Tumor-bearing Nude Mice Research Articles

Oncolytic vaccinia virus harboring aphrocallistes vastus lectin exerts anti-tumor effects by directly oncolysis and inducing immune response through enhancing ROS in human ovarian cancer

Aphrocallistes vastus lectin (AVL) is a Ca2+ dependent C-type lectin produced by sponges. Previous studies have demonstrated that oncolytic vaccinia virus harboring AVL (oncoVV-AVL) effectively triggers cell death in various tumors. However, the effects of oncoVV-AVL on human ovarian cancer (OV) remain unknown. This study aims to investigate the mechanism-of-action of oncoVV-AVL in human OV cell lines and in tumor-bearing nude mice. We found that oncoVV-AVL could directly induce apoptosis and autophagy in ovarian cancer cells. Additionally, our results showed that oncoVV-AVL increased the serum levels of mouse IFN-γ (mIFN-γ), leading to the activation of M1-polarized macrophages. Conversely, NADPH, a reducing agent by providing reducing equivalents, reduced the production of mIFN-γ, and suppressed M1-polarization of macrophage. Based on these findings, we propose that oncoVV-AVL not only contributes to direct cytolysis, but also enhances host immune response by promoting ROS levels.

Combination of monensin and erlotinib synergistically inhibited the growth and cancer stem cell properties of triple-negative breast cancer by simultaneously inhibiting EGFR and PI3K signaling pathways.

Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are recognized as a highly challenging subset of cells, renowned for their heightened propensity for relapse and unfavorable prognosis. Monensin, an ionophoric antibiotic, has been reported to exhibit significant therapeutic efficacy against various cancers, especially CSCs. Erlotinib is classified as one of the EGFR-TKIs and has been previously identified as a promising therapeutic target for TNBC. Our research aims to assess the effectiveness of combination of monensin and erlotinib as a potential treatment strategy for TNBC. The combination of monensin and erlotinib was assessed for its potential anticancer activity through various in vitro assays, including cytotoxicity assay, colony formation assay, wound healing assay, transwell assay, mammosphere formation assay, and proportion of CSCs assay. Additionally, an in vivo study using tumor-bearing nude mice was conducted to evaluate the inhibitory effect of the monensin and erlotinib combination on tumor growth. The results indicated that combination of monensin with erlotinib synergistically inhibited cell proliferation, the migration rate, the invasion ability and decreased the CSCs proportion, and CSC markers SOX2 and CD133 in vivo and in vitro. Furthermore, the primary proteins involved in the signaling pathways of the EGFR/ERK and PI3K/AKT are simultaneously inhibited by the combination treatment of monensin and erlotinib in vivo and in vitro. The simultaneous inhibition of the EGFR/ERK and PI3K/AKT/mTOR signaling pathways by the combination of monensin and erlotinib exhibited a synergistic effect on suppressing tumor proliferation and cancer cell stemness in TNBC.

Construction and Evaluation of BAL-PTX Co-Loaded Lipid Nanosystem for Promoting the Anti-Lung Cancer Efficacy of Paclitaxel and Reducing the Toxicity of Chemotherapeutic Drugs.

The present study aimed to develop a lipid nanoplatform, denoted as "BAL-PTX-LN", co-loaded with chiral baicalin derivatives (BAL) and paclitaxel (PTX) to promote the anti-lung cancer efficacy of paclitaxel and reduce the toxicity of chemotherapeutic drugs. BAL-PTX-LN was optimized through central composite design based on a single-factor experiments. BAL-PTX-LN was evaluated by TEM, particle size, encapsulation efficiency, hemolysis rate, release kinetics and stability. And was evaluated by pharmacokinetics and the antitumor efficacy studied both in vitro and in vivo. The in vivo safety profile of the formulation was assessed using hematoxylin and eosin (HE) staining. BAL-PTX-LN exhibited spherical morphology with a particle size of 134.36 ± 3.18 nm, PDI of 0.24 ± 0.02, and with an encapsulation efficiency exceeding 90%, BAL-PTX-LN remained stable after 180 days storage. In vitro release studies revealed a zero-order kinetic model of PTX from the liposomal formulation. No hemolysis was observed in the preparation group. Pharmacokinetic analysis of PTX in the BAL-PTX-LN group revealed an approximately three-fold higher bioavailability and twice longer t1/2 compared to the bulk drug group. Furthermore, the IC50 of BAL-PTX-LN decreased by 2.35 times (13.48 μg/mL vs 31.722 μg/mL) and the apoptosis rate increased by 1.82 times (29.38% vs 16.13%) at 24 h compared to the PTX group. In tumor-bearing nude mice, the BAL-PTX-LN formulation exhibited a two-fold higher tumor inhibition rate compared to the PTX group (62.83% vs 29.95%), accompanied by a ten-fold decrease in Ki67 expression (4.26% vs 45.88%). Interestingly, HE staining revealed no pathological changes in tissues from the BAL-PTX-LN group, whereas tissues from the PTX group exhibited pathological changes and tumor cell infiltration. BAL-PTX-LN improves the therapeutic effect of poorly soluble chemotherapeutic drugs on lung cancer, which is anticipated to emerge as a viable therapeutic agent for lung cancer in clinical applications.

