MCF-7 Xenograft Tumors Research Articles

NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid

BackgroundNAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7.MethodsNSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control.ResultsBCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens (n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression.ConclusionsOur findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors.

Discovery of ERD-1233 as a Potent and Orally Efficacious Estrogen Receptor PROTAC Degrader for the Treatment of ER+ Human Breast Cancer.

Despite the development of highly effective therapies for the treatment of estrogen receptor α (ERα)-positive human breast cancer, clinical resistance to current therapies requires the development of novel therapeutic strategies. Herein, we report the discovery of ERD-1233 as a potent and orally efficacious ERα degrader designed using the PROTAC technology. ERD-1233 was developed based on Lasofoxifene as the ER binding moiety and a novel cereblon ligand through extensive optimization of the linker. ERD-1233 potently and effectively reduces the ERα protein in vitro and achieves excellent oral bioavailability in mice and rats. Oral administration of ERD-1233 effectively reduces ER protein in ER+ tumors and achieves tumor regression in the ER wild-type MCF-7 xenograft tumor model and strong tumor growth inhibition in the ESR1Y537S mutated model in mice. Our data demonstrate that ERD-1233 is a promising ER PROTAC degrader for extensive evaluation as a new therapy for the treatment of ER+ human breast cancer.

Discovery of Novel ERα and Aromatase Dual-Targeting PROTAC Degraders to Overcome Endocrine-Resistant Breast Cancer.

Estrogen receptor α (ERα) plays a pivotal role in the proliferation, differentiation, and migration of breast cancer (BC) cells, and aromatase (ARO) is a crucial enzyme in estrogen synthesis. Hence, it is necessary to inhibit estrogen production or the activity of ERα for the treatment of estrogen receptor-positive (ER+) BC. Herein, we present a new category of dual-targeting PROTAC degraders designed to specifically target ERα and ARO. Among them, compound 18c bifunctionally degrades and inhibits ERα/ARO, thus effectively suppressing the proliferation of MCF-7 cells while showing negligible cytotoxicity to normal cells. In vivo, 18c promotes the degradation of ERα and ARO and inhibits the growth of MCF-7 xenograft tumors. Finally, compound 18c demonstrates promising antiproliferative and ERα degradation activity against the ERαMUT cells. These findings suggest that 18c, being the inaugural dual-targeting degrader for ERα and ARO, warrants further advancement for the management of BC and the surmounting of endocrine resistance.

