Cell Xenograft Research Articles

Enhancing carboplatin sensitivity in ovarian cancer cells by blocking the mercapturic acid pathway transporter.

Ral-binding/interacting protein (RLIP) acts as a transporter that responds to stress and provides protection, specifically against glutathione-electrophile conjugates and xenobiotic toxins. Its increased presence in malignant cells, especially in cancer, emphasizes its crucial antiapoptotic function. This is achieved by selectively regulating the cellular levels of proapoptotic oxidized lipid byproducts. Suppressing the progression of tumors in human xenografts can be achieved by effectively inhibiting RLIP, a transporter in the mercapturic acid pathway, without involving chemotherapy. Utilizing ovarian cancer (OC) cell lines (MDAH2774, OVCAR4, and OVCAR8), we observed that agents targeting RLIP, such as RLIP antisense and RLIP antibodies, not only substantially impeded the viability of OC cells but also remarkably increased their sensitivity to carboplatin. To delve further into the cytotoxic synergy between RLIP antisense, RLIP antibodies, and carboplatin, we conducted investigations in both cell culture and xenografts of OC cells. The outcomes revealed that RLIP depletion via phosphorothioate antisense led to rapid and sustained remissions in established subcutaneous human ovary xenografts. Furthermore, RLIP inhibition by RLIP antibodies exhibited comparable efficacy to antisense and enhanced the effectiveness of carboplatin in MDAH2774 OC xenografts. These investigations underscore RLIP as a central carrier crucial for supporting the survival of cancer cells, positioning it as a suitable focus for cancer treatment.

SMYD5 methylation of rpL40 links ribosomal output to gastric cancer.

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical-proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5-rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K-mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer.

Anti-HDGF antibody targets EGFR tyrosine kinase inhibitor-tolerant cells in NSCLC patient-derived xenografts.

Constitutively active mutant epidermal growth factor receptor (EGFR) is one of the major oncogenic drivers in NSCLC. Targeted therapy using EGFR tyrosine kinase inhibitor (TKI) is a firstline option in patients that have metastatic or recurring disease. However, despite the high response rate to TKI, most patients have a partial response, and the disease eventually progresses in 10 to 19 months. It is believed that drug-tolerant cells that survive TKI exposure during the progression-free period facilitate the emergence of acquired resistance. Thus, targeting the drug-tolerant cells could improve the treatment of NSCLC with EGFR mutations. We demonstrated here that EGFR mutant patient-derived xenograft (PDX) tumors responded partially to osimertinib despite near complete inhibition of EGFR activation. Signaling in AKT/mTOR and MAPK pathways could be reactivated shortly after initial inhibition. As a result, many tumor cells escaped drug killing and regained growth following about 35 days of continuous osimertinib dosing. However, when an antibody to hepatoma-derived growth factor (HDGF) was given concurrently with osimertinib, tumors showed complete or near-complete responses.There was significant prolongation of progression-free survival (PFS) of tumor-bearing mice as well. Immunohistochemistry and western blot analysis of tumors collected in the early stages of treatment suggest that increased suppression of the AKT/mTOR and MAPK pathways could be a mechanism that results in enhanced efficacy of osimertinib when it is combined with anti-HDGF antibody.

Tumor-associated fibroblasts promotes proliferation and migration of prostate cancer cells by suppressing FBXL3 via upregulating hsa-miR-18b-5p