Lipid-Based Nanocarrier by Targeting with LHRH Peptide: A Promising Approach for Prostate Cancer Radio-Imaging and Therapy.

Prostate cancer is a prevalently detected malignancy with a dismal prognosis. Luteinizing-hormone-releasing-hormone (LHRH) receptors are overexpressed in such cancer cells, to which the LHRH-decapeptide can specifically bind. A lipid-polyethylene glycol-conjugated new LHRH-decapeptide analogue (D-P-HLH) was synthesized and characterized. D-P-HLH-coated and anticancer drug doxorubicin (DX)-loaded solid lipid nanoparticles (F-DX-SLN) were formulated by the cold homogenization technique and characterized by Fourier transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, differential scanning calorimetry, dynamic light scattering, electron microscopy, entrapment efficiency, and drug-release profile studies. F-DX-SLN allows site-specific DX delivery by reducing the side effects of chemotherapy. Cancer cells could precisely take up F-DX-SLN by targeting specific receptors, boosting the cytotoxicity at the tumor site. The efficacy of F-DX-SLN on PC3/SKBR3 cells by the MTT assay revealed that F-DX-SLN was more cytotoxic than DX and/or DX-SLN. Flow cytometry and confocal microscopic studies further support F-DX-SLNs' increased intracellular absorption capability in targeting LHRH overexpressed cancer cells. F-DX-SLN ensured high apoptotic potential, noticeably larger mitochondrial transmembrane depolarization action, as well as the activation of caspases, a longer half-life, and greater plasma concentration. F-DX-SLN/DX-SLN was radiolabeled with technetium-99m; scintigraphic imaging studies established its tumor selectivity in PC3 tumor-bearing nude mice. The efficacy of the formulations in cancer treatment, in vivo therapeutic efficacy tests, and histopathological studies were also conducted. Results clearly indicate that F-DX-SLN exhibits sustained and superior targeted administration of anticancer drugs, thus opening up the possibility of a drug delivery system with precise control and targeting effects. F-DX-SLN could also provide a nanotheranostic approach with improved efficacy for prostate cancer therapy.

DIPG-38. VAL-083 IN COMBINATION WITH AZD1775 FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline gliomas (DMGs) are malignant pediatric brain tumors characterized by H3K27M mutation and global genome hypomethylation. This results in enhanced transcription of MGMT, a DNA-repair enzyme, rendering alkylating chemotherapeutics ineffective. Therefore, we investigated the efficacy of a functionally different DNA damaging agent, Val-083, that induces double strand breaks and triggers S/G2 phase cell cycle arrest for homologous DNA repair. We further evaluated its combined efficacy with Wee1 (G2/M checkpoint) inhibitor, AZD1775, to override cell cycle arrest and propel cells with DNA damage into premature mitosis and apoptosis. METHODS DMG cells were treated with increasing concentration of Val-083 and/or AZD1775 and their viability was evaluated using CellTiter-Glo luminescent assay. Next, the drugs’ mechanisms of action were investigated by immunoblotting for biomarkers involved in DNA damage response and cell cycle arrest. Flow cytometry was used to investigate cell cycle profile and apoptosis. Additionally, in vivo tumor growth inhibition and survival benefit were evaluated using zebrafish and mouse xenograft DMG models. RESULTS The IC50 values of Val-083 range between 0.69 and 4.38 µM while the IC50 values of AZD1775 range between 0.22 to 0.98 µM. Combination of both drugs showed high areas of synergy on multiple DMG lines, DNA double strand breaks and significant increase in cell population at the S (p<0.01) and G2/M (p<0.05) phases. Importantly, on zebrafish xenograft tumor model, combination treatment significantly (p<0.001) inhibited tumor growth in comparison to control and single treatment groups. Furthermore, the combined treatment demonstrated significant (p<0.01) survival benefit (median survival (MS) = 62 days) on tumor-bearing nude mice models, in comparison to vehicle (MS= 44 days), Val-083 (MS= 54.5 days) and AZD1775 (MS= 47 days) treatment groups. CONCLUSION Val-083 in combination with AZD1775 demonstrate promising efficacy in DMG preclinical models, providing a strong rationale for positioning these arms for clinical testing.

