Inhibited Cell Motility Research Articles

Drug-Free Mesoporous Silica Nanoparticles Enable Suppression of Cancer Metastasis and Confer Survival Advantages to Mice with Tumor Xenografts.

Despite advancements in nanomedicine for drug delivery, many drug-loaded nanoparticles reduce tumor sizes but often fail to prevent metastasis. Mesoporous silica nanoparticles (MSNs) have attracted attention as promising nanocarriers. Here, we demonstrated that MSN-PEG/TA 25, with proper surface modifications, exhibited unique antimetastatic properties. In vivo studies showed that overall tumor metastasis decreased in 4T1 xenografts mice treated with MSN-PEG/TA 25 with a notable reduction in lung tumor metastasis. In vitro assays, including wound-healing, Boyden chamber, tube-formation, and real-time cell analyses, showed that MSN-PEG/TA 25 could modulate cell migration of 4T1 breast cancer cells and interrupt tube formation by human umbilical vein endothelial cells (HUVECs), key factors in suppressing cancer metastasis. The synergistic effect of MSN-PEG/TA 25 combined with liposomal-encapsulated doxorubicin (Lipo-Dox) significantly boosted mouse survival rates, outperforming Lipo-Dox monotherapy. We attributed the improved survival to the antimetastatic capabilities of MSN-PEG/TA 25. Moreover, Dox-loaded MSN-PEG/TA 25 suppressed primary tumors while retaining the antimetastatic effect, thereby enhancing therapeutic outcomes and overall survival. Western blot and qPCR analyses revealed that MSN-PEG/TA 25 interfered with the phosphorylation of ERK, FAK, and paxillin, thus impacting focal adhesion turnover and inhibiting cell motility. Our findings suggest that drug-free MSN-PEG/TA 25 is highly efficient for cancer treatment via suppressing metastatic activity and angiogenesis.

CH6824025, potent and selective DDR1 inhibitor, reduces kidney fibrosis in UUO mice.

Discoidin domain receptor 1 (DDR1) is a collagen receptor with tyrosine kinase activity, and its expression is enhanced in various disease conditions. Although previous research suggests that DDR1 contributes to renal disease progression, DDR1 inhibitors for renal fibrosis have yet to be developed. In this study, we used unilateral ureteral obstruction (UUO) mice to investigate whether CH6824025, a strong and selective DDR1 phosphorylation inhibitor, can improve renal fibrosis. Furthermore, we performed 10x Visium spatial transcriptomics (ST) analysis on the kidney. CH6824025 suppressed the phosphorylation of DDR1 in the kidney, and the amount of hydroxyproline, the Sirius red- and the F4/80-positive area, and the mRNA expression of fibrosis and inflammation-related genes in the kidney were significantly decreased. 10x Visium ST analysis suggested that DDR1 is mainly expressed in distal nephrons under normal conditions, but that its expression appears to increase in the injured proximal tubules in UUO mice. Comparing mRNA expression in DDR1 positive spots in the Vehicle and the CH6824025 group, oxidative phosphorylation and mitochondrial dysfunction might be improved, and pathways involved in fibrosis tended to be inhibited in the CH6824025 administration group. Downstream analysis would suggest that mRNA expression changes in the CH6824025 group contribute to the inhibition of cell movement. Taken together, our findings suggest that CH6824025 inhibited kidney fibrosis in UUO mice, which might be due to the inhibition of the migration of inflammatory cells to the injury site and the reduction of inflammation. DDR1 inhibitors are expected to be a promising treatment for renal fibrosis. Significance Statement The novel DDR1 inhibitor CH6824025 could ameliorate fibrosis and inflammation in UUO mice. CH6824025 would inhibit cell motility (e.g., migration) that prevents the progression of fibrosis and improves mitochondrial function in UUO mice. CH6824025 could provide a significant benefit to patients with kidney fibrosis.

