Concentrations Of Doxorubicin Research Articles

Pharmacokinetic-Pharmacodynamic Analysis of pH-Responsive Doxorubicin-Releasing Micelles with Anticancer Activity.

This study aimed to investigate the effect of in vivo pH-responsive doxorubicin (DOX) release and the targetability of pilot molecules in folic acid (FA)-modified micelles using a pharmacokinetic-pharmacodynamic (PK-PD) model. The time profiles of intratumoral DOX concentrations in Walker256 tumor-bearing rats were monitored using a microdialysis probe, followed by compartmental analysis, to evaluate intratumoral tissue pharmacokinetics. Maximal DOX was released from micelles 350 min after the administration of pH-responsive DOX-releasing micelles. However, FA modification of the micelles shortened the time to peak drug concentration to 150 min. Additionally, FA modification resulted in a 27-fold increase in the tumor inflow rate constant. Walker256 tumor-bearing rats were subsequently treated with DOX, pH-responsive DOX-releasing micelles, and pH-responsive DOX-releasing FA-modified micelles to monitor the tumor growth-time profiles. An intratumoral threshold concentration of DOX (55-64 ng/g tumor) was introduced into the drug efficacy compartment to construct a PD model, followed by PK-PD analysis of the tumor growth-time profiles. Similar results of threshold concentration and drug potency of DOX were obtained across all three formulations. Cell proliferation was delayed as the drug delivery ability of DOX was improved. The PK model, which was developed using the microdialysis method, revealed the intratumoral pH-responsive DOX distribution profiles. This facilitated the estimation of intratumoral PK parameters. The PD model with threshold concentrations contributed to the estimation of PD parameters in the three formulations, with consistent mechanisms observed. We believe that our PK-PD model can objectively assess the contributions of pH-responsive release ability and pilot molecule targetability to pharmacological effects.

DOX-DNA Interactions on the Nanoscale: In Situ Studies Using Tip-Enhanced Raman Scattering.

Chemotherapeutic anthracyclines, like doxorubicin (DOX), are drugs endowed with cytostatic activity and are widely used in antitumor therapy. Their molecular mechanism of action involves the formation of a stable anthracycline-DNA complex, which prevents cell division and results in cell death. It is known that elevated DOX concentrations induce DNA chain loops and overlaps. Here, for the first time, tip-enhanced Raman scattering was used to identify and localize intercalated DOX in isolated double-stranded calf thymus DNA, and the correlated near-field spectroscopic and morphologic experiments locate the DOX molecules in the DNA and provide further information regarding specific DOX-nucleobase interactions. Thus, the study provides a tool specifically for identifying intercalation markers and generally analyzing drug-DNA interactions. The structure of such complexes down to the molecular level provides mechanistic information about cytotoxicity and the development of potential anticancer drugs.

Sulfonated Azocalix[4]arene-Modified Metal-Organic Framework Nanosheets for Doxorubicin Removal from Serum.

Chemotherapy is one of the most commonly used methods for treating cancer, but its side effects severely limit its application and impair treatment effectiveness. Removing off-target chemotherapy drugs from the serum promptly through adsorption is the most direct approach to minimize their side effects. In this study, we synthesized a series of adsorption materials to remove the chemotherapy drug doxorubicin by modifying MOF nanosheets with sulfonated azocalix[4]arenes. The strong affinity of sulfonated azocalix[4]arenes for doxorubicin results in high adsorption strength (Langmuir adsorption constant = 2.45-5.73 L mg-1) and more complete removal of the drug. The extensive external surface area of the 2D nanosheets facilitates the exposure of a large number of accessible adsorption sites, which capture DOX molecules without internal diffusion, leading to a high adsorption rate (pseudo-second-order rate constant = 0.0058-0.0065 g mg-1 min-1). These adsorbents perform effectively in physiological environments and exhibit low cytotoxicity and good hemocompatibility. These features make them suitable for removing doxorubicin from serum during "drug capture" procedures. The optimal adsorbent can remove 91% of the clinical concentration of doxorubicin within 5 min.