Synthesis and Preclinical Evaluation of Novel 68Ga-DOTA-RBB as Potential PET Radiotracer for Imaging CDK4/6 in Tumors.

Many malignant tumors, including breast cancer, exhibit amplification and overexpression of cyclin-dependent kinase 4 and 6 (CDK4/6). Ribociclib, approved and used in clinical treatment, acts as a highly selective CDK4/6 inhibitor for ER+/HER2- breast cancer. By modifying ribociclib with the chelator DOTA, we designed and synthesized a novel CDK4/6-positive PET imaging agent, which was radiolabeled by 68Ga for radioactive tagging. The radiotracer demonstrates high radiochemical purity, excellent stability in vitro and in vivo, and favorable pharmacokinetic characteristics. Cell uptake experiments using MCF-7 cells indicate that an excess of ribociclib (RBB) can inhibit cellular uptake of 68Ga-DOTA-RBB. Imaging and biodistribution experiments in MCF-7 tumor-bearing nude mice show significant radioactive accumulation in the tumor. However, preadministration of excess ribociclib results in a substantial reduction in radioactive accumulation within the tumor. On the basis of our explorations, 68Ga-DOTA-RBB, as a targeted imaging agent for CDK4/6-positive tumors, holds significant potential application values.

Pleurotus pulmonarius polysaccharides inhibit glioma growth through the Hippo signaling pathway and regulate the structure of gut microbiota

Pleurotus pulmonarius (Fr.) is an edible and medicinal fungus. Here, we isolated polysaccharides from P. pulmonarius and evaluated their physicochemical properties, anti-tumor activity, and gut microbiota regulation. Freeze-dried powder of P. pulmonarius fruiting body polysaccharide (PFP) is a heteropolysaccharide, mainly composed of galactose (295.52 μg/mg). PFP significantly inhibited glioma cell survival in vitro and U251 glioma cell tumor growth in nude mice (inhibition rate, 53.70%). PFP inhibited YAP1 and TAZ protein expression levels, upregulated those of MST1 and LAST1, regulated Hippo signaling, stimulated E-cadherin, and downregulated N-cadherin proteins, inhibiting tumor migration. PFP downregulated Bcl-2 protein expression, upregulated that of Cleaved Caspase 3, and induced tumor cell apoptosis. PFP regulated gut microbiota structure in tumor-bearing nude mice and increased the relative abundance of beneficial bacteria. In summary, PFP is a natural, safe, and non-toxic anti-cancer drug, and a potential probiotic. Our data provide theoretical guidance for the treatment of glioma.

Eugenol suppresses VEGF-dependent angiogenesis by JAK2/STAT3 pathway in non-small cell lung cancer

Background: Non-small cell lung cancer (NSCLC) is a highly vascularized solid tumor, and tumor angiogenesis is closely associated with the metastasis and progression of NSCLC. Antiangiogenic drugs can target the tumor microenvironment to degrade existing tumor blood vessels while inhibiting tumor angiogenesis and have become one of the indispensable treatments for patients with advanced NSCLC. Although various new drugs have been tested in different settings of NSCLC, none of them have shown the desired therapeutic effects so far. Therefore, the search for new and effective therapeutic modalities has become a new goal for treating NSCLC. Objective: Eugenol is a phenolic aromatic compound derived from Eugenia caryophyllata, Cinnamomum cassia, etc., which has historically been used for various medical purposes. Studies have shown that eugenol exhibits significant anticancer effects against several types of cancer; however, its therapeutic effect on angiogenesis remains a mystery. In this study, the in vitro and in vivo antiangiogenic effects of eugenol in NSCLC and the underlying molecular mechanism were explored, which could provide a promising strategy for the treatment of NSCLC. Methods: The effects of eugenol on the proliferative capacity of human umbilical vein endothelial cells (HUVECs) and A549 cells were examined by methyl thiazolyl tetrazolium assay. The migration and invasion of eugenol-treated HUVECs were evaluated by wounding-healing and transwell assay, and the angiogenesis was measured by tube formation assay. The expression of angiogenesis-related genes and proteins, as well as the JAK2/STAT3 pathway, was evaluated by real-time quantitative PCR and western blot. Flow cytometry was performed to detect the effect of eugenol on the apoptotic profile of A549 cells. Finally, the A549 tumor-bearing nude mice were constructed to evaluate the in vivo anti-NSCLC activity of eugenol. Results: Eugenol inhibited the migration, invasion, and tube formation of HUVECs. Meanwhile, eugenol blocked the phosphorylation of vascular endothelial growth factor reporter-2 and inhibited the expression of other angiogenesis-related proteins. In addition, eugenol suppressed the expression of p-JAK2 and p-STAT3 in HUVECs and A549 cells. Eugenol also suppressed the proliferation of A549 cells by promoting apoptosis and inhibited tumor growth and microvessel formation in A549 cell xenograft-bearing nude mice. Conclusions: Eugenol could be a potential lead compound for the treatment of NSCLC by blocking the vascular endothelial growth factor/vascular endothelial growth factor reporter-2 and JAK2/STAT3 signaling pathways.