WYC-209 suppresses gastric cancer by down-regulating FGF18 via inactivating the STAT3 signaling pathway

BackgroundGastric cancer (GC) is regarded as a major health burden all over the world. WYC-209 inhibits the growth and metastasis of tumor-repopulating cells (TRCs). However, its effectiveness on GC was unexplored. Herein, this study aims to investigate the effect of WYC-209 on GC and elucidate its underlying mechanism. MethodsWe examined the effects of WYC-209 on cell survival, migration, invasion, and colony-forming capacities of two GC cell lines (AGS and HGC-27). Subsequently, RNA-seq and enrichment analyses were performed to screen the differentially expressed genes (DEGs) and the enriched signaling pathways. To further explore the underlying mechanism, loss- and gain-function experiments, Chromatin immunoprecipitation, and luciferase reporter were conducted. Finally, xenograft models were constructed to examine the effects of WYC-209 in vivo. ResultsWYC-209 significantly inhibited cell motility in vitro and tumor growth in vivo. RNA-seq performed in AGS cells after WYC-209 treatment revealed that the inhibition effect of WYC-209 on GCs may be associated with the down-regulation of fibroblast growth factor-18 (FGF18), and pleasantly, FGF18 overexpression abrogated the suppression effect of the drug. In addition, we found that WYC-209 attenuated the activation of the Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway, and impeded the FGF18 levels expressed in GCs. Importantly, the WYC-209 treatment circumvented the binding of STAT3 to the FGF18 promoter, suggested that WYC-209 down-regulated FGF18 expression via the STAT3 signaling pathway. ConclusionTogether, our findings presented the promise of WYC-209 in suppressing GC by down-regulating FGF18 expression through inactivating the STAT3 signaling pathway.

The benzoylphenylurea derivative BPU17 acts as an inhibitor of prohibitin and exhibits antifibrotic activity

Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT.

Up-regulation of LPCAT1 is correlated with poor prognosis and promotes tumor progression in glioblastoma.

Glioblastoma (GBM) is a cancer with high malignancy because of its rapid proliferation and high metastatic ability. LPCAT1 is reported to play a tumor-promoting role in multiple cancers, but its precise molecular mechanism in GBM remains to be further explored. We aim to explore the biological role of LPCAT1 in GBM. In this study, the expression of LPCAT1 and its correlation with clinicopathological characteristics of GBM patients were analyzed based on The Cancer Genome Atlas (TCGA) dataset. Kaplan-Meier approach was adopted for plotting survival curves for patients showing different expression levels of LPCAT1. Meanwhile, LPCAT1 expression within 50 GBM tumor tissues and 30 non-tumor clinical samples was analyzed by qRT-PCR and western blot assays, respectively. Later, LPCAT1's effect on GBM tumorigenesis was analyzed in vivo and in vitro by CCK8, EdU proliferation, clone forming, scratch, TUNEL assays, and subcutaneous xenograft experiments. As a result, LPCAT1 expression elevated within GBM tumor tissues and cells. Overexpression of LPCAT1 enhanced GBM cell growth, invasion and migration, while accelerating cell cycle progression. LPCAT1 silencing significantly inhibited cell motility and proliferation in vivo and in vitro, and arrested U251 cells at G0/G1 phase. Moreover, LPCAT1 might play a role in GBM progression by activating the p-AKT-MYC signaling pathway. LPCAT1 activated AKT, which were synchronously up-regulated MYC to accelerate cancer progression. Knockdown of LPCAT1 induced the opposite changes to repress the viability and motility of GBM cells. LPCAT1 contributed to the progression of GBM by participating in the p-AKT-MYC axis.

Inhibitory Effect of Pyra-Metho-Carnil on the Differentiation and Maturation of Macrophages in Normal and Cancer Microenvironments.

In a previous study, we have demonstrated heightened Pyra-Metho-Carnil (PMC) efficacy in nude mice with intact innate immunity that lack T and B cells. This has prompted hypothesizing that PMC may target macrophages that promote cancer growth. In this study, we conducted co-culture experiments with macrophages derived from THP-1 human monocyte cell lines and spheroids representing normal and cancer microenvironments. We then performed RNA sequencing and flow cytometry analysis to elucidate the mechanisms by which PMC affects macrophage differentiation and maturation. THP-1 cells were differentiated by phorbol 12-myristate 13-acetate (PMA) and matured by PMA and lipopolysaccharide (LPS) either with or without PMC. Co-cultures were performed using stimulated THP-1 cells and HKe3-wild-type KRAS or HKe3-mutant (mt) KRAS spheroids. We then performed RNA-seq analysis of THP-1 cells stimulated by PMA (either with or without PMC) and flow cytometry analysis of mice peripheral blood obtained after PMC administration. THP-1 cells matured by PMA and LPS specifically increased the area of HKe3-mtKRAS cancer spheroids and the addition of PMC to THP-1 cells was found to inhibit cancer spheroid growth. RNA-seq data suggested that PMC treatment of THP-1 cells stimulated with PMA suppressed cell motility regulatory functions via down-regulation of the NF[Formula: see text]B pathway. Furthermore, flow cytometry results showed that PMC treatment suppressed monocyte maturation in B6 mice. The high level of in vivo tumor suppression caused by PMC may be due to inhibition of the differentiation and maturation of tumor-associated macrophages via the NF[Formula: see text]B signaling pathway.