Abstract PO4-28-01: Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC

Abstract Background: Chemotherapeutic resistance observed in triple-negative breast cancer (TNBC) poses a substantial clinical hurdle, emphasizing the imperative to enhance our comprehension of the underlying mechanisms with the aim of potentially mitigating or reversing this phenomenon. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were established through continuous exposure to increasing concentrations of doxorubicin. RNA-Seq was conducted using the Illumina platform, while bioinformatics analyses were performed using CLC genomics workbench 20.2 and iDEP.91. Colony forming unit (CFU), flow cytometry and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. Western blotting was used to confirm the expression and phosphorylation of protein targets. Ingenuity pathway analysis (IPA) and STRING database v 11.5 were used for network and pathway analyses, while Kaplan-Meier Plotter was used for survival analyses. Results: Herein, we provide a comprehensive functional and transcriptomic characterization of DoxR TNBC models, revealing multiple affected signaling networks and functional categories. The defense and immune response, response to stress, response to cytokine and external stimulus and cytokine-mediated signaling pathways were among the top activated, while suppression of cholesterol, sterol and lipid biosynthesis and metabolism were the hallmarks of DoxR cells. Upstream regulator analysis revealed IL-1B cytokine network activation in DoxR cells. Concordantly, disease and function analysis on the downregulated genes in DoxR TNBC cells revealed the most pronounced enrichment in pathways associated with cell invasion, colony formation, as well as lipid and carbohydrate metabolism. A number of upregulated genes were validated by qRT-PCR, which correlated with unfavorable overall survival (OS). Functionally, DoxR cells exhibited remarkable suppression of cell proliferation, as evidenced by diminished CFU potential, sphere formation and growth under 3D organoid culture, and cell migration. Although DoxR TNBC cells exhibited a slow cell cycling under normal culture conditions, wild-type TNBC cells exhibited remarkable arrest in the G2M phase when exposed to doxorubicin, which was less evident in DoxR TNBC models, suggesting DoxR cells undergo a quiescent state in response to chemotherapeutic challenge. In agreement with those data, increased phosphorylation of CHK2 and p53 proteins was remarkable in DoxR cells, thus promoting cellular dormancy. Conclusion: Our data revealed cellular dormancy and suppression of lipid metabolism as the hallmark associated with doxorubicin-resistant TNBC cells. On the other hand, cytokine signaling and stress response were the most enriched functional categories in DoxR TNBC cells. Therapeutic targeting to reverse the quiescent state or suppression of cytokine signaling networks could enhance the efficacy of doxorubicin-based therapeutics. Citation Format: Vishnubalaji Radhakrishnan, Nehad Alajez. Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC [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 PO4-28-01.

The Potential of the Fibronectin Inhibitor Arg-Gly-Asp-Ser in the Development of Therapies for Glioblastoma.

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. An important feature that supports GBM aggressiveness is the unique composition of its extracellular matrix (ECM). Particularly, fibronectin plays an important role in cancer cell adhesion, differentiation, proliferation, and chemoresistance. Thus, herein, a hydrogel with mechanical properties compatible with the brain and the ability to disrupt the dynamic and reciprocal interaction between fibronectin and tumor cells was produced. High-molecular-weight hyaluronic acid (HMW-HA) functionalized with the inhibitory fibronectin peptide Arg-Gly-Asp-Ser (RGDS) was used to produce the polymeric matrix. Liposomes encapsulating doxorubicin (DOX) were also included in the hydrogel to kill GBM cells. The resulting hydrogel containing liposomes with therapeutic DOX concentrations presented rheological properties like a healthy brain. In vitro assays demonstrated that unmodified HMW-HA hydrogels only caused GBM cell killing after DOX incorporation. Conversely, RGDS-functionalized hydrogels displayed per se cytotoxicity. As GBM cells produce several proteolytic enzymes capable of disrupting the peptide-HA bond, we selected MMP-2 to illustrate this phenomenon. Therefore, RGDS internalization can induce GBM cell apoptosis. Importantly, RGDS-functionalized hydrogel incorporating DOX efficiently damaged GBM cells without affecting astrocyte viability, proving its safety. Overall, the results demonstrate the potential of the RGDS-functionalized hydrogel to develop safe and effective GBM treatments.