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

To investigate the mechanism by which fragile X mental retardation protein (FMRP) regulates ferroptosis evasion in colorectal cancer (CRC) cells. We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database. A lentiviral FMRP overexpression vector (Lv-FMRP) and 3 knockdown vectors (siFMRP-1, siFMRP-2, and siFMRP-3) were constructed, and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay; the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe2+ kits, mitochondrial membrane potential changes were detected using JC-1 fluorescence staining, and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting. The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice. Compared with normal colonic mucosal epithelial NCM460 cells, the CRC cell lines had significantly higher FMRP expression level. Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen, oxidative stress-induced cell death, mitochondrial respiration, and glutathione metabolism pathways. In the cell experiments, FMRP knockdown significantly inhibited proliferation of HCT116 cells, lowered cellular GSH content, increased MDA and ROS levels, Fe2+ fluorescence intensity, and mitochondrial membrane potential, and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK, MEK, MAPK, and RAS proteins; FMRP overexpression resulted in the opposite changes in the cells. In the tumor-bearing nude mice, HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft. The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.

Dual-Targeted Novel Temozolomide Nanocapsules Encapsulating siPKM2 Inhibit Aerobic Glycolysis to Sensitize Glioblastoma to Chemotherapy.

Chemotherapy of glioblastoma (GBM) has not yielded success due to inefficient blood-brain barrier (BBB) penetration and poor glioma tissue accumulation. Aerobic glycolysis, as the main mode of energy supply for GBM, safeguards the rapid growth of GBM while affecting the efficacy of radiotherapy and chemotherapy. Therefore, to effectively inhibit aerobic glycolysis, increase drug delivery efficiency and sensitivity, a novel temozolomide (TMZ) nanocapsule (ApoE-MT/siPKM2 NC) is successfully designed and prepared for the combined delivery of pyruvate kinase M2 siRNA (siPKM2) and TMZ. This drug delivery platform uses siPKM2 as the inner core and methacrylate-TMZ (MT) as the shell component to achieve inhibition of glioma energy metabolism while enhancing the killing effect of TMZ. By modifying apolipoprotein E (ApoE), dual targeting of the BBB and GBM is achieved in a "two birds with one stone" style. The glutathione (GSH) responsive crosslinker containing disulfide bonds ensures "directional blasting" cleavage of the nanocapsules to release MT and siPKM2 in the high GSH environment of glioma cells. In addition, in vivo experiments verify that ApoE-MT/siPKM2 NC has good targeting ability and prolongs the survival of tumor-bearing nude mice. In summary, this drug delivery system provides a new strategy for metabolic therapy sensitization chemotherapy.

Beta cyclodextrin conjugated Au[sbnd]Fe3O4 Janus nanoparticles with enhanced chemo-photothermal therapy performance

The strategic integration of multi-functionalities within a singular nanoplatform has received growing attention for enhancing treatment efficacy, particularly in chemo-photothermal therapy. This study introduces a comprehensive concept of Janus nanoparticles (JNPs) composed of Au and Fe3O4 nanostructures intricately bonded with β-cyclodextrins (β-CD) to encapsulate 5-Fluorouracil (5-FU) and Ibuprofen (IBU). This strategic structure is engineered to exploit the synergistic effects of chemo-photothermal therapy, underscored by their exceptional biocompatibility and photothermal conversion efficiency (∼32.88 %). Furthermore, these β-CD-conjugated JNPs enhance photodynamic therapy by generating singlet oxygen (1O2) species, offering a multi-modality approach to cancer eradication. Computer simulation results were in good agreement with in vitro and in vivo assays. Through these studies, we were able to prove the improved tumor ablation ability of the drug-loaded β-CD-conjugated JNPs, without inducing adverse effects in tumor-bearing nude mice. The findings underscore a formidable tumor ablation potency of β-CD-conjugated Au-Fe3O4 JNPs, heralding a new era in achieving nuanced, highly effective, and side-effect-free cancer treatment modalities. Statement of significanceThe emergence of multifunctional nanoparticles marks a pivotal stride in cancer therapy research. This investigation unveils Janus nanoparticles (JNPs) amalgamating gold (Au), iron oxide (Fe3O4), and β-cyclodextrins (β-CD), encapsulating 5-Fluorouracil (5-FU) and Ibuprofen (IBU) for synergistic chemo-photothermal therapy. Demonstrating both biocompatibility and potent photothermal properties (∼32.88 %), these JNPs present a promising avenue for cancer treatment. Noteworthy is their heightened photodynamic efficiency and remarkable tumor ablation capabilities observed in vitro and in vivo, devoid of adverse effects. Furthermore, computational simulations validate their interactions with cancer cells, bolstering their utility as an emerging therapeutic modality. This endeavor pioneers a secure and efficacious strategy for cancer therapy, underscoring the significance of β-CD-conjugated Au-Fe3O4 JNPs as innovative nanoplatforms with profound implications for the advancement of cancer therapy.