To explore the mechanism of tumor-associated fibroblasts (CAFs) for regulating proliferation and migration of prostate cancer (PCa) cells. We conducted a bioinformatics analysis to identify miRNAs with high expression in PCa. The proliferation, migration and hsa-miR-18b-5p expression levels were observed in PCa cells co-cultured with CAFs. We further examined hsa-miR-18b-5p expression level in 20 pairs of PCa and adjacent tissue samples and in different PCa cell lines and normal epithelial cells using RT-qPCR. In PCa cell lines C4-2 and LNCAPNC, the effects of transfection with a hsa-miR-18b-5p inhibitor on cell proliferation, migration, invasion, drug resistance, apoptosis and cell cycle were evaluated, and the effects of has-miR-18b-5p knockdown on C4-2 cell xenograft growth and mouse survival were observed in nude mice. Dual luciferase reporter gene assay was used to validate the targeting relationship between hsa-miR-18b-5p and its target genes, whose expressions were detected in PCa cells using RT-qPCR and Western blotting. The expression of hsa-miR-18b-5p was significantly increased in the co-culture of CAFs and PCa cell lines, which exhibited significantly enhanced proliferation and migration abilities. Transfection with has-miR-18b-5p inhibitor strongly attenuated the effect of CAFs for promoting proliferation and migration of PCa cells, and in C4-2 and LNCAP cells cultured alone, inhibition of hsa-miR-18b-5p obviously suppressed cell proliferation, migration, invasion, and drug resistance. In the tumor-bearing mice, hsa-miR-18b-5p knockdown in the transplanted cells significantly inhibited xenograft growth and increased the survival time of the mice. Target gene prediction suggested that FBXL3 was a potential target of hsa-miR-18b-5p, and dual luciferase reporter gene confirmed a binding site between them. In C4-2 and LNCAP cells, hsa-miR-18b-5p knockdown resulted in significantly increased expression levels of FBXL3. CAFs promotes proliferation and migration of PCa cells by up-regulating hsa-miR-18b-5p to suppress FBXL3 expression.

Pixantrone as a novel MCM2 inhibitor for ovarian cancer treatment

BackgroundMini-chromosome maintenance protein 2 (MCM2) is a potential target for the development of cancer therapeutics. However, small molecule inhibitors targeting MCM2 need further investigation. MethodsMolecular dynamics simulation was performed to identify active pockets in the MCM2 protein structure (6EYC). The active pocket was used as a docking model to discover MCM2 inhibitors by using structure-based virtual screening and surface plasmon resonance (SPR) assay. Furthermore, the efficacy of pixantrone targeting MCM2 in ovarian cancer was evaluated in vitro and in vivo. ResultsPixantrone was identified as a novel inhibitor of MCM2 by virtual screening. SPR binding affinity analysis confirmed the direct binding of pixantrone to MCM2 protein. Pixantrone significantly reduced the viability of ovarian cancer cells A2780 and SKOV3 in a dose- and time-dependent manner. In addition, pixantrone inhibited DNA replication, and induced cell cycle arrest and apoptosis in ovarian cancer cells via targeting MCM2. Knockdown of MCM2 could attenuate the inhibitory activity of pixantrone in ovarian cancer cells. Furthermore, pixantrone significantly suppressed ovarian cancer growth in the A2780 cell xenograft mouse model and showed favorable safety. ConclusionThese findings suggest that pixantrone may be a promising drug for ovarian cancer patients by targeting MCM2 in the clinic.

A brain organoid/ALL coculture model reveals the AP-1 pathway as critically associated with CNS involvement of BCP-ALL

AbstractCentral nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2-dimensional cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3-dimensional coculture model combining human brain organoids and BCP-ALL cells. We noticed significantly higher engraftment of BCP-ALL cell lines and patient-derived xenograft (PDX) cells in cerebral organoids than non-ALL cells. To validate translatability between organoid coculture and in vivo murine models, we confirmed that targeting CNS leukemia–relevant pathways such as CD79a/Igα or C-X-C motif chemokine receptor 4–stromal cell-derived factor 1 reduced the invasion of BCP-ALL cells into organoids. RNA sequencing and functional validations of organoid-invading leukemia cells compared with the noninvaded fraction revealed significant upregulation of activator protein 1 (AP-1) transcription factor–complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in PDX ALL cells recovered from the CNS compared with spleen blasts of mice that had received transplantation with TCF3::PBX1+ PDX cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1 gene, jun proto-oncogene, in patients initially diagnosed as CNS-positive BCP-ALL compared with CNS-negative cases as well as CNS-relapse vs non–CNS-relapse cases in a cohort of 100 patients with BCP-ALL. Our results suggest CNS organoids as a novel model to investigate CNS involvement and identify the AP-1 pathway as a critical driver of CNS disease in BCP-ALL.