Abstract PO4-27-12: Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen, aromatase inhibitors, AI, or fulvestrant) in ER+ breast cancer (BC) could be due to survival of breast cancer stem cells (BCSCs). BCSCs contribute to relapse of ER+ BC. We discovered a novel and potent anti-BCSC gene, Death Associated Protein 6 (DAXX) through a pre-surgical biomarker window study combining ET plus a Notch inhibitor [MK-0752, a g-secretase inhibitor (GSI)]. We also found that ET enhances the BCSC population by decreasing DAXX protein levels. In this current study, we measured DAXX protein levels in ER+ PDX tumors and human tumor tissues treated with ET. We determined the mechanism by which ET regulates DAXX expression at the level of protein stability. The goal of this study was to identify novel therapeutic strategies to prevent ET-mediated degradation of the DAXX protein, eliminate BCSCs, and prevent ER+ recurrence. Methods: ER+ BC cell lines (MCF-7 and T47D), an ER+ PDX BCM 5097 tumor line, and human breast cancer tumor samples were used. The ER+ PDX tumor (BCM 5097) was implanted into NSG mice and passaged into female athymic, nude mice-supplemented with an estradiol capsule. After one week, the estradiol capsule was removed from 10 mice, to mimic the use of AI. The remaining 10 mice retained the estradiol capsule. Tumor area was measured weekly up to 50 weeks. Western blotting detected ERa, the stem cell factor Notch4, and DAXX levels. Immunohistochemistry (IHC) detected DAXX protein levels. ER+ human tumor samples were collected prior to and after neoadjuvant treatment with ET (N=11) or chemotherapy (N=14) and stained for the DAXX protein. Correlations were made between DAXX and Ki67. Full length and deletion mutants of DAXX were expressed in ER+ BC cells. BCSCs were measured using the mammosphere forming assay. Liquid chromatography followed by mass spectrometry were used to detect phosphorylated residues of DAXX in response to ET. Kinases that phosphorylate these residues on DAXX were identified using PhosphoSite.org. ER+ cells were treated with kinase inhibitors for AURKA (alisertib), AURKB (barasertib), CK1 (CK-IN-1), or CK2 (CX-4945) and DAXX protein was detected by western blotting. BCSC survival was measured using mammosphere forming assay. Based on results from the kinase inhibitor screen, barasertib was selected for pre-clinical testing in mice. ER+ MCF-7 xenograft tumors were treated with vehicle or barasertib in vivo up to 120 days. Results: DAXX protein levels decreased in ER+ PDX tumors in response to ET. Similar trends were detected in human tumor tissue after ET or chemotherapy alone. DAXX protein levels inversely correlated with Ki67 in ER+ human tumor samples. Analysis of DAXX deletion mutants demonstrated that amino acids 400-740 of the DAXX protein were required to inhibit BCSC survival. This region of the DAXX protein had high levels of phosphorylated residues in response to ET. Only the AURKB (barasertib) inhibitor increased DAXX protein expression and inhibited BCSCs in a DAXX-dependent manner. Barasertib inhibited growth of ER+ MCF-7 tumor xenografts compared to the vehicle control. After treatment ceased, tumor recurrence was measured up to 120 days. Regrowth of tumors was partially delayed in response to barasertib. Conclusions: ET decreased DAXX protein levels in ER+ PDX and human tumors. Downregulation of the DAXX protein by ET was through activation of AURKB and hyper-phosphorylation of DAXX which resulted in protein degradation and enhanced survival of BCSCs. Therefore, Inhibition of AURKB using barasertib partially restored DAXX expression, inhibited BCSCs, and delayed tumor recurrence. A combination approach of ET plus an AURKB inhibitor might be a novel therapeutic strategy to prevent ER+ breast cancer relapse by increasing DAXX in order to eradicate BCSCs. Support: Breast Cancer Research Foundation Citation Format: Clodia Osipo, Debra Wyatt, Kathy Albain. Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-12.

Polymer-lipid hybrid nanoparticles of exemestane for improved oral bioavailability and anti-tumor efficacy: An extensive preclinical investigation

Exemestane (EXE), an irreversible aromatase inhibitor, is primarily used as a first-line therapy for estrogen receptor-positive breast cancer patients. However, complex physicochemical characteristics of EXE limit its oral bioavailability (<10%) and anti-breast cancer efficacy. The present study aimed to develop a novel nanocarrier system to improve the oral bioavailability and anti-breast cancer efficacy of EXE. In this perspective, EXE-loaded TPGS-based polymer lipid hybrid nanoparticles (EXE-TPGS-PLHNPs) were prepared by the nanoprecipitation method and evaluated for their potential in improving oral bioavailability, safety, and therapeutic efficacy in the animal model. EXE-TPGS-PLHNPs showed significantly higher intestinal permeation in comparison to EXE-PLHNPs (without TPGS) and free EXE. After oral administration, EXE-TPGS-PLHNPs and EXE-PLHNPs revealed 3.58 and 4.69 times higher oral bioavailability in Wistar rats compared to the conventional EXE suspension. The results of the acute toxicity experiment suggested that the developed nanocarrier was safe for oral administration. Furthermore, EXE-TPGS-PLHNPs and EXE-PLHNPs represented much better anti-breast cancer activity in Balb/c mice bearing MCF-7 tumor xenograft with tumor inhibition rate of 72.72% and 61.94% respectively in comparison with the conventional EXE suspension (30.79%) after 21 days of oral chemotherapy. In addition, insignificant changes in the histopathological examination of vital organs and hematological analysis further confirm the safety of the developed PLHNPs. Therefore, the findings of the present investigation advocated that the encapsulation of EXE in PLHNPs can be a promising approach for oral chemotherapy of breast cancer.