Folic acid-tripeptide-conjugated synthetic biodegradable nanoparticle-loaded with Ormeloxifene potentially inhibited breast cancer xenograft tumor

ObjectivesOrmeloxifene (OLF) is an estrogen receptor modulator used in breast cancer treatment with highly favorable pharmacodynamics and pharmacokinetic properties. Folic acid (FA) receptor overexpresses in breast cancer cells. Hence, the study was intended to evaluate the therapeutic potential of OLF-encapsulated FA-peptide-conjugated nanoparticles (FA-Pep-OLF-NP) for breast cancer treatment. MethodsWe prepared folic acid tripeptide conjugated OLF-encapsulated PLGA nanoparticles. The surface conjugation of folic acid-tripeptide was assessed by FTIR and XPS analysis. Selectivity, cytotoxicity, apoptosis, mitochondrial depolarization, reactive oxygen species, and cell cycle analysis of the experimental nanoparticles were tested on human breast cancer cells, MDA-MB-231 and MCF-7. The therapeutic efficacy of FA-Pep-OLF-NP and unconjugated nanoparticle OLF-NP was compared in the MDA-MB-231-xenograft tumor animal model. ResultsOLF-NP and FA-Pep-OLF-NP exhibited 4.8 % and 4.5 % w/w of drug loading, respectively. The particles were spherical with a smooth surface and uniform size distribution. FA-Pep-OLF-NP revealed higher cytotoxicity, mitochondrial depolarization, and apoptosis potential in MDA-MB-231 cells compared to MCF-7 cells. Tumor uptake and tumor volume reduction by FA-Pep-OLF-NP and OLF-NP were established in MDA-MB-231 tumor-bearing nude mice. ConclusionFA-Pep-OLF-NP showed preferential delivery of OLF to the breast cancer cells and in breast tumors compared to OLF-NP and showed more drug accumulation in breast cancer xenograft models. FA-Pep-OLF-NP delayed the progress of folate receptor overexpressed breast cancer.

A GAD1 inhibitor suppresses osteosarcoma growth through the Wnt/β-catenin signaling pathway

BackgroundAs a marker of the GABAergic system, the expression of glutamate decarboxylase 1 (GAD1) is mainly restricted to the central nervous system. Emerging studies have shown that aberrant expression of GAD1 in tumor tissues may promote tumor cell growth. The role of GAD1 in the development of osteosarcoma (OS) remains unclear, so this study sought to investigate the expression status of GAD1 and the effect of its specific inhibitor 3-mercaptopropionic acid (3-MPA) on OS. MethodsThe R2 database was used to analyze the relationship between the expression of GAD1 and clinical prognosis in OS patients. Immunohistochemistry was used to compare the expression profile of GAD1 between OS and matched neighboring tissues. The potential antitumor effects of 3-MPA on cell viability, colony formation and the cell cycle were examined. Moreover, the in vivo effect of 3-MPA on tumor growth was investigated using tumor-bearing nude mice. ResultsThe expression level of GAD1 was aberrantly upregulated in OS tissues, but almost no expression of GAD1 was found in matched neighboring tissues. Western blotting analyses showed upregulation of GAD1 in OS cells compared to human osteoblast cells. In vitro and in vivo, 3-MPA significantly suppressed the growth of OS. Regarding the mechanism, 3-MPA inhibited β-catenin and cyclin D1 in OS cells, thereby inactivating the Wnt/β-catenin pathway. ConclusionsOS displays increased expression of the GABAergic neuronal marker GAD1, and 3-MPA significantly reduces OS growth by inhibiting the Wnt/β-catenin pathway.