Cold atmospheric plasma-activated medium for potential ovarian cancer therapy.

Cold atmospheric plasma (CAP) has emerged as an innovative tool with broad medical applications, including ovarian cancer (OC) treatment. By bringing CAP in close proximity to liquids such as water or cell culture media, solutions containing reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated, called plasma-activated media (PAM). In this systematic review, we conduct an in-depth analysis of studies focusing on PAM interactions with biological substrates. We elucidate the diverse mechanisms involved in the activation of different media and the complex network of chemical reactions underlying the generation and consumption of the prominent reactive species. Furthermore, we highlight the promises of PAM in advancing biomedical applications, such as its stability for extended periods under appropriate storage conditions. We also examine the application of PAM as an anti-cancer and anti-metastatic treatment for OC, with a particular emphasis on its ability to induce apoptosis via distinct signaling pathways, inhibit cell growth, suppress cell motility, and enhance the therapeutic effects of chemotherapy. Finally, the future outlook of PAM therapy in biomedical applications is speculated, with emphasis on the safety issues relevant to clinical translation.

In vitro comparison of zinc-based, chlorhexidine, and essential oil mouth rinses.

Chlorhexidine (CHX)-based mouth rinses are frequently prescribed following periodontal surgeries. A more recently available brand of zinc-based mouth rinses advertises one of its mouth rinses as a substitute for chlorhexidine. The purpose of this study was to evaluate, in vitro, the effects of this brand of zinc-based mouth rinses on cell survival, cell motility, and gene expression of human gingival fibroblasts (HGFs). HGFs were exposed to essential oil (EO), CHX, and three types of one brand of zinc-based mouth rinses designed to treat breath malodor (ZnA), dry mouth (ZnB), and gingivitis (ZnC). Each mouth rinse was tested over a range of concentrations for its effects on HGF survival and motility. Gene expression of cytokines, interleukins, and growth factors were evaluated via reverse transcriptase-polymerase chain reaction (RT-PCR), as a means to assess potential influences on inflammation and wound healing. Cell survival was significantly decreased for CHX and ZnC at 10% dilutions (p<0.05). For all time points, cells exposed to ZnC displayed the greatest reduction in cell motility (p<0.05). The various mouth rinses examined differentially altered the expression of growth factor transcripts. ZnC particularly enhanced the expression of BMP-2 and FGF-2. ZnC was more cytotoxic and inhibited cell motility to a greater extent than any of the other mouth rinses. Therefore, using ZnC as an alternative to CHX could potentially have negative effects on wound healing after periodontal surgery. However, further investigation is required to confirm the clinical relevance of these in vitro findings. One type of zinc-based mouth rinse designed to replace chlorhexidine (often prescribed after oral surgeries) demonstrated the greatest oral cell death and reduction in cell movement when compared to other zinc-based mouth rinses. These zinc-based mouth rinses also reduced the amounts of proteins involved in regulating inflammation, potentially reducing the destruction of bone holding the teeth in place. They also changed the amounts of several molecules involved in tissue healing. It is unknown if this will speed or slow the healing of the soft tissues of the mouth.

Phytoconstituent-derived zingerone nanoparticles disrupt the cell adhesion mechanism and suppress cell motility in melanoma B16F10 cells