GMI-based biosensor for the detection and quantification of doxorubicin anticancer drugs labeled to Fe3O4 superparamagnetic nanoparticles

We present the results of research on a Co-based giant magnetoimpedance (GMI) biosensor for Fe3O4-labelled doxorubicin (DOX) anticancer drugs. Co62Fe5Ni4Si15B14 ribbons were used as biosensing material for test solutions containing different concentrations of DOX. The plain ribbon, ribbon+Fe3O4 and ribbon+DOX2 samples exhibited low coercivity (Hc), high saturation magnetization (Ms) and low anisotropy (Hk). The GMI ratio (ΔZ/Z) increased from 33% to 38% with the increase of doxorubicin content in the ribbon. It was found that the ΔZ/Z ratio first increased rapidly up to a frequency of ∼2 MHz, and then decreased slowly as the frequency increased. While the magnetic field dependent ΔZ/Z has a single peak at 1 MHz, it has a double peak at 5 MHz. It was found that the peak height of ΔZ/Z in the ribbons with test solution of 20 ng/ml and 50 ng/ml increased by ∼3% and ∼6%, respectively, compared to the ribbon without test solution. Then the ΔZ/Z ratio decreased very rapidly with the increase of the applied external magnetic fields. The frequency-dependent GMI sensitivity (η) increased with increasing frequency and reached a maximum value at a critical frequency (∼1.6 MHz), after which it decreased. The detection sensitivity of the GMI biosensor (ξ) increased rapidly with increasing doxorubicin concentration up to ∼80 ng/ml and then remained almost unchanged. The results show that doxorubicin (DOX+Fe3O4), used as an anticancer drug, can be effectively detected even at low concentrations using the GMI based sensors fabricated in this study.

Development of a biophotonic fiber sensor using direct-taper and anti-taper techniques with seven-core and four-core fiber for the detection of doxorubicin in cancer treatment

Doxorubicin (DOX) is an important drug for cancer treatment, but its clinical application is limited due to its toxicity and side effects. Therefore, detecting the concentration of DOX during treatment is crucial for enhancing efficacy and reducing side effects. In this study, the authors developed a biophotonic fiber sensor based on localized surface plasmon resonance (LSPR) with the multimode fiber (MMF)-four core fiber (FCF)-seven core fiber (SCF)-MMF-based direct-taper and anti-taper structures for the specific detection of DOX. Compared to other detection methods, it has the advantages of high sensitivity, low cost, and strong anti-interference ability. In this experiment, multi-walled carbon nanotubes (MWCNTs), cerium-oxide nanorods (CeO2-NRs), and gold nanoparticles (AuNPs) were immobilized on the probe surface to enhance the sensor's biocompatibility. MWCNTs and CeO2-NRs provided more binding sites for the fixation of AuNPs. By immobilizing AuNPs on the surface, the LSPR was stimulated by the evanescent field to detect DOX. The sensor surface was functionalized with DOX aptamers for specific detection, enhancing its specificity. The experiments demonstrated that within a linear detection range of 0-10 µM, the sensitivity of the sensor is 0.77 nm/µM, and the limit of detection (LoD) is 0.42 µM. Additionally, the probe's repeatability, reproducibility, stability, and selectivity were evaluated, indicating that the probe has high potential for detecting DOX during cancer treatment.

Enhancement of doxorubicin production in Streptomyces peucetius by genetic engineering and process optimization

Doxorubicin is an important class of anthracycline antitumor antibiotics produced by Streptomyces peucetius. The doxorubicin fermentation yield of the wild-type strain was very low, so it could not be produced directly by fermentation at an industrial scale due to the high cost. In the present study, S. peucetius SIPI-7-14 was obtained from SIPI-14 through several rounds of doxorubicin resistance screening. Then, the ketoreductase gene dnrU was knocked out to reduce (13S)-13-dihydrodaunorubicin production, and the resistance gene drrC was overexpressed to further enhance resistance to doxorubicin. The resulting engineered strain S. peucetius △U1/drrC produced 1128 mg/L doxorubicin, a 102.1% increase compared to that of SIPI-14. Then, fermentation medium was optimized using the response surface method. In the optimized fermentation medium, the yield of doxorubicin was increased to 1406 mg/L in shake flask on the 7th day. Furthermore, batch culture was carried out in a 10 L fermenter, and the concentration of doxorubicin reached 1461 mg/L after 7 days of culture, which was the highest yield reported to date, indicating the potential for industrial production of doxorubicin by fermentation.