Discovery of novel covalent selective estrogen receptor degraders against endocrine-resistant breast cancer

Endocrine-resistance remains a major challenge in estrogen receptor α positive (ERα+) breast cancer (BC) treatment and constitutively active somatic mutations in ERα are a common mechanism. There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrine-resistance. Given aberrant ERα activity, we herein report the identification of novel covalent selective estrogen receptor degraders (cSERDs) possessing the advantages of both covalent and degradation strategies. A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERα+ breast cancer cell lines including mutant ERα. Crystal structure of ERα‒29c complex alongside intact mass spectrometry revealed that 29c disrupted ERα protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11, thus enforcing a unique antagonist conformation and driving the ERα degradation. These significant effects of the cSERD on ERα homeostasis, unlike typical ERα degraders that occur directly via long side chains perturbing the morphology of H12, demonstrating a distinct mechanism of action (MoA). In vivo, 29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity. This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.

The Synergistic Effect of Ruthenium Complex Δ-Ru1 and Doxorubicin in a Mouse Breast Cancer Model.

Doxorubicin is a significant drug for the treatment of breast cancer, but its cardiotoxicity is an obvious obstacle. Previously, we confirmed that ruthenium complex (Δ-Ru1) and doxorubicin (Δ-Ru1/Dox) combination had a synergistic effect in MCF-7 cells, but its biological effect in vivo is unknown. To find a way to overcome the toxicity of doxorubicin and build MCF-7 xenograft tumor mouse model to test whether this potential combination has better efficacy and less toxicity. The tumor model of nude mice was established to verify the synergistic antitumor effect of the drug combination in vivo. H&E staining was used to detect the toxicity of major organs in mice. Sirius red staining and transmission electron microscopy were used to detect cardiotoxicity. Prussian blue was used to measure iron accumulation in heart tissue. TUNEL staining was used to detect the antitumor effect in vivo. Immunohistochemical staining was used to detect the expression of iron death-related pathway proteins. High-throughput sequencing techniques were used to determine the molecular mechanism of ferroptosis. Histopathological analysis of tumor tissues indicated that the Δ-Ru1/Dox combination significantly promoted tumor cell apoptosis. Doxorubicin damaged cardiac tissue by inducing fibrosis and iron accumulation, but it was reversed by the Δ-Ru1/Dox combination treatment. Further exploration found that doxorubicin could regulate iron accumulation in the ferroptosis pathway and the expression of lipid peroxidation-related proteins, including upregulation of Tf, DMT1, and HO-1, and downregulation of Nrf2, SLC7A11, and GPX4. Δ-Ru1/Dox combination synergistically inhibits tumor growth, and it can significantly reduce and alleviate the toxic side effects of doxorubicin, especially cardiac injury.

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC.

Asiaticoside Increases Caspase-9 Activity in MCF-7 Cells and Inhibits TNF-α and IL-6 Expression in Nude Mouse Xenografts via the NF-κB Pathway.

Background: We hypothesized that the antitumor effects of asiaticoside on breast cancer are driven by its ability to decrease the expression of tumor inflammation-promoting genes and increase apoptotic signaling. In this study, we aimed to better understand the mechanisms of action of asiaticoside as a chemical modulator or as a chemopreventive agent in breast cancer. Methods: MCF-7 cells were cultured and treated with 0, 20, 40, and 80 μM asiaticoside for 48 h. Fluorometric caspase-9, apoptosis, and gene expression analyses were conducted. For the xenograft experiments, we divided nude mice into the following 5 groups (10 animals per group): group I, control mice; group II, untreated tumor-bearing nude mice; group III, tumor-bearing nude mice treated with asiaticoside at weeks 1-2 and 4-7 and injected with MCF-7 cells at week 3; group IV, tumor-bearing nude mice injected with MCF-7 cells at week 3 and treated with asiaticoside beginning at week 6; and group V, nude mice treated with asiaticoside, as a drug control. After treatment, weight measurements were performed weekly. Tumor growth was determined and analyzed using histology and DNA and RNA isolation. Results: In MCF-7 cells, we found that asiaticoside increased caspase-9 activity. In the xenograft experiment, we found that TNF-α and IL-6 expression decreased (p < 0.001) via the NF-κB pathway. Conclusion: Overall, our data suggest that asiaticoside produces promising effects on tumor growth, progression, and tumor-associated inflammation in MCF-7 cells as well as a nude mouse MCF-7 tumor xenograft model.