Loss of monopolar spindle-binding protein 3B expression promotes colorectal cancer invasiveness by activation of target of rapamycin kinase/autophagy signaling.

Monopolar spindle-binding protein 3B (MOB3B) functions as a signal transducer and altered MOB3B expression is associated with the development of human cancers. To investigate the role of MOB3B in colorectal cancer (CRC). This study collected 102 CRC tissue samples for immunohistochemical detection of MOB3B expression for association with CRC prognosis. After overexpression and knockdown of MOB3B expression were induced in CRC cell lines, changes in cell viability, migration, invasion, and gene expression were assayed. Tumor cell autophagy was detected using transmission electron microscopy, while nude mouse xenograft experiments were performed to confirm the in-vitro results. MOB3B expression was reduced in CRC vs normal tissues and loss of MOB3B expression was associated with poor CRC prognosis. Overexpression of MOB3B protein in vitro attenuated the cell viability as well as the migration and invasion capacities of CRC cells, whereas knockdown of MOB3B expression had the opposite effects in CRC cells. At the molecular level, microtubule-associated protein light chain 3 II/I expression was elevated, whereas the expression of matrix metalloproteinase (MMP)2, MMP9, sequestosome 1, and phosphorylated mechanistic target of rapamycin kinase (mTOR) was downregulated in MOB3B-overexpressing RKO cells. In contrast, the opposite results were observed in tumor cells with MOB3B knockdown. The nude mouse data confirmed these in-vitro findings, i.e., MOB3B expression suppressed CRC cell xenograft growth, whereas knockdown of MOB3B expression promoted the growth of CRC cell xenografts. Loss of MOB3B expression promotes CRC development and malignant behaviors, suggesting a potential tumor suppressive role of MOB3B in CRC by inhibition of mTOR/autophagy signaling.

Intratumoral Delivery of Genetically Engineered Anti-IL-6 Trans-signaling Therapeutics.

Interleukin-6 (IL-6) is a highly pro-inflammatory cytokine involved in the etiopathology of several inflammatory diseases and cancer. As so, the inhibition of IL-6 signaling pathways has emerged as an attractive therapeutic avenue for the treatment of several chronic diseases. Since IL-6 trans-signaling was described as the pathological branch of IL-6, selective inhibitors were developed. Next-generation variants with increased trans-signaling specificity and potency emerged as great candidates for the treatment of several diseases, with reduced off-target effects. The highly time-consuming and costly processes involving recombinant protein production, however, have hampered the progress of anti-cytokine pharmaceuticals in clinic so far. Herein, we developed gene therapeutic modalities of IL-6-trans-signaling inhibitors as alternatives for sustained recombinant protein secretion. By using an IL-6-dependent lymphoma cell line and xenograft tumor model, we demonstrated the superior inhibitory potential of second-generation anti-IL-6 trans-signaling therapeutic. We compared the efficiency of distinct gene delivery modalities using a bioluminescent biomarker probe and observed consistent protein production via cell-based delivery. When delivered intratumorally, genetically engineered sgp130FlyRFc-secreting cells significantly reduced tumor burden and increased animal survival, representing a promising therapeutic avenue to be explored in clinically relevant gene delivery applications.

γ-Tocotrienol enhances autophagy of gastric cancer cells by the regulation of GSK3β/β-Catenin pathway.