In vitro evaluation of doxorubicin release from diopside particles on MG-63 and HF spheroids as a 3D model of tumor and healthy tissues

Local drug delivery systems based on bioceramics ensure safe and effective treatment of bone defects and anticancer therapy. A promising drug delivery scaffold material for bone treatment applications is diopside (CaMgSi2O6) which is bioactive, degradable, and possesses drug-release ability. Currently, in vitro assessment of drug release from biomaterials is performed mostly on a 2D cell monolayer. However, to interpret and integrate biochemical signals, cells need a 3D microenvironment that provides cell-cell and cell-extracellular matrix interactions. In this regard, 3D cell models are gaining popularity. In this work, we proposed the protocol for evaluation of the effect of doxorubicin released from diopside on MG-63 cells and primary human fibroblasts in 3D culture conditions. Tissue spheroids with similar diameters were incubated with doxorubicin-loaded diopside for 72 h, the amount of diopside was calculated in accordance with the required doxorubicin concentration. We demonstrated that doxorubicin is gradually released from diopside and exhibits an activity similar to that of the pure drug at the same total concentration. It is important to note that doxorubicin was more potent on MG-63 spheroids compared to HF spheroids, which confirmed the reliability of spheroids as 3D models of tumor and healthy tissues.

Combination of Doxorubicin and Antiangiogenic Agents in Drug-Eluting Beads: In Vitro Loading and Release Dynamics in View of a Novel Therapeutic Approach for Hepatocellular Carcinoma.

Antiangiogenic agents have been used for many years as a first-line systemic treatment for advanced HCC. Embolization with cytostatic drugs on the other hand is the first-line treatment for intermediate HCC. The two types of drugs have not been combined for intraarterial delivery yet. The loading and release dynamics and the in vitro effect of their combination are tested in this experimental study. Drug-eluting beads were loaded with doxorubicin, sunitinib and sunitinib analogue piperazine (SAP) alone and with their combinations. Diameter change, loading, release, and effect in cellular proliferation were assessed. The average microsphere diameter after loading was 473.7µm (μm) for Doxorubicin, 388.4μm for Sunitinib, 515.5μm for SAP, 414.8μm for the combination Doxorubicin/Sunitinib and 468.8μm for the combination Doxorubicin /SAP. Drug release in 0.9%NaCl was 10% for Doxorubicin, 49% for Sunitinib, 25% for SAP, 20%/18% for the combination Doxorubicin/Sunitinib, and 18%/23% for the combination Doxorubicin/SAP whereas in human plasma it was 56%, 27%, 13%, 76%/63% and 62%/15%, respectively. The mean concentration of Doxorubicin that led to inhibition of 50% of cellular proliferation in an HCC Huh7 cell line was 163.1nM (nM), for Sunitinib 10.3 micromolar (μΜ), for SAP 16.7μΜ, for Doxorubicin/Sunitinib 222.4nM and for Doxorubicin/SAP 275nM. Doxorubicin may be combined with antiangiogenic drugs with satisfactory in vitro loading and release outcomes and effect on cellular lines.

SiRNA-Mediated Knockdown of ABCB1 Enhances the Efficacy of Doxorubicin and Vinorelbine in Breast Cancer Cells.

Breast cancer remains a leading cause of cancer-related deaths among women, primarily attributed to the formidable challenge of multidrug resistance, often driven by the overexpression of the ABCB1 gene. This study aimed to assess the synergistic effects of siRNA, doxorubicin, and vinorelbine on ABCB1 gene expression and cell viability in doxorubicin-resistant MCF-7/ADR breast cancer cells, with siRNA targeting ABCB1 to reduce its expression and doxorubicin/ vinorelbine to eradicate cancer cells. Our methodology involved culturing MCF-7 and MCF-7/ADR cells in standard cell culture conditions. The synthesized siRNA sequences transfected cells with siRNA at final concentrations of 10, 20, and 30 nM and assessed cell viability using the MTT assay was performed. Real-time PCR was employed to quantify ABCB1 mRNA expression levels. Results indicated that MCF-7/ADR cells exhibited substantial resistance to vinorelbine and doxorubicin compared to MCF-7 cells, displaying resistance at 12.50 μM and 25.00 μM for vinorelbine and 6.25 μM and 25.00 μM for doxorubicin. Remarkably, siRNA treatment effectively reversed drug resistance in MCF-7/ADR cells across all concentrations of vinorelbine and doxorubicin tested. When combined, siRNA, doxorubicin, and vinorelbine yielded a significantly greater reduction in cell viability compared to individual drug treatments, particularly at a 20 μM siRNA concentration. This combination therapy also significantly suppressed ABCB1 gene expression by a factor of 41.48 in MCF-7 cells relative to MCF-7/ADR cells. these findings suggest that combining siRNA, doxorubicin, and vinorelbine holds promise as a therapeutic strategy to overcome ABCB1-mediated multidrug resistance in breast cancer. Further investigations and clinical trials are warranted to evaluate its clinical efficacy rigorously.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Abstract CT188: A Phase I trial of AVA6000, a Fibroblast Activation Protein (FAP)-released and tumor microenvironment (TME)-targeted doxorubicin peptide drug conjugate in patients with FAP-positive solid tumors