Mitochondria-targeting sonosensitizer-loaded extracellular vesicles for chemo-sonodynamic therapy.

Sonodynamic therapy (SDT) has emerged as an effective therapeutic modality as it employs ultrasound (US) to eradicate deep-seated tumors noninvasively. However, the therapeutic efficacy of SDT in clinical settings remains limited owing to the low aqueous stability and poor pharmacokinetic properties of sonosensitizers. In this study, extracellular vesicles (EVs), which have low systemic toxicity, were used as clinically available nanocarriers to effectively transfer a sonosensitizer to cancer cells. Chlorin e6 (Ce6), a sonosensitizer, was conjugated to a mitochondria-targeting triphenylphosphonium (TPP) moiety and loaded into EVs to enhance the efficacy of SDT, because mitochondria are critical subcellular organelles that regulate cell survival and death. Additionally, piperlongumine (PL), a pro-oxidant and cancer-specific chemotherapeutic agent, was co-encapsulated into EVs to achieve efficient and selective anticancer activity. The EVs substantially amplified the cellular internalization of TPP-conjugated Ce6 (TPP-Ce6), resulting in the enhanced generation of intracellular reactive oxygen species (ROS) in MCF-7 human breast cancer cells upon US exposure. Importantly, EVs encapsulating TPP-Ce6 effectively destroyed the mitochondria under irradiation with US, leading to efficient anticancer activity. The co-encapsulation of pro-oxidant PL into EVs significantly enhanced the SDT efficacy in MCF-7 cells through the excessive generation of ROS. Moreover, the EV co-encapsulating TPP-Ce6 and PL [EV(TPP-Ce6/PL)] exhibited cancer-specific cell death owing to the cancer-selective apoptosis triggered by PL. In vivo study using MCF-7 tumor-xenograft mice revealed that EV(TPP-Ce6/PL) effectively accumulated in tumors after intravenous injection. Notably, treatment with EV(TPP-Ce6/PL) and US inhibited tumor growth significantly without causing systemic toxicity. This study demonstrated the feasibility of using EV(TPP-Ce6/PL) for biocompatible and cancer-specific chemo-SDT.

Choosing the Right Protocol to Establish MCF-7 Tumor Xenograft in Nude Mice.

Xenografts of various human cancers in nude mice provide a helpful model in cancer research. This study aimed to develop a xenograft mouse model of MCF-7 breast cancer using injectable estradiol valerate. Thirty healthy female C57 nu/nu mice were engrafted with three protocols to establish an MCF-7 tumor. Injectable estradiol valerate (10 mg/ml) was used as a substitute for estradiol pellets. The development of tumors was recorded daily, and data were statistically analyzed. Histology of bladder, kidney, and tumors was used to estimate tumor establishment and probable urinary adverse effects. According to the findings, the duration of MCF-7 tumor growth was the lowest for protocol B (tumor tissue). Also, this protocol had the highest xenograft yield within the shortest time duration (37 days for protocol B vs. 73 days for protocol A) without causing urinary adverse effects. Our findings revealed that estradiol valerate, which is way less expensive than estradiol pellets, can be used as a tumor proliferator to establish MCF-7 tumors with the highest yield when MCF-7 tumors have been used for xenograft.

Polysaccharide dextran-based conjugate for selective co-delivery of two synergistic drugs docetaxel and docosahexaenoic acid to tumor cells