HOXB5 promotes the progression and metastasis of osteosarcoma cells by activating the JAK2/STAT3 signalling pathway

ObjectiveTo investigate the involvement of the homeobox gene B5 (HOXB5) in the progression and metastasis of osteosarcoma. MethodsThe expression of HOXB5 in human osteosarcoma tissues and its correlation with clinical indicators were investigated using bioinformatics analysis and immunohistochemical labelling. Human osteosarcoma cells (HOS, MG63, U2OS, and Saos-2) and normal human osteoblasts (hFOB1.19) were cultivated. The expression of HOXB5 in these cells was detected using western blotting (WB) and RT‒PCR. Two cell lines exhibiting elevated HOXB5 expression were chosen and divided into three groups: the blank group (mock), control group (control) and transfection group (shHOXB5). The transfection group was infected with lentivirus expressing shRNAs targeting HOXB5. The transfection efficiency was detected by WB. Cell proliferation suppression was measured by CCK-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays; the percentage of apoptotic cells was determined by flow cytometry; and cell migration and invasion were detected via the Transwell chamber test. WB was utilized to determine the protein expression of genes linked to metastasis (MMP2, MMP9), apoptosis (Bax, Bcl-2), and the JAK2/STAT3 pathway (JAK2, p-JAK2, STAT3, p-STAT3). ResultsIn osteosarcoma tissues, HOXB5 expression was elevated and strongly correlated with distant metastasis. Silencing HOXB5 reduced the proliferation, migration and invasion of osteosarcoma cells; prevented the progression and metastasis of tumours in tumour-bearing nude mice; and reduced the activation of key proteins in the JAK2/STAT3 signalling pathway. ConclusionThrough the JAK2/STAT3 signalling pathway, HOXB5 plays a crucial role in the malignant progression of osteosarcoma and is a promising target for osteosarcoma treatment.

Small animal PET imaging with the 68Ga-labeled pH (low) insertion peptide-like peptide YJL-4 in a triple-negative breast cancer mouse model

BackgroundThe aim of this study was to prepare a novel 68Ga-labeled pH (low) insertion peptide (pHLIP)-like peptide, YJL-4, and determine its value for the early diagnosis of triple-negative breast cancer (TNBC) via in vivo imaging of tumor-bearing nude mice. The novel peptide YJL-4 was designed using a template-assisted method and synthesized by solid-phase peptide synthesis. After modification with the chelator 1,4,7‑triazacyclononane-N,N′,N″-triacetic acid (NOTA), the peptide was labeled with 68Ga. Then, the biodistribution of 68Ga-YJL-4 in tumor-bearing nude mice was investigated, and the mice were imaged by small animal positron emission tomography (PET).ResultsThe radiochemical yield and radiochemical purity of 68Ga-YJL-4 were 89.5 ± 0.16% and 97.95 ± 0.06%, respectively. The biodistribution of 68Ga-YJL-4 in tumors (5.94 ± 1.27% ID/g, 6.72 ± 1.69% ID/g and 4.54 ± 0.58% ID/g at 1, 2 and 4 h after injection, respectively) was significantly greater than that of the control peptide in tumors at the corresponding time points (P < 0.01). Of the measured off-target organs, 68Ga-YJL-4 was highly distributed in the liver and blood. The small animal PET imaging results were consistent with the biodistribution results. The tumors were visualized by PET at 2 and 4 h after the injection of 68Ga-YJL-4. No tumors were observed in the control group.ConclusionsThe novel pHLIP family peptide YJL-4 can adopt an α-helical structure for easy insertion into the cell membrane in an acidic environment. 68Ga-YJL-4 was produced in high radiochemical yield with good stability and can target TNBC tissue. Moreover, the strong concentration of radioactive 68Ga-YJL-4 in the abdomen does not hinder the imaging of early TNBC.

Pre-coating cRGD-modified bovine serum albumin enhanced the anti-tumor angiogenesis of siVEGF-loaded chitosan-based nanoparticles by manipulating the protein corona composition

Chitosan-based nanoparticles inevitably adsorb numerous proteins in the bloodstream, forming a protein corona that significantly influences their functionality. This study employed a pre-coated protein corona using cyclic Arg-Gly-Asp peptide (cRGD)-modified bovine serum albumin (BcR) to confer tumor-targeting capabilities on siVEGF-loaded chitosan-based nanoparticles (CsR/siVEGF NPs) and actively manipulated the serum protein corona composition to enhance their anti-tumor angiogenesis. Consequently, BcR effectively binds to the nanoparticles' surface, generating nanocarriers of appropriate size and stability that enhance the inhibition of endothelial cell proliferation, migration, invasion, and tube formation, as well as suppress tumor proliferation and angiogenesis in tumor-bearing nude mice. Proteomic analysis indicated a significant enrichment of serotransferrin, albumin, and proteasome subunit alpha type-1 in the protein corona of BcR-precoated NPs formed in the serum of tumor-bearing nude mice. Additionally, there was a decrease in proteins associated with complement activation, immunoglobulins, blood coagulation, and acute-phase responses. This modification resulted in an enhanced impact on anti-tumor angiogenesis, along with a reduction in opsonization and inflammatory responses. Therefore, pre-coating of nanoparticles with a functionalized albumin corona to manipulate the composition of serum protein corona emerges as an innovative approach to improve the delivery effectiveness of chitosan-based carriers for siVEGF, targeting the inhibition of tumor angiogenesis.

