driving recurrence. It is imperative to develop an effective strategy to specifically eliminate this cell population in order to reduce recurrence and improve survival rates. Importantly, these cells have a defined cellular niche that contains potentially immunogenic neoantigens, making them suitable targets for immune-based therapies. Our primary objective is to utilize SCC antigens in the form of RNA or peptides to stimulate the immune system and enable it to identify and target treatment-resistant clones. By focusing on these crucial drivers of recurrence, our aim is to achieve strong and effective tumor control. Our research findings indicate that our approach effectively primes T cells to identify and target treatment-resistant cells in glioma. Our results demonstrated that T cells specific to slow-cycling cells (SCC-T cells) showed greater activation when exposed to treatment-resistant tumor cells, as evidenced by increased differentiation of CD8 cells and enhanced development of effector and memory cells. Furthermore, SCC-T cells exhibited the highest level of anti-tumor activity, resulting in a significant reduction in tumor cell proliferation and an increase in apoptosis. Taken together, these results highlight the effectiveness and superiority of our SCC-based immunotherapy platform in targeting treatment-resistant glioblastoma cells.

Long-Term Tumor Control Following Targeted Alpha Therapy (TAT) of Low-Grade Gliomas (LGGs): A New Treatment Paradigm?

The median survival time has been reported to vary between 5 and 8 years in low-grade (WHO grade 2) astrocytoma, and between 10 and 15 years for grade 2 oligodendroglioma. Targeted alpha therapy (TAT), using the modified peptide vector [213Bi]Bi/[225Ac]Ac-DOTA-substance P, has been developed to treat glioblastoma (GBM), a prevalent malignant brain tumor. In order to assess the risk of late neurotoxicity, assuming that reduced tumor cell proliferation and invasion should directly translate into good responses in low-grade gliomas (LGGs), a limited number of patients with diffuse invasive astrocytoma (n = 8) and oligodendroglioma (n = 3) were offered TAT. In two oligodendroglioma patients, TAT was applied as a second-line treatment for tumor progression, 10 years after targeted beta therapy using [90Y]Y-DOTA-substance P. The radiopharmaceutical was locally injected directly into the tumor via a stereotactic insertion of a capsule-catheter system. The activity used for radiolabeling was 2-2.5 GBq of Bismuth-213 and 17 to 35 MBq of Actinium-225, mostly applied in a single fraction. The recurrence-free survival times were in the range of 2 to 16 years (median 11 years) in low-grade astrocytoma (n = 8), in which TAT was administered following a biopsy or tumor debulking. Regarding oligodendroglioma, the recurrence-free survival time was 24 years in the first case treated, and 4 and 5 years in the two second-line cases. In conclusion, TAT leads to long-term tumor control in the majority of patients with LGG, and recurrence has so far not manifested in patients with low-grade (grade 2) astrocytomas who received TAT as a first-line therapy. We conclude that targeted alpha therapy has the potential to become a new treatment paradigm in LGG.

GPR4 Knockout Attenuates Intestinal Inflammation and Forestalls the Development of Colitis-Associated Colorectal Cancer in Murine Models.

GPR4 is a proton-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and has been shown to potentiate intestinal inflammation in murine colitis models. Herein, we evaluated the proinflammatory role of GPR4 in the development of colitis-associated colorectal cancer (CAC) using the dextran sulfate sodium (DSS) and azoxymethane (AOM) mouse models in wild-type and GPR4 knockout mice. We found that GPR4 contributed to chronic intestinal inflammation and heightened DSS/AOM-induced intestinal tumor burden. Tumor blood vessel density was markedly reduced in mice deficient in GPR4, which correlated with increased tumor necrosis and reduced tumor cell proliferation. These data demonstrate that GPR4 ablation alleviates intestinal inflammation and reduces tumor angiogenesis, development, and progression in the AOM/DSS mouse model.