Abstract Background: AVA6000 is a peptide drug conjugate, consisting of a peptide moiety that is specifically cleaved by FAP in the TME that is bound to doxorubicin. FAP is selectively overexpressed in many solid tumors. The peptide moiety linker (pre|CISIONTM) prevents cellular entry of doxorubicin unless cleaved by FAP, thus enabling targeted delivery of doxorubicin to tumors. Design: The safety, PK and preliminary efficacy of AVA6000 was studied in a first-in-man, multicenter dose escalation phase I trial. AVA6000 was administered intravenously q3w using a standard 3+3 design in patients with locally advanced or metastatic solid tumors reported to be FAP-positive. PK (AUC) based dosing was implemented to calculate maximum cycles of AVA6000 with the cumulative limit of 550 mg/m2. Results: Forty patients (median age 65 years, range 30-79), with median 3 prior lines of therapy (range, 0-7) received escalating AVA6000 doses from 80mg/m2 (51 mg/m2 doxorubicin; molar equivalent, 0.675) to 385 mg/m2 (260 mg/m2 doxorubicin equivalent). Tumor types included soft tissue sarcoma (30%), colorectal cancer (27.5%), pancreatic cancer (20%), cancers of the biliary tract (7.5%). The safety profile was favorable, the most frequent adverse events (any grade) being fatigue (50%), alopecia (42.5%), and nausea (32.5%), with rare grade 3-4 hematologic toxicities of neutropenia (7.5%), anemia, thrombocytopenia, and WBC decreased (5% each). Grade 3 non-hematologic toxicities included mucositis, fatigue and hematemesis (n=1 each). No grade 4 non-hematologic toxicities were reported. Two DLTs were observed of grade 2 cardiac failure (120 mg/m2; LVEF decrease 61 to 39%) and grade 4 neutropenia/thrombocytopenia (200 mg/m2). AVA6000 distributes rapidly with a t1/2 of 45 min. The Cmax of released doxorubicin was reduced as compared to standard dose doxorubicin (range 78-93% reduction) across dose cohorts. Tumor biopsy data demonstrate concentration of doxorubicin in the TME of mean 860 ng/gm (range 76-2310 ng/gm, n=9). In contrast, blood samples taken with the biopsy demonstrate a circulating free doxorubicin of 8.3 ng/ml (range 2.4-15.9), indicating concentration of doxorubicin in the tumor relative to plasma. Using RECISTv.1.1, one PR (-65%) with duration of 6 months was observed in a patient with undifferentiated pleomorphic sarcoma at 160 mg/m2, and a mixed response in a patient with angiosarcoma (SLD -22%) at 200 mg/m2. The disease control rate was 50%, at 12 wks. In each case, SD > 4 months or PR was associated with high FAP enzyme activity in the on-study tumor biopsy compared to observed disease progression as best response (n=6). Conclusions: AVA6000 delivers high concentration of doxorubicin to the TME relative to plasma which results in antitumor activity in tumors with high FAP activity. A q2w dose escalation arm is ongoing. Citation Format: Udai Banerji, Natalie Cook, Alan Anthoney, Ruth Plummer, William D. Tap, Jeffry T. Evans, Lee D. Cranmer, Christopher Plummer, Paul Loadman, Gezim Lahu, Huw S. Jones, Nelson Kinnersley, Fiona McLaughlin, Chris Twelves. A Phase I trial of AVA6000, a Fibroblast Activation Protein (FAP)-released and tumor microenvironment (TME)-targeted doxorubicin peptide drug conjugate in patients with FAP-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT188.