Most chemotherapeutic agents are nonspecific distribution and cause systemic toxicities. Polysaccharide-based conjugates are promising strategies to overcome these drawbacks. To this end, two synergistic drugs docetaxel (DTX) and docosahexaenoic acid (DHA) were independently covalently bonded through individual linkers to dextran (100 kDa) to produce a novel dual-drug conjugate dextran–DHA–DTX. The single-drug conjugates dextran–DHA and dextran–DTX were also prepared for comparison. Fluorescent dye Cy7.5-based conjugates dextran–Cy7.5 and dextran–DHA–Cy7.5 were synthesized for cellular uptake study. The dual-drug conjugate dextran–DHA–DTX self-assembled into nanoparticles with the diameter of 102.3 ± 8.3 nm and demonstrated enhanced water solubility and improved pharmacokinetic profiles. Cellular uptake results showed that the dual-drug conjugate entered cells more than the parent DTX by determining the intracellular DTX contents via HPLC/MS analysis and by determining the fluorescent intensity of dextran-Cy7.5 and dextran–DHA–Cy7.5. Importantly, the dual-drug conjugate dextran–DHA–DTX significantly accumulated in tumor tissues and dramatically reduced the DTX concentrations in normal tissues. The dual-drug conjugate completely eradicated all the MCF-7 xenograft tumors without obvious side effects and showed more superior antitumor activity than parent DTX and single-drug conjugate dextran–DTX and dextran–DHA. Both in vitro and in vivo studies showed that DHA enhanced the antitumor activity of dextran–DTX. The polysaccharide dextran-based dual-drug conjugates may represent an effective way to improve the chemotherapeutic agents.

A Novel Dextran-Based Dual Drug Conjugate Targeted Tumors with High Biodistribution Ratio of Tumors to Normal Tissues.

PurposeMost chemotherapeutic agents possess poor water solubility and show more significant accumulations in normal tissues than in tumor tissues, resulting in serious side effects. To this end, a novel dextran-based dual drug delivery system with high biodistribution ratio of tumors to normal tissues was developed.MethodsA bi-functionalized dextran was developed, and several negatively charged dextran-based dual conjugates containing two different types of drugs, docetaxel and docosahexaenoic acid (DTX and DHA, respectively) were synthesized. The structures of these conjugates were characterized using nuclear magnetic resonance and liquid chromatography/mass spectrometry (1H-NMR and LC/MS, respectively) analysis. Cell growth inhibition, apoptosis, cell cycle distribution, and cellular uptake were measured in vitro. Drug biodistribution and pharmacokinetics were investigated in mice bearing 4T1 tumors using LC/MS analysis. Drug biodistribution was also explored by in vivo imaging. The effects of these conjugates on tumor growth were evaluated in three mice models.ResultsThe dextran–docosahexaenoic acid (DHA)– docetaxel (DTX) conjugates caused a significant enhancement of DTX water solubility and improvement in pharmacokinetic characteristics. The optimized dextran–DHA–DTX conjugate A treatment produced a 2.1- to 15.5-fold increase in intra-tumoral DTX amounts for up to 96 h compared to parent DTX treatment. Meanwhile, the concentrations of DTX released from conjugate A in normal tissues were much lower than those of the parent DTX. This study demonstrated that DHA could lead to an improvement in the efficacy of the conjugates and that the conjugate with the shortest linker displayed more activity than conjugates with longer linkers. Moreover, conjugate A completely eradicated all MCF-7 xenograft tumors without causing any obvious side effects and totally outperformed both the conventional DTX formulation and Abraxane in mice.ConclusionThese dextran-based dual drug conjugates may represent an innovative tumor targeting drug delivery system that can selectively deliver anticancer agents to tumors.

Robust ERα-Targeted Near-Infrared Fluorescence Probe for Selective Hydrazine Imaging in Breast Cancer.

Breast cancer is the most common malignancy in women and may become worse when a high concentration of hydrazine is absorbed from the environment or drug metabolite. Therefore, rapid and sensitive detection of hydrazine in vivo is beneficial for people's health. In this work, a novel estrogen receptor α (ERα)-targeted near-infrared fluorescence probe was designed to detect hydrazine levels. The probe showed good ERα affinity and an excellent fluorescence response toward hydrazine. Selectivity experiments demonstrated that the probe had a strong anti-interference ability. Mechanistic studies, including mass spectrometry (MS) and density functional theory (DFT) calculation, indicated that intermolecular charge transfer (ICT) progress was hindered when the probe reacted with hydrazine, resulting in fluorescent quenching. In addition, the probe could selectively bind to MCF-7 breast cancer cells with excellent biocompatibility. The in vivo and ex vivo imaging studies demonstrated that the probe could rapidly visualize hydrazine with high contrast in MCF-7 xenograft tumors. Therefore, this probe can serve as a potential tool to robustly monitor hydrazine levels in vivo.