γ-Tocotrienol (γ-T3) is a major subtype of vitamin E, mainly extracted from palm trees, barley, walnuts, and other plants. γ-T3 has effects on anti-inflammation, anti-oxidation, and potential chemoprevention against malignancies. It is still uncompleted to understand the effect of γ-T3 on the inhibitory mechanism of cancer. This study aimed to investigate whether γ-T3 enhanced autophagy in gastric cancer and the underlying molecular mechanism. The results showed that γ-T3 (0-90 μmol/L) inhibited the proliferation of gastric cancer MKN45 cells and AGS cells, and arrested the cell cycle at the G0/G1 phase in a dose-dependent manner. Autophagy was increased in MKN45 cells treated with γ-T3 (0-45 μmol/L), especially at a dose of 30 μmol/L for 24 h. These effects were reversed by 3-methyladeninepretreatment. Furthermore, γ-T3 (30 μmol/L) also significantly downregulated the expression of pGSK-3β (ser9) and β-catenin protein in MKN45 cells, and γ-T3 (20 mg/kg b.w.) effectively decreased the growth of MKN45 cell xenografts in BABL/c mice. GSK-3β inhibitor-CHIR-99021 reversed the negative regulation of GSK-3β/β-Catenin signaling and autophagy. Our findings indicated that γ-T3 enhances autophagy in gastric cancer cells mediated by GSK-3β/β-Catenin signaling, which provides new insights into the role of γ-T3 enhancing autophagy in gastric cancer.

Brusatol improves the efficacy of an anti-mouse-PD-1 antibody via inhibiting programmed cell death 1 ligand 1 expression in a murine head and neck squamous cell carcinoma model

ObjectiveCombing PD-1/PD-L1 immune checkpoint inhibitors with natural products has exhibited better efficacy than monotherapy. Hence, the purpose of this research was to examine the anti-cancer effects of brusatol, a natural quassinoid-terpenoid derived from Brucea javanica, when used in conjunction with an anti-mouse-PD-1 antibody in a murine head and neck squamous cell carcinoma (HNSCC) model and elucidate underlying mechanisms. DesignA murine HNSCC model and an SCC-15 cell xenograft nude mouse model were established to investigate the anti-cancer effects of brusatol and anti-PD-1 antibody. Mechanistic studies were performed using immunohistochemistry. Cell proliferation, migration, colony formation, and invasion were evaluated by MTT, migration, colony formation, and transwell invasion assays. PD-L1 levels in oral squamous cell carcinoma (OSCC) cells were assessed through qRT-PCR, flow cytometry, and western blotting assays. The impact of brusatol on Jurkat T cell function was assessed by an OSCC/Jurkat co-culture assay. ResultsBrusatol improved tumor suppression by anti-PD-1 antibody in HNSCC mouse models. Mechanistic studies revealed brusatol inhibited tumor cell growth and angiogenesis, induced apoptosis, increased T lymphocyte infiltration, and reduced PD-L1 expression in tumors. Furthermore, in vitro assays confirmed brusatol inhibited PD-L1 expression in OSCC cells and suppressed cell migration, colony formation, and invasion. Co-culture assays indicated that brusatol's PD-L1 inhibition enhanced Jurkat T cell-mediated OSCC cell death and reversed the inhibitory effect induced by OSCC cells. ConclusionsBrusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy.

Interfering with aggregated α-synuclein in advanced melanoma leads to a major upregulation of MHC class II proteins.

Melanoma is the most serious and deadly form of skin cancer and with progression to advanced melanoma, the intrinsically disordered protein α-synuclein is upregulated to high levels. While toxic to dopaminergic neurons in Parkinson's disease, α-synuclein is highly beneficial for primary and metastatic melanoma cells. To gain detailed insights into this exact opposite role of α-synuclein in advanced melanoma, we performed proteomic studies of high-level α-synuclein-expressing human melanoma cell lines that were treated with the diphenyl-pyrazole small-molecule compound anle138b, which binds to and interferes with the oligomeric structure of α-synuclein. We also performed proteomic and transcriptomic studies of human melanoma xenografts that were treated systemically with the anle138b compound. The results reveal that interfering with oligomerized α-synuclein in the melanoma cells in these tumor xenografts led to a substantial upregulation and expression of major histocompatibility complex proteins, which are pertinent to enhancing anti-melanoma immune responses.