Combining phytochemicals and nanotechnology to improve the unfavorable innate properties of phytochemicals and develop them into potent nanomedicines to enhance antitumor efficacy has become a novel strategy for cancer chemoprevention. Melanoma is the most aggressive, metastatic, and deadly disease of the primary cutaneous neoplasms. In this study, we fabricated phytoconstituent-derived zingerone nanoparticles (NPs) and validated their effects on cell adhesion and motility in melanoma B16F10 cells. Our data indicated that zingerone NPs significantly induced cytotoxicity and anti-colony formation and inhibited cell migration and invasion. Moreover, zingerone NPs dramatically interfered with the cytoskeletal reorganization and markedly delayed the period of cell adhesion. Our results also revealed that zingerone NPs-mediated downregulation of MMPs (matrix metalloproteinases) activity is associated with inhibiting cell adhesion and motility. We further evaluated the effects of zingerone NPs on Src/FAK /Paxillin signaling, our data showed that zingerone NPs significantly inhibited the protein activities of Src, FAK, and Paxillin, indicating that they play important roles in zingerone NP-mediated anti-motility and anti-invasion in melanoma cells. Accordingly, the phytoconstituent-zingerone NPs can strengthen the inhibition of tumor growth, invasion, and metastasis in malignant melanoma. Altogether, these multi-pharmacological benefits of zingerone NPs will effectively achieve the purpose of melanoma prevention and invasion inhibition.

Ru(II)-based complexes containing 2-thiouracil derivatives suppress liver cancer stem cells by targeting NF-κB and Akt/mTOR signaling

Cancer stem cells (CSCs) are defined as a rare population of cancer cells related to tumor initiation and maintenance. These cells are primarily responsible for tumor growth, invasion, metastasis, recurrence, and resistance to chemotherapy. In this paper, we demonstrated the ability of Ru(II)-based complexes containing 2-thiouracil derivatives with the chemical formulas trans-[Ru(2TU)(PPh3)2(bipy)]PF6 (1) and trans-[Ru(6m2TU)(PPh3)2(bipy)]PF6 (2) (where 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil) to suppress liver CSCs by targeting NF-κB and Akt/mTOR signaling. Complexes 1 and 2 displayed potent cytotoxic effects on cancer cell lines and suppressed liver CSCs from HepG2 cells. Increased phosphatidylserine exposure, loss of mitochondrial transmembrane potential, increased PARP (Asp214) cleavage, DNA fragmentation, chromatin condensation and cytoplasmic shrinkage were detected in HepG2 cells treated with these complexes. Mechanistically, complexes 1 and 2 target NF-κB and Akt/mTOR signaling in HepG2 cells. Cell motility inhibition was also detected in HepG2 cells treated with these complexes. Complexes 1 and 2 also inhibited tumor progression in mice with HepG2 cell xenografts and exhibited tolerable systemic toxicity. Taken together, these results indicate that these complexes are new anti-HCC drug candidates that can suppress liver CSCs.

A Novel 2-Methoxyestradiol Derivative: Disrupting Mitosis Inhibiting Cell Motility and Inducing Apoptosis in HeLa Cells In Vitro.

The clinical application of 2-methoxyestradiol (2ME) in cancer therapy has been limited by its low solubility and rapid metabolism. Derivatives of 2ME have been synthesised to enhance bioavailability and decrease hepatic metabolism. Compound 4a, an analog of 2ME, has demonstrated exceptional pharmacological activity, in addition to promising pharmacokinetic profile. Our study, therefore, aimed at exploring the anticancer effects of 4a on the cervical cancer cell line, HeLa. Compound 4a exhibited a significant and dose-dependent antimetastatic and antiinvasive impact on HeLa cells, as determined by wound-healing and Boyden chamber assays, respectively. Hoechst/Propidium iodide (HOPI) double staining showcased a substantial induction of apoptosis via 4a, with minimal necrotic effect. Flow cytometry revealed a significant G2/M phase arrest, accompanied by a noteworthy rise in the sub-G1 cell population, indicating apoptosis, 18 h post-treatment. Moreover, a cell-independent tubulin polymerisation assay illustrated compound 4a's ability to stabilise microtubules by promoting tubulin polymerisation. Molecular modelling experiments depicted that 4a interacts with the colchicine-binding site, nestled between the α and β tubulin dimers. Furthermore, 4a displayed an affinity for binding to and activating ER-α, as demonstrated by the luciferase reporter assay. These findings underscore the potential of 4a in inhibiting HPV18+ cervical cancer proliferation and cellular motility.