A novel form of docetaxel polymeric micelles demonstrates anti-tumor and ascites-inhibitory activities in animal models as monotherapy or in combination with anti-angiogenic agents.

Malignant ascites (MA) is caused by intraperitoneal spread of solid tumor cells and results in a poor quality of life. Chemotherapy is a common first-line treatment for patients with MA. Taxotere ® (DTX) is widely used in solid tumor therapies. However, the low water solubility and side effects caused by additives in the formulation restrict the clinical application of docetaxel. HT001 is a clinical stage docetaxel micelle developed to overcome the solubility issue with improved safety profiles. To support clinical development and expand clinical application of HT001, this study used in vitro and in vivo approaches to investigate the anti-tumor effects of HT001 when applied as monotherapy or in combination with anti-angiogenic agents. HT001 demonstrated comparable anti-proliferative activities as docetaxel in a broad range of cancer cell lines in vitro. Furthermore, HT001 suppressed tumor growth in a dose-dependent manner in A549, MCF-7, and SKOV-3 xenograft tumor mouse models in vivo. In a hepatocellular carcinoma H22 malignant ascites-bearing mouse model, HT001 presented a dose-dependent inhibition of ascites production, prolonged animal survival, and reduced VEGF levels. When dosed at 20 mg/kg, the HT001-treated group exhibited curative results, with no ascites formation in 80% of mice at the end of the study while all the mice in the vehicle control group succumbed. Similar results were obtained in HT001 treatment of mice bearing malignant ascites produced by human ovarian cancer ES-2 cells. Notably, the combination of HT001 with Endostar not only significantly reduced ascites production but also prolonged survival of H22 ascites-bearing mice. HT001 showed similar PK and tissue distribution profiles as DTX in non-rodent hosts. Collectively, these results demonstrate potent anti-tumor activity of HT001 in multiple solid tumor models or malignant ascites models, and reveal synergistic effects with anti-angiogenic agents, supporting the clinical development and clinical expansion plans for HT001.

Identification of phenolics from miracle berry (Synsepalum dulcificum) leaf extract and its antiangiogenesis and anticancer activities.

Miracle berry is well-known for its ability to convert sour foods to sweet. In this study, the secondary metabolites of miracle berry leaves (MBL) were identified by UPLC-DAD-MS, and its antiangiogenesis and anticancer activities were evaluated by using a zebrafish model and the MCF-7 xenograft mouse model, respectively. The result showed that 18 phenolic compounds were identified in MBL extract, and dominated by the derivatives of quercetin and myricetin. The MBL extract showed low toxicity and high antiangiogenesis activity, it significantly inhibited the subintestinal vein vessels development in zebrafish at very low concentration. Furthermore, the MBL extract could promote the apoptosis of tumor cells and significantly inhibit the growth of MCF-7 xenograft tumor. In addition, the analysis of metabolites revealed that the MBL extract inhibited tumor growth by activating the metabolic pathways of unsaturated fatty acids and purines. Overall, this study suggests that MBL extract can be used as a natural anticancer adjuvant in the fields of functional foods.

Abstract 1865: Tumor microenvironment ameliorating effect of E7130 sensitized ER+ breast cancer against fulvestrant