Targeting exhausted cytotoxic T cells through CTLA-4 inhibition promotes elimination of neoplastic cells in human myelofibrosis xenografts.

Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. In the context of myeloid neoplasms, there is a growing recognition of the dysregulation of immune response and T-cell function as significant contributors to disease progression and immune evasion. We investigated cytotoxic T-cell exhaustion in MF to restore immune response against malignant cells. Increased expression of inhibitory receptors like CTLA-4 was observed on cytotoxic T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. CTLA-4 ligands CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T-cell activation in MF patients. Unlike healthy donors, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, anti-CTLA-4 treatment promoted elimination of neoplastic monocytes and granulocytes in a co-culture system with cytotoxic T cells. To test CTLA-4 inhibition in vivo, patient-derived xenografts were generated by transplanting MF CD34+ cells and by infusing homologous T cells in NSGS mice. CTLA-4 blockade reduced human myeloid chimerism and led to T-cell expansion in spleen and bone marrow. Overall, these findings shed light on T-cell dysfunction in MF and suggest that CTLA-4 blockade can boost the cytotoxic T cell-mediated immune response against tumor cells.

YAP represses the TEAD-NF-κB complex and inhibits the growth of clear cell renal cell carcinoma.

The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.

Redistribution of Defective Mitochondria-mediated Dihydroorotate Dehydrogenase Imparts 5-Fluorouracil Resistance in Colorectal Cancer

Although 5-fluorouracil (5-FU) is the primary chemotherapy treatment for colorectal cancer (CRC), its efficacy is limited by drug resistance. Ferroptosis activation is a promising treatment for 5-FU-resistant cancer cells; however, potential therapeutic targets remain elusive. This study investigated ferroptosis vulnerability and dihydroorotate dehydrogenase (DHODH) activity using stable, 5-FU-resistant CRC cell lines and xenograft models. Ferroptosis was characterized by measuring malondialdehyde levels, assessing lipid metabolism and peroxidation, and using mitochondrial imaging and assays. DHODH function is investigated through gene knockdown experiments, tumor behavior assays, mitochondrial import reactions, intramitochondrial localization, enzymatic activity analyses, and metabolomics assessments. Intracellular lipid accumulation and mitochondrial DHODH deficiency led to lipid peroxidation overload, weakening the defense system of 5-FU-resistant CRC cells against ferroptosis. DHODH, primarily located within the inner mitochondrial membrane, played a crucial role in driving intracellular pyrimidine biosynthesis and was redistributed to the cytosol in 5-FU-resistant CRC cells. Cytosolic DHODH, like its mitochondrial counterpart, exhibited dihydroorotate catalytic activity and participated in pyrimidine biosynthesis. This amplified intracellular pyrimidine pools, thereby impeding the efficacy of 5-FU treatment through molecular competition. These findings contribute to the understanding of 5-FU resistance mechanisms and suggest that ferroptosis and DHODH are promising therapeutic targets for patients with CRC exhibiting resistance to 5-FU.

A novel role of AIM2 inflammasome-mediated pyroptosis in radiofrequency ablation of hepatocellular carcinoma