Swine pseudorabies virus attenuated vaccine reprograms the kidney cancer tumor microenvironment and synergizes with PD-1 blockade.

The global incidence rate of kidney cancer (KC) has been steadily increasing over the past 30 years. With the aging global population, kidney cancer has become an escalating concern that necessitates vigilant surveillance. Nowadays, surgical intervention remains the optimal therapeutic approach for kidney cancer, while the availability of efficacious treatments for advanced tumors remains limited. Oncolytic viruses, an emerging form of immunotherapy, have demonstrated encouraging anti-neoplastic properties and are progressively garnering public acceptance. However, research on oncolytic viruses in kidney cancer is relatively limited. Furthermore, given the high complexity and heterogeneity of kidney cancer, it is crucial to identify an optimal oncolytic virus agent that is better suited for its treatment. The present study investigates the oncolytic activity of the Pseudorabies virus live attenuated vaccine (PRV-LAV) against KC. The findings clearly demonstrate that PRV-LAV exhibits robust oncolytic activity targeting KC cell lines. Furthermore, the therapeutic efficacy of PRV-LAV was confirmed in both a subcutaneous tumor-bearing nude mouse model and a syngeneic mouse model of KC. Combined RNA-seq analysis and flow cytometry revealed that PRV-LAV treatment substantially enhances the infiltration of a diverse range of lymphocytes, including T cells, B cells, macrophages, and NK cells. Additionally, PRV-LAV treatment enhances T cell activation and exerts antitumor effects. Importantly, the combination of PRV-LAV with anti-PD-1 antibodies, an approved drug for KC treatment, synergistically enhances the efficacy against KC. Overall, the discovery of PRV-LAV as an effective oncolytic virus holds significant importance for improving the treatment efficacy and survival rates of KC patients.

Abstract 606: The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with rapid disease progression and poor patient survival. Therapeutic options for SCLC remain a challenge due to limited understanding of both the biology of the disease and the major determinants of treatment response. Key transcription factors associated with unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Previously, we demonstrated that onvansertib, a highly specific inhibitor of the PLK1 Ser/Thr kinase, was effective in SCLC preclinical models, leading to its ongoing clinical development for relapsed/refractory SCLC patients (NCT05450965). Here we further explored potential strategies to enhance onvansertib activity by assessing its in vitro and in vivo activity in combination treatments. Methods: Human SCLC cell lines were treated for 72 hr with onvansertib in combination with paclitaxel, lurbinectedin or the ATR inhibitors BAY1895344 and AZD6738. Effect on cell proliferation was analyzed by WST1 and CellTiterGlo assays. In vivo activity of onvansertib in combination with paclitaxel was tested in 3 SCLC patient-derived xenograft (PDX) models either cisplatin-sensitive (TKO5- ASCL1 subtype) or cisplatin-resistant (TKO2-ASCL1 subtype, TKO8-NEUROD1 subtype). Tumor-bearing nude mice were treated with vehicle, onvansertib (50 mg/kg, oral, 4 times/week), paclitaxel (15 mg/kg, IP, once a week) or the combination for 5 weeks. Mice were followed for tumor growth and survival (defined as time to reach tumor volume greater than 2000 mm3). Results: The combination of onvansertib and paclitaxel inhibited cell proliferation synergistically in cell lines from 3 SCLC subtypes (ASCL1, NEUROD1, and YAP1), while the combinations with lurbinectedin or ATR inhibitors were not synergistic. We further examined the combination of onvansertib and paclitaxel in SCLC PDXs. The combination exhibited potent anti-tumor activity, resulting in significantly greater tumor growth inhibition and increase in survival compared to the monotherapies in all 3 PDXs. The combination was well tolerated with no observed body weight loss. While onvansertib and paclitaxel had negligible single agent activity in cisplatin-resistant TK02 PDX and TKO8 PDX models, the combination induced durable complete responses in 25% and 100% of treated mice respectively. Conclusion: The PLK1 inhibitor onvansertib in combination with paclitaxel showed synergy in multiple SCLC cell lines in vitro and potent anti-tumor activity in cisplatin-sensitive and -resistant SCLC PDX models in vivo. Tumor tissue analysis is underway to detail the mechanisms of this interaction. Citation Format: John C. Schmitz, Guojing Zhang, Abbe Pannucci, Tod Smeal, Chu-Chiao Wu, Maya Ridinger, Taofeek K. Owonikoko. The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung 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 606.