Abstract Introduction and objectives: E7130 is a novel anti-cancer agent created from a total synthetic study of the natural compound norhalichondrin B, and is evaluated in a clinical study currently (NCT03444701). Previous pre-clinical study reported that E7130 had both cancer cell proliferation inhibitory activity and tumor microenvironment ameliorating activities featuring vascular remodeling effect and anti-CAF (cancer-associated fibroblasts) effect. Intratumoral hypoxia induces tumor malignancy and causes resistance against anti-cancer drugs including anti-estrogen agents (eg fulvestrant). It is hypothesized that vascular remodeling effect of E7130 mitigates hypoxia-induced resistant mechanism of ER (estrogen receptor) + breast cancer against anti-estrogen agents, resulting in improving anti-tumor activity of fulvestrant in combination with E7130. Experimental procedure: ERα expression levels in human ER+ breast cancer cell lines under hypoxic or normoxic culture condition were examined by FCM (flow cytometry) analysis. The histological changes after treatment with E7130 were examined by immunohistochemical analyses in the MCF-7 subcutaneous xenograft model. Anti-tumor activity of E7130 in combination with fulvestrant were examined in concurrent and in sequential treatment in the MCF-7 model. Summary: In vitro hypoxic culture condition down-regulated ERα expression in MCF-7 and T-47D compared with normoxic condition. E7130 had no effect on the ERα expression level in the cancer cells in vitro. On the contrary, E7130 increased ERα expression level in the cancer cells concomitantly with an increase of CD31-positive endothelial cell number (ie vascular remodeling effect) in MCF-7 xenograft tumor. These results suggested that E7130 up-regulated ERα expression in cancer cells not by cell-autonomous manner but by mitigating intratumoral hypoxia via its vascular remodeling effect. Concurrent combination therapy of E7130 at 90 μg/kg with fulvestrant at 250 mg/kg significantly inhibited the tumor growth compared with each monotherapy. In sequential combination therapy, pretreatment of E7130 followed by fulvestrant showed superior anti-tumor activity to posttreatment of E7130 following fulvestrant. Severe body weight loss was not observed in any treatment groups. Conclusions: The vascular remodeling effect of E7130 increased ERα expression in cancer cells in vivo, and sensitized ER+ breast cancer against fulvestrant to achieve promising combinational anti-tumor activity. Our data provides compelling evidence that E7130 attenuates tumor malignancy by its tumor microenvironment ameliorating effects and improves current anti-cancer therapy in combination with other drugs. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Taro Semba, Kimiyo Tabata, Hiroki Jozawa, Osamu Asano, Yoshito Kishi. Tumor microenvironment ameliorating effect of E7130 sensitized ER+ breast cancer against fulvestrant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1865.

Discovery of Novel Bicyclic Phenylselenyl-Containing Hybrids: An Orally Bioavailable, Potential, and Multiacting Class of Estrogen Receptor Modulators against Endocrine-Resistant Breast Cancer.

Breast cancer (BC) is a multifactorial disease and is prone to drug resistance during treatment. In this study, we described a new class of multifunctional estrogen receptor (ER) modulators ground on a prerogative indirect antagonism skeleton (OBHS, oxabicycloheptene sulfonate) of ER containing a phenylselenyl group. Compound 34b showed significant antiproliferative activities against tamoxifen-sensitive (MCF-7) and -resistant (LCC2) cells. Moreover, hexokinase 1 (HK1) was identified as a direct target of 34b. Further mechanism investigations proved that 34b induced apoptosis, which was associated with mitochondrial dysfunction caused by the synergistic effects of downregulating mitochondrial-bound HK1 protein and promoting reactive oxygen species generation. In vivo, 34b had a favorable pharmacokinetic profile with a bioavailability of 23.20% and exhibited more potent tumor suppression than tamoxifen both in MCF-7 and LCC2 tumor xenograft models. Collectively, our studies showed that 34b is a promising new multifunctional candidate compound for ERα+ BC treatment, particularly for tamoxifen-resistant BC.

Microcrater-Arrayed Chemiluminescence Cell Chip to Boost Anti-Cancer Drug Administration in Zebrafish Tumor Xenograft Model.