Introduction and ObjectivesThe absence of melanoma 2 (AIM2) protein triggers the activation of the inflammasome cascade. It is unclear whether AIM2 plays a role in hepatocellular carcinoma (HCC) and radiofrequency ablation (RFA), which uses radiofrequency waves to treat tumors. In this study, we investigated if RFA could induce pyroptosis, also called cell inflammatory necrosis, in HCC through AIM2-inflammasome signaling in vivo and in vitro. Materials and MethodsBALB/c nude mice were used to generate HepG2 or SMMC-7721 cell-derived tumor xenografts. HCC cells with knockdown or overexpression of AIM2 were created using short hairpin RNA (shRNA) and expression vector transfection, respectively, for functional and mechanistic studies. Downstream effects were examined using flow cytometry, qRT-PCR, ELISAs, and other molecular assays. ResultsRFA significantly suppressed tumor growth in HCC cell xenografts. Flow cytometry analysis revealed that RFA could induce pyroptosis. Furthermore, AIM2, NLRP3, caspase-1, γ-H2AX, and DNA-PKc had significantly greater expression levels in liver tissues from mice treated with RFA compared with those of the controls. Additionally, interleukin (IL)-1β and IL-18 expression levels were significantly higher in the HCC cell-derived xenograft mice treated with RFA compared with those without RFA. Notably, a significantly greater effect was achieved in the RFA complete ablation group versus the partial ablation group. Knockdown or overexpression of AIM2 in HCC cells demonstrated that AIM2 exerted a role in RFA-induced pyroptosis. ConclusionsRFA can suppress HCC tumor growth by inducing pyroptosis via AIM2. Therefore, therapeutically intervening with AIM2-mediated inflammasome signaling may help improve RFA treatment outcomes for HCC patients.

KCa channel targeting impairs DNA repair and invasiveness of patient-derived glioblastoma stem cells in culture and orthotopic mouse xenografts which only in part is predictable by KCa expression levels.

Prognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain-infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem-like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal-to-proneural mRNA signature of patient derived GSC-enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IKCa and BKCa Ca2+-activated K+ channels. Therefore, we analyzed here the effect of IKCa- and BKCa-targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC-derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K+ channel targeting. As a result, the IKCa- and BKCa-blocker TRAM-34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IKCa- and BKCa-expressing pGSC cultures, respectively. In addition, BKCa- but not IKCa-blockade slowed-down gel invasion in vitro. Moreover, systemic administration of TRAM-34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that KCa-blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy.

Rhodiolin inhibits the PI3K/AKT/mTOR signaling pathway via the glycolytic enzyme GPI in human papillary thyroid cancer

BackgroundPapillary thyroid carcinoma (PTC) is an endocrine malignant tumor of the head and neck. Surgery and chemotherapy are PTC treatments, but have adverse effects. Exploration of new non-toxic anti-PTC drugs for PTC treatment is an unmet need. MethodsWe aimed to identify anti-PTC drugs that could inhibit PTC-cell proliferation through high-throughput screening of a library of well-characterized naturally occurring small-molecule compounds. Then, the anti-PTC function of rhodiolin was validated by in vitro cell models and xenograft tumor models ResultsWe initially demonstrated that rhodiolin inhibited the growth and induced the apoptosis of PTC cells significantly in vitro and in vivo. At the metabolic level, rhodiolin blocked glycolysis through glucose 6-phosphate isomerase (GPI), which suggested that glycolytic inhibition may be involved in mediating the anti-PTC function of rhodiolin. Transcriptomics analysis combined with bioinformatics analysis identified rhodiolin treatment to inhibit phosphorylation of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. Collectively, our findings demonstrated that rhodiolin inhibited the proliferation and induced the apoptosis of PTC cells by blocking glycolysis through the glycolytic enzyme GPI, thereby inhibiting phosphorylation of the PI3K/Akt/mTOR signaling pathway. ConclusionOur study demonstrates the potential use of rhodiolin in inhibiting the proliferation and inducing the apoptosis of PTC cells. Inhibition of phosphorylation of the PI3K/Akt/mTOR signaling pathway mediated by GPI plays an extremely important part in the ant-PTC function of rhodiolin. These results suggest that rhodiolin is a promising drug in the treatment of PTC progression. Our results provide a novel target and cell signaling pathway for PTC therapy from the perspective of energy metabolism, which could provide new perspectives and new drug choices for PTC therapy. In addition to that, our study will help to make up for the lack of drug research for PTC.

Combining CD38 antibody with CD47 blockade is a promising strategy for treating hematologic malignancies expressing CD38.

CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.