Lung cancer cell-intrinsic IL-15 promotes cell migration and sensitizes murine lung tumors to anti-PD-L1 therapy

BackgroundIL-15 plays a vital role in enhancing NK cell- and T-cell-mediated antitumor immune responses; however, the direct effect of IL-15 on tumor cells has not been fully elucidated. Herein, we investigated the effect of IL-15 on lung adenocarcinoma cells.MethodsSilencing and overexpression techniques were used to modify endogenous IL-15 expression in tumor cells. Transwell assays were used to assess tumor cell migration and invasion; a live-cell analysis system was used to evaluate cell motility; cellular morphological changes were quantified by confocal fluorescence microscopy; the molecular mechanisms underlying the effect of IL-15 on tumor cells were analyzed by western blotting; and RhoA and Cdc42 activities were evaluated by a pulldown assay. NCG and C57BL/6 mouse models were used to evaluate the functions of IL-15 in vivo.ResultsCancer cell-intrinsic IL-15 promoted cell motility and migration in vitro and metastasis in vivo via activation of the AKT-mTORC1 pathway; however, exogenous IL-15 inhibited cell motility and migration via suppression of the RhoA-MLC2 axis. Mechanistic analysis revealed that both the intracellular and extracellular IL-15-mediated effects required the expression of IL-15Rα by tumor cells. Detailed analyses revealed that the IL-2/IL-15Rβ and IL-2Rγ chains were undetected in the complex formed by intracellular IL-15 and IL-15Rα. However, when exogenous IL-15 engaged tumor cells, a complex containing the IL-15Rα, IL-2/IL-15Rβ, and IL-2Rγ chains was formed, indicating that the differential actions of intracellular and extracellular IL-15 on tumor cells might be caused by their distinctive modes of IL-15 receptor engagement. Using a Lewis lung carcinoma (LLC) metastasis model, we showed that although IL-15 overexpression facilitated the lung metastasis of LLC cells, IL-15-overexpressing LLC tumors were more sensitive to anti-PD-L1 therapy than were IL-15-wild-type LLC tumors via an enhanced antitumor immune response, as evidenced by their increased CD8+ T-cell infiltration compared to that of their counterparts.ConclusionsCancer cell-intrinsic IL-15 and exogenous IL-15 differentially regulate cell motility and migration. Thus, cancer cell-intrinsic IL-15 acts as a double-edged sword in tumor progression. Additionally, high levels of IL-15 expressed by tumor cells might improve the responsiveness of tumors to immunotherapies.

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

The suppression of cell motility through the reduction of FAK activity and expression of cell adhesion proteins by hAMSCs secretome in MDA-MB-231 breast cancer cells.

Breast cancer is a leading cause of death in women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through the reduction of focal adhesion kinase (FAK) activity, SHP-2, and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression in MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using 6-well plate transwell. After 72h, hAMSCs-treated MDA-MB-231 breast cancer cells, the activity of focal adhesion kinase (FAK) and the expression of SHP-2 and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression were analyzed using western blot. The shape and migration of cells were also analyzed. Based on our results, a significant reduction in tumor cell motility through downregulation of the tyrosine phosphorylation level of FAK (at Y397 and Y576/577 sites) and cell adhesion expression in MDA-MB-231 breast cancer cells was demonstrated. Our findings indicate that hAMSCS secretome has therapeutic effects on cancer cell migration through downregulation of FAK activity and expression of cell adhesion proteins.

Abstract 2048: Integrated tumor models for immune oncology: Using live cell imaging for prediction of treatment efficacy in vitro and in vivo