Simple SummaryPersonalized drug cocktails have been widely implemented in cancer treatment, due to their merits of using a drug synergistic combination rather than a single effector. Through the incorporation of a cell microarray chip, the usage of both cell amount and reagent can be optimized and effectively reduced in cost. Herein, we present a laser direct writing strategy to fabricate a microcrater-arrayed (µCA) chip incorporated with an automatic liquid handling platform. Each cell droplet with a critical volume of 200 nL containing 100 SK-N-DZ or MCF-7 cells was utilized. The drug synergy screening took less than 30 s for a total of 81 tests. The results show that the potent drug prediction of the µCA chip is more accurate than the conventional 96-well plate assay, which are all compared using zebrafish DiI-labelled tumor xenograft models. Taken together, these findings may impact high-throughput drug screening and personalized drug medicine.Purpose: The aim of this study was to develop a rapid and automatic drug screening platform using microcrater-arrayed (µCA) cell chips. Methods: The µCA chip was fabricated using a laser direct writing technique. The fabrication time required for one 9 × 9 microarray wax chip was as quick as 1 min. On a nanodroplet handling platform, the chip was pre-coated with anti-cancer drugs, including cyclophosphamide, cisplatin, doxorubicin, oncovin, etoposide, and 5-fluorouracil, and their associated mixtures. Cell droplets containing 100 SK-N-DZ or MCF-7 cells were then loaded onto the chip. Cell viability was examined directly through a chemiluminescence assay on the chip using the CellTiter-Glo assay. Results: The time needed for the drug screening assay was demonstrated to be less than 30 s for a total of 81 tests. The prediction of optimal drug synergy from the µCA chip was found by matching it to that of the zebrafish MCF-7 tumor xenograft model, instead of the conventional 96-well plate assay. In addition, the critical reagent volume and cell number for each µCA chip test were 200 nL and 100 cells, respectively, which were significantly lower than 100 µL and 4000 cells, which were achieved using the 96-well assay. Conclusion: Our study for the µCA chip platform could improve the high-throughput drug synergy screening targeting the applications of tumor cell biology.

Evaluation of 3-l- and 3-d-[18F]Fluorophenylalanines as PET Tracers for Tumor Imaging.

Simple SummaryThe early detection and treatment of malignant brain tumors can significantly improve the survival time and life quality of affected patients. Whereas positron emission tomography (PET) with O-(2-[18F]fluoroethyl)tyrosine ([18F]FET) offers improved diagnostic accuracy compared to other imaging methods, there is still a need for PET tracers with better tumor-specificity. A higher protein incorporation rate, as well as a higher affinity for the amino acid transporter LAT1, could provide probes with superior image quality compared to [18F]FET. The aim of the present study was a preclinical evaluation of the two enantiomeric phenylalanine (Phe) analogues, 3-l- and 3-d-[18F]fluorophenylalanine ([18F]FPhes), as possible alternatives to [18F]FET. Based on promising in vitro evaluation results, the radiolabeled amino acids were studied in vivo in two subcutaneous and one orthotopic rodent tumor xenograft models using µPET. The results show that 3-l- and 3-d-[18F]FPhe enable high-quality visualization of tumors with certain advantages over [18F]FET, making them promising candidates for further preclinical and clinical evaluations. Purpose: The preclinical evaluation of 3-l- and 3-d-[18F]FPhe in comparison to [18F]FET, an established tracer for tumor imaging. Methods: In vitro studies were conducted with MCF-7, PC-3, and U87 MG human tumor cell lines. In vivo µPET studies were conducted in healthy rats with/without the inhibition of peripheral aromatic l-amino acid decarboxylase by benserazide pretreatment (n = 3 each), in mice bearing subcutaneous MCF-7 or PC-3 tumor xenografts (n = 10), and in rats bearing orthotopic U87 MG tumor xenografts (n = 14). Tracer accumulation was quantified by SUVmax, SUVmean and tumor-to-brain ratios (TBrR). Results: The uptake of 3-l-[18F]FPhe in MCF-7 and PC-3 cells was significantly higher relative to [18F]FET. The uptake of all three tracers was significantly reduced by the suppression of amino acid transport systems L or ASC. 3-l-[18F]FPhe but not 3-d-[18F]FPhe exhibited protein incorporation. In benserazide-treated healthy rats, brain uptake after 42–120 min was significantly higher for 3-d-[18F]FPhe vs. 3-l-[18F]FPhe. [18F]FET showed significantly higher uptake into subcutaneous MCF-7 tumors (52–60 min p.i.), while early uptake into orthotopic U87 MG tumors was significantly higher for 3-l-[18F]FPhe (SUVmax: 3-l-[18F]FPhe, 107.6 ± 11.3; 3-d-[18F]FPhe, 86.0 ± 4.3; [18F]FET, 90.2 ± 7.7). Increased tumoral expression of LAT1 and ASCT2 was confirmed immunohistologically. Conclusion: Both novel tracers enable accurate tumor delineation with an imaging quality comparable to [18F]FET.