Silencing MUC1 to regulate intracellular lipid metabolism: Overcoming sunitinib resistance and inhibiting metastasis in renal cell carcinoma

Renal cell carcinoma (RCC) has been extensively studied and is a paradigmatic example of a malignancy distinguished by metabolic reprogramming. Reprogramming lipid metabolism is crucial for determining the response and resistance to chemotherapy. To overcome this, we developed a modified nanomicelle delivery system comprising polyethyleneimine (PEI) as a positive charge donor and ibuprofen as the hydrophobic end. The overall system, denoted as SPPI, is PEGylated, conjugated with sialic acid, and plays a targeting role. This system successfully delivered siRNA to cancer cells by overcoming systemic and cellular barriers. The resulting conjugates form uniform, spherical nanomicelles (37.8 ± 0.9 nm; 25.5 ± 1.4 mV) with targeting ability, stability, efficient delivery, and low toxicity. The complicated and indirect way cholesterol affects ATP-binding cassette transporters was investigated. Administering SPPI/siMUC1 resulted in significant suppression of MUC1 expression and a 3.25-fold decrease in cholesterol synthesis compared to the control, and it subsequently reversed resistance to sunitinib by modulating P-GP, ABCG2, and MRP1 activities. Importantly, SPPI/siMUC1 nanomicelles demonstrated outstanding growth inhibition (72.2 %) of sunitinib-resistant 786-O cell xenografts and remarkable inhibitory effects on tumor metastasis (45.0 %) without observable systemic toxicity according to tissue analysis and weight change evaluation. The efficacy and safety of SPPI/siMUC1 in the treatment of ccRCC were demonstrated, especially after drug resistance develops. Thus, this study introduces a novel strategy for addressing drug resistance in metabolically active cancers by targeting the lipid metabolism pathway.

LGG-49. EVALUATING THE EFFECTS OF MIRDAMETINIB IN NF1-MUTANT LOW-GRADE GLIOMAS USING BOTH CULTURED CELLS AND A NOVEL ZEBRAFISH XENOGRAFT MODEL

Abstract BACKGROUND Pediatric low-grade gliomas (LGGs) with NF1 mutations represent a significant portion of childhood brain tumors. NF1 mutations lead to the dysregulation of the MAPK kinase and mTOR pathways, contributing to tumor growth. However, obtaining cell lines and xenograft animal models for NF1-mutated LGGs remains challenging due to their slow growth and less aggressive nature. Methods and RESULTS We examined the therapeutic efficacy of a novel MEK inhibitor mirdametinib in targeting the MAPK pathway within JHH-NF1-PA1 cells, a cell line derived from a low-grade optic pathway glioma originating from a patient with type 1 neurofibromatosis. Mirdametinib decreased p-ERK protein expression levels on western blot within four hours. We confirmed that mirdametinib significantly suppressed tumor cell proliferation by approximately 60% (BrdU staining (P<0.05), CellTiter Blue assay). β-galactosidase staining revealed approximately a 50% induction of cellular senescence in JHH-NF1-PA1 cells following mirdametinib treatment (P<0.05). We confirmed our findings using murine Nf1-null low grade glioma cell lines 1861 and 1491. To establish an in vivo model for drug testing, we utilized zebrafish due to their transparency, rapid reproduction, and short development time. Injecting RFP-labeled JHH-NF1-PA1 cells into the zebrafish at the 1000-cell stage, we monitored tumor growth using TECAN fluorescence detection from the third to eighth days post-injection. The fluorescence signals obtained from TECAN were validated by fluorescence microscopy. Western blot analysis confirmed a decrease in p-ERK levels within the zebrafish following mirdametinib treatment. We are evaluating the effect of mirdametinib on NF1 low grade glioma cell survival, migration and senescence in the zebrafish brain. CONCLUSION Our findings show that mirdametinib inhibits cell proliferation and induces senescence in NF1-mutated LGGs. Moreover, our zebrafish model provides a valuable tool for evaluating the efficacy of MEK inhibitors in vivo, particularly in cases where traditional xenograft models are difficult to establish.