Abstract 6023: 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity

Abstract Despite significant developments over the last few decades, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Prostate specific membrane antigen (PSMA), a prostate cancer-specific membrane marker which directly correlates with androgen independence, metastasis, and disease progression, has been well-established as a radioligand target for the diagnosis and treatment of mCRPC. Lutetium-177 (177Lu)-PSMA-617 (PLUVICTOTM) was approved in 2022 for the treatment of progressive PSMA-positive mCRPC. However, only 30% of patients showed a radiological response in the registrational trial. Meanwhile, recent clinical experience has shown very limited efficacy in low PSMA expressing patients. These data collectively request for further improvement in the clinical benefit of PSMA-targeted radiotherapy. Actinium-225 (225Ac), an alpha emitter, is significantly more potent on cancer cell killing and has a shorter range in tissue penetration when compared to the beta emitter 177Lu. Such a profile supports the development of 225Ac based radiotherapies. Using our proprietary Clear-XTM technology platform, we developed 225Ac-FL-020, a novel 225Ac-based PSMA radioligand therapy candidate which has demonstrated prominent pre-clinical properties. We have assessed the binding affinity against PSMA of the non-labeled vector FL-020 in vitro. The in vivo biodistribution profile of FL-020 was characterized by SPECT/CT imaging and biodistribution using Indium-111 (111In)-FL-020 in PSMA high LNCaP tumor-bearing nude mice. In addition, anti-tumor activities of 225Ac-FL-020 were evaluated in the LNCaP xenograft model and directly compared to 225Ac-PSMA-617. FL-020 was found bound to LNCaP cells with an IC50 value of 51.55 nM. Meanwhile, the off-target screening showed that less than 50% inhibition of binding or activity was observed by FL-020 at 10 µM against 85 targets including receptors, ion channels, enzymes, and transporters, indicating the high selectivity of FL-020. Moreover, 111In-FL-020 displayed a very promising in vivo distribution profile with high and sustained tumor uptake and fast systemic clearance. Furthermore, 225Ac-FL-020 exhibited superior anti-tumor activity compared to 225Ac-PSMA-617 at the same dose level (10 KBq/mouse) in the LNCaP xenograft model with a favorable safety profile as indicated by body weight and hematological parameters. A mechanistic study was also conducted in 225Ac-FL-020-treated LNCaP tumor samples where DNA double-strand breaks and tumor cell apoptosis were observed, confirming the MOA of alpha emitters. Taken together, these results collectively demonstrate that 225Ac-FL-020 is a potent and selective PSMA-targeting radioligand therapy candidate with superior anti-tumor activity and a favorable safety profile warranting further clinical development. Citation Format: Fa Liu, Jiangwei Zhang, Junyu Yang, Kirstine T. Thrane, Mathias W. Hallund, Rikke V. Grønlund, Nicholas C. Wong. 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity [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 6023.

Abstract 656: A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells

Abstract Human telomerase reverse transcriptase (hTERT) is overexpressed in most human cancers. hTERT is considered as an important target for cancer therapy. In the present study, a new peptide HS1002 were synthesized with sequence of pGlu-His-Trp-Ser-Tyr-Arg-Leu-Arg-Phe-Ile-Pro-NHEt based on the GnRH agonist and hTERT. The aim of this study was to evaluate HS1002 for its anticancer activity by dual-targeting to GnRH/hTERT in prostate cancer cells. Upon comparison with different prostate cancer cell lines, HS1002 exhibited highest cytotoxicity against LNCaP cells. The hTERT expression correlated with telomerase activity in LNCaP cells, was significantly inhibited by HS1002, resulting in reduction of proliferation, metastasis and increase in apoptosis. Additionally, HS1002 suppressed c-Myc and AKT/ERK pathway in LNCaP cells. In the tumor-bearing nude mice model, HS1002 significantly inhibited tumor growth and downregulated the expression of hTERT and Ki-67 in tumor tissue as evidenced by western blot and immunohistochemistry analysis, respectively. Moreover, repeated subcutaneous injection of HS1002 resulted in the reduction of both serum testosterone levels and seminal vesicle weight. HS1002 also increased cytosolic calcium influx and CRE-luciferase activity in GnRHR-overexpressing HEK293 cells compared to mock-transfected transfected cells. Furthermore, a potent cytotoxicity in LNCaP cells was exhibited by HS1002-activated peripheral blood mononuclear cell (PBMC) with interleukin (IL)-2 using LDH release assay. In addition, a flow-cytometric analysis confirmed an elevation in the production of granzyme B and IFN-γ in CD8+ T cells in the MC38 syngeneic mouse model by HS1002. These data indicate that the inhibition of hTERT expression by HS1002 suppresses prostate cancer cell growth and induced anti-cancer immunity. Therefore, HS1002 might be used as a novel therapeutic drug for prostate cancer. Citation Format: Jae Hyeon Park, Ju Ri Kim, Hwa Young Cha, Hyung Sik Kim. A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells [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 656.