Abstract Background: The preclinical evaluation of novel cancer treatments demands comprehensive model systems in vitro that provide meaningful data before entering in vivo studies. Here we evaluate the capabilities of live cell imaging systems to evaluate novel immune therapies. Using integrated immune and tumor cell models in vitro we demonstrate, that these model systems can generate reliable data of pharmacodynamic activity of biologicals, small molecules or combinatorial approaches for further preclinical in vivo characterization. Methods: Target tumor cell killing was assessed in vitro with immune cells (T- and NK-cells) and engagers. Tumor cells were transduced with a fluorescent marker to discriminate tumor cells from immune cells. The technology was used to determine inhibition of cell motility (re-invasion) after scratching of tumor cell monolayers. Cells were monitored using the IncuCyte. Dose-response-curves of single treatments and all combinations were generated in parallel. Active therapies were selected for further in vivo validation of immune cell killing. Humanized mice were generated by injection of CD34+ HSC or human immune cell subsets. Immune cell engraftment was monitored by FACS. To analyze the effect biologicals or small molecules, tumor cells were transplanted into these humanized mice. Tumor development and therapeutic effects were monitored by BLI measurements. Results and conclusion: Tumor cell killing by immune cells and monolayer scratch assay in 96 well format were successfully monitored in the IncuCyte. Here, data can be generated over time without the need of new samples at every time point compared with conventional end point measurements. Using antibodies directing immune cells to attack target cells extensive cell killing was observed over time. These data predicted in vivo treatment outcome in mice co-engrafted with human immune cells. After successful humanization of mice, immune cells can be directed to kill target tumor cells. Small molecule combinations were tested in vitro utilizing the metastasis/2D scratch assay. After setup of dose-response curves for two molecules combinatorial treatments were tested. Here we found a synergistic increase in efficacy. These combinations were tested in vivo to evaluate their abilities to inhibit cell motility and distant metastasis. Here we show that the in vitro assays predicted correctly the highest efficacy of combined treatments compared to mono treatments.The IncuCyte System provides data that translate our integrated model systems into in vivo studies. We have shown that activated immune cells can kill target tumor cells in vitro. These data have been validated in vivo using immune cell humanized mice. Further, immune cells, biologicals and small molecule based treatments can be tested either alone or in combination, allowing the preselection of active combinations for further development. Citation Format: Dennis Kobelt, Maria Stecklum, Simone Rhein, Wolfgang Walther, Jens Hoffmann. Integrated tumor models for immune oncology: Using live cell imaging for prediction of treatment efficacy in vitro and in vivo [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 2048.

Abstract 303: Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer

Abstract G protein-independent pathway (β-arrestin dependent signaling cascade) is initiated by activated GPCRs phosphorylated by G protein-coupled receptor kinases (GRKs), triggering multiple signaling pathways. The purpose of this research is to reveal the role of GRK2 on the tumor microenvironment (TME). Through in vitro assays, we showed that GRK2 overexpression led to loss of cell-matrix and cell-cell adhesion. The cell spreading assay further indicated that the loss of focal adhesion (FA) assembly and cell size decreased in GRK2-overexpressing cells. In vivo experiments demonstrated that tumor formation and metastasis were suppressed in xenograft mouse models implanted with GRK2-overexpressing ovarian cancer cells. In addition, label-free imaging detection of the collagen pattern suggested that GRK2 overexpression caused bundle loss but the formation of thicker, twisted collagen fibers in xenograft tumor samples. Together with the transcriptome and proteomic data, we hypothesize that GRK2 suppresses cell motility and metastasis by reducing FA and extracellular matrix (ECM) remodeling in ovarian cancer. This research revised the mechanisms underlying GRK2-mediated ovarian cancer metastasis and suggested the potential to target the GRK2-meditated pathway for cancer suppression. Citation Format: Weng I Lei, Shan Yu, Chi Teng Lei, Zhiming Zhang, Tzu Ming Liu, Wakam Chang, Leo Tsz On Lee. Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian 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 303.

Combined inhibition of HER2 and VEGFR synergistically improves therapeutic efficacy via PI3K-AKT pathway in advanced ovarian cancer

BackgroundOvarian cancer (OC) is a prevalent malignancy in the female reproductive system, and developing effective targeted therapies for this disease remains challenging. The aim of this study was to use clinically-relevant OC models to evaluate the therapeutic effectiveness of RC48, an antibody-drug conjugate (ADC) targeting HER2, either alone or in combination with the VEGFR inhibitor Cediranib Maleate (CM), for the treatment of advanced OC.MethodsOC tumor specimens and cell lines were analyzed to determine HER2 and VEGFR expression by Western blot, immunocytochemistry and immunofluorescence. Moreover, the OC cell lines, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models were treated with RC48 and/or CM and then subjected to cell proliferation, viability, apoptosis, and tumor growth analyses to evaluate the feasibility of combination therapy for OC both in vitro and in vivo. Additionally, RNA-Seq was performed to investigate the critical mechanism underlying the combination therapy of RC48 and CM.ResultsOur results demonstrated that RC48 alone effectively targeted and inhibited the growth of HER2-positive OC tumors in both cell lines and PDX models. Furthermore, the combination of RC48 and CM synergistically induced tumor regression in human OC cell lines, as well as CDX and PDX models. Mechanistically, we observed that the combination treatment inhibited the growth of OC cells involved inducing apoptosis and suppressing cell motility. RNA-seq analysis provided further mechanistic insights and revealed that co-administration of RC48 and CM downregulated multiple cancer-related pathways, including the AKT/mTOR pathway, cell cycle, and cell proliferation. Notably, our data further confirmed that the PI3K-AKT pathway played a key role in the inhibition of proliferation triggered by combinational treatment of RC48 and CM in OC cells.ConclusionsThese findings provide a preclinical framework supporting the potential of dual targeting HER2 and VEGFR as a promising therapeutic strategy to improve outcomes in patients with OC.