Targeting IL-23 for the interception of obesity-associated colorectal cancer

Inflammation and obesity are two major factors that promote Colorectal cancer (CRC). Our recent data suggests that interleukin (IL)-23, is significantly elevated in CRC tumors and correlates with patient obesity, tumor grade and survival. Thus, we hypothesize that obesity and CRC may be linked via inflammation and IL-23 may be a potential target for intervention in high-risk patients. TCGA dataset and patient sera were evaluated for IL-23A levels. IL-23A [IL-23 p19−/−] knockout (KO) mice were crossed to Apcmin/+ mice and progeny were fed low-fat or high-fat diets. At termination intestines were evaluated for tumorigenesis. Tumors, serum, and fecal contents were analyzed for protein biomarkers, cytokines, and microbiome profile respectively. IL-23A levels are elevated in the sera of patients with obesity and colon tumors. Genetic ablation of IL-23A significantly suppressed colonic tumor multiplicity (76–96 %) and incidence (72–95 %) in male and female mice. Similarly, small-intestinal tumor multiplicity and size were also significantly reduced in IL-23A KO mice. IL-23A knockdown in Apcmin/+ mice fed high-fat diet, also resulted in significant suppression of colonic (50–58 %) and SI (41–48 %) tumor multiplicity. Cytokine profiling showed reduction in several circulating pro-inflammatory cytokines including loss of IL-23A. Biomarker analysis suggested reduced tumor cell proliferation and immune modulation with an increase in tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the IL-23A KO mice compared to controls. Fecal microbiome analysis revealed potentially beneficial changes in the bacterial population profile. In summary, our data indicates a tumor promoting role for IL-23 in CRC including diet-induced obesity. With several IL-23 targeted therapies in clinical trials, there is a great potential for targeting this cytokine for CRC prevention and therapy.

Antioxidant Phytocomplexes Extracted from Pomegranate (Punica granatum L.) Using Hydrodynamic Cavitation Show Potential Anticancer Activity In Vitro.

Hydrodynamic cavitation (HC), as an effective, efficient, and scalable extraction technique for natural products, could enable the affordable production of valuable antioxidant extracts from plant resources. For the first time, whole pomegranate (Punica granatum L.) fruits, rich in bioactive phytochemicals endowed with anti-cancer properties, were extracted in water using HC. Aqueous fractions sequentially collected during the process (M1-M5) were lyophilized (L), filtered (A), or used as such, i.e., crude (C), and analyzed for their biochemical profile and in vitro antioxidant power. The fractions M3 and M4 from the L and C series showed the highest antiradical activity and phytochemical content. While the lyophilized form is preferable for application purposes, sample L-M3, which was produced faster and with lower energy consumption than M4, was used to assess the potential antiproliferative effect on human breast cancer line (AU565-PAR) and peripheral blood mononuclear (PBMC) cells from healthy donors. In a pilot study, cell growth, death, and redox state were assessed, showing that L-M3 significantly reduced tumor cell proliferation and intracellular oxygen reactive species. No effect on PBMCs was detected. Thus, the antioxidant phytocomplex extracted from pomegranate quickly (15 min), at room temperature (30 °C), and efficiently showed potential anticancer activity without harming healthy cells.

HYbrid DegRAding Copolymer (HYDRAC): Heterobifunctional polymers for targeted degradation of undruggable proteins.

22 Background: Of the ~20,000 proteins encoded by the human genome, only a small subset can be modulated via traditional pharmacological approaches. Included in the list of “undruggable” proteins are ones with pivotal roles in cancer. We aim to expand the arsenal of compounds able to affect the activity of these therapeutically relevant targets by introducing a modular proteomimetic platform technology designed to carry side chains consisting of targeting warheads and recruiters of cellular protein degradation machinery. We term these compounds HYbrid DegRAding Copolymers (HYDRACs) in reference to the multiplexing of different protein binders and degradation inducing sequences. Peptides arranged as HYDRACs exhibit favorable emergent properties including resistance to proteolysis, high levels of cell uptake, and payload-specific activity. Initial studies were conducted focusing on a undruggable transcription factor MYC, which is widely dysregulated in human cancers. Methods: A library of HYDRACs and control polymers were synthesized and were cell-penetrating with antiproliferative effects at sub-micromolar IC50s in a formulation- and MYC-dependent manner across multiple cell lines. Results: HYDRAC treatment resulted in significant decreases in MYC protein levels, which was rescued by proteasome inhibitor MG132 and neddylation inhibitor MLN4924, without changes in mRNA levels. RNAseq showed selective overexpression of MYC pathway genes only in HYDRAC treated cells, confirming on-target effects. Circular dichroism showed strong HYDRAC-MYC interactions, which was supported by the presence of MYC following pull-down of biotin-terminated HYDRACs. Removal of the RRRG degron, scrambling the H1 sequence, and disassociation of the two domains on separate polymers all abrogated MYC-degradation. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth and reduced tumor cell proliferation following HYDRAC treatment. Mice bearing luminescent Luc-MV-411 tumors treated with Cy5.5-labeled HDRACs showed selective tumor accumulation with detectable fluorescent signal in the tumor up to 72 hours following a single IP administration. The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained ability to degrade MYC. HYDRACs were also able to selectively degrade other protein targets including alpha-syn and RAS. Conclusions: We present a platform technology that addresses challenges inherent to small molecule drug design. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for development of modulators of "undruggable" targets. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for target protein degradation.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFβ coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.