SATB1 mediated tumor colonization and β-catenin nuclear localization are associated with colorectal cancer progression

ABSTRACT Colorectal cancer (CRC) is a malignancy with high incidence and poor prognosis. It is urgent to identify valuable biomarkers for early diagnosis and potent therapeutic targets. It has been reported that SATB1 is associated with the malignant progression in CRC. To explore the role of SATB1 in CRC progression and the underlying mechanism, we evaluated the expression of SATB1 in the paired CRC tissues with immunohistochemistry. The results showed that the expression of SATB1 in lymph node metastasis was higher than that in primary lesion, and that in distant organ metastasis was higher than that in primary lesion. The retrospective analysis showed that patients with high expression of SATB1 had a significantly worse prognosis than those with negative and moderate expression. In vitro experiments that employing SATB1 over-expressing and depleted CRC cell lines confirmed that SATB1 contributes to cell proliferation and colonization, while inhibiting cell motility. Furthermore, the tissue immunofluorescence assay, Co-IP and Western blot were conducted to reveal that SATB1 induced translocation of β-catenin and formed a protein complex with it in the nuclei. In conclusion, SATB1 mediated tumor colonization and β-catenin nuclear localization are associated with the malignant progression and poor prognosis of CRC.

Overexpression of Klotho gene using CRISPR/Cas9 induces apoptosis and inhibits cell motility in the human colorectal cancer cells.

Despite advances in early detection and treatment, colorectal cancer remains one of the leading causes of cancer-related deaths. The klotho (KL) gene plays a critical role in the development and progression of colorectal cancer. This study investigates the role of the KL gene in colorectal cancer by using the CRISPR/Cas9 system to overexpress and knock out (KO) the KL gene in human colorectal cancer cells (Caco-2). The effects of the changes were assessed by gene expression analysis, flow cytometry, scratch wound closure assays, colony formation assays, and immunofluorescence staining. Our results showed that overexpression of the KL gene increased apoptosis and decreased cell motility in cancer cells, whereas knockout of the KL gene had the opposite role. The present study elucidates the mechanisms underlying this role and highlights the potential of the CRISPR/Cas9 system as a gene editing tool in cancer research. Our data suggest that activation of the KL gene may serve as a novel therapeutic strategy and biomarker for studies in colorectal cancer.

Brunonianines A-C, C20-diterpenoid alkaloids with cyano group from Delphinium brunonianum Royle

Cyano tends to have better biological activity, but it is rarely reported in natural products, especially in the C20-diterpene alkaloids. Herein, three unprecedented C20-diterpenoid alkaloids, brunonianines A-C (1-3), possessing rare cyano functional group as well as an atisine backbone constructed from a phenethyl substituent and a tetrahydropyran ring, along with four C19-alkaloids (4–7) and one amide alkaloids (8), were isolated from the whole plant of Delphinium brunonianum Royle. Compounds 1–3 are also the first atisine type diterpenoid alkaloids with cyano group obtained from nature. The structures of the previously undescribed compounds were elucidated by HR-ESI-MS, 1D/2D NMR spectroscopic data and electronic circular dichroism calculations and single-crystal X-ray diffraction. Reasonable speculations have also been made regarding the biogenic synthetic pathways of compounds 1–3. In addition, the inhibitory activity of all compounds was also tested against four tumor lines: A549, Caco-2, H460 and Skov-3, where compound 2 (IC50 2.20 ± 0.21 μM) showed better inhibitory activity against Skov-3 cells than the hydroxycamptothecin. Using flow cytometry, cell staining, migration and invasion analysis, and Western blot, compound 2 was found to arrest cells in the G2/M phase and was able to effectively inhibit cell motility to achieve potent anti-tumor effects. In addition, compound 2 can effectively induce apoptosis by activating the Bax/Bcl-2/Caspase-3 signaling pathway.