Blood-Brain Barrier Opening with Ultrasound Augments Doxorubicin and Anti-PD-1 Therapy Efficacy, Intracranial Immune Activation, and Prolongs Survival in Preclinical Glioma Models

INTRODUCTION: The limited drug penetration across the blood-brain barrier (BBB) represents a challenge for the treatment of glioblastoma patients. A novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) overcomes this challenge by opening the BBB. In this study, we hypothesize that LIPU/MB enhances the therapeutic effect of both liposomal doxorubicin and anti-PD-1 therapy in gliomas by eliciting local immune responses. METHODS: We utilized LIPU/MB to deliver doxorubicin and anti-PD-1 blocking antibodies to murine models and evaluated drug concentrations in the brain. Flow cytometry evaluated IFN-g production by glioma-associated microglia and macrophages in murine gliomas treated with doxorubicin and anti-PD-1 therapy delivered by LIPU/MB. Furthermore, we assessed the efficacy of this drug combination delivered by LIPU/MB in glioma-bearing mice. RESULTS: LIPU/MB induced a 4-fold increase in doxorubicin concentrations in sonicated brains compared to those not sonicated (p = 0.003). In addition, doxorubicin delivered with LIPU/MB elicited an IFN-g phenotype (p < 0.0001) as well as MHC I and PD-L1 expression in glioma-associated microglia and macrophages (p < 0.05). Increased brain concentrations of doxorubicin led to long-term survivors in the majority of glioma-bearing mice. On the other hand, LIPU/MB-mediated BBB opening increased brain concentrations of anti-PD-1 therapy in mice (6.3-fold increase, p < 0.01). The combination of doxorubicin and anti-PD-1 therapy delivered with LIPU/MB resulted in long-term survival (p < 0.0001). This effect was abrogated in CD8-/- mice suggesting that T cells are important for the efficacy of doxorubicin and anti-PD-1 therapy delivered with LIPU/MB. CONCLUSIONS: This preclinical study demonstrates the immense potential of LIPU/MB technology in enhancing intracranial immune responses and drug delivery for glioblastoma treatment using doxorubicin and anti-PD-1 therapy, paving the way for more effective treatment strategies for this brain cancer.

The fabrication method impacts biological properties of microfibrous three-dimensional in vitro cell culture scaffolds and their applications in drug screening

Two-dimensional (2D) cell culture models cannot accurately emulate the three-dimensional (3D) in vivo microenvironment; hence, the shift from compound screening in traditional 2D well plates to 3D cell culture models is forthcoming. New scaffold designs must comply with the 3Rs principle to reduce the use of animal-derived products, such as natural proteins. In line with this motivation, we engineered and compared various fibrous scaffolds featuring diverse fibre networks (coarse and fine fibres) and fibre surface properties (porous vs. smooth fibres), and tested them in in vitro cell culture. We fabricated coarse solid fibre scaffolds via a melt-based electrohydrodynamic process, which resulted in fibres with a diameter of 10.6 ± 1.9 µm and a porosity of 87.1 ± 0.9%. Using solution-based cryo-electrohydrodynamic process, we fabricated fine porous fibre and fine solid fibre scaffolds that exhibited fibres of 2.01 ± 0.5 µm and 1.72 ± 0.6 µm in diameter, with porosities of 96.5 ± 0.3% and 94.4 ± 0.5%, respectively. The scaffolds underwent only chemical modification via an alkaline treatment to enhance their hydrophilicity. We used mouse fibroblast L929 cells and human triple-negative breast cancer MDA-MB-231 cells as cell models, and used the MTT assay to measure their viability. After seven days of cell culture, all scaffolds were thoroughly penetrated. However, the coarse scaffolds created by the melt-based electrohydrodynamic process and the porous fibre scaffolds produced by the solution-based cryo-electrohydrodynamic process outperformed the simpler morphology of smooth fine fibres. Furthermore, the 50% half-maximum effective concentration (EC50) of doxorubicin tested in human glioblastoma (U87-MG) and human triple-negative breast cancer (MDA-MB-231) cell lines in 3D cell culture models was approximately twice as high as that achieved in conventional 2D cell cultures. Therefore, we infer that poly (ɛ-caprolactone) (PCL) microfibre scaffolds, devoid of animal-derived product surface treatment, may provide a favourable 3D milieu for various cell types for in vitro studies.

Abstract 7224: Investigation of the efficacy of duocarmycin SA in combination with FDA approved drugs in acute myeloid leukemia cells in vitro

Abstract Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by the arrest of hematopoietic stem cell differentiation, clonal expansion of myeloid hematopoietic precursors and poor patient survival outcomes. Despite therapeutic advancements, the prognosis of AML patients remains poor and improving patient survival outcomes continues to be a challenge. Therefore, the objective of this study was to investigate the efficacy of Duocarmycin SA (DSA- an antitumor antibiotic that induces DNA alkylation) in combination with FDA approved drugs currently used in the treatment of AML. In this study, DSA was evaluated in combination with doxorubicin (an anthracycline that disrupts topoisomerase II mediated DNA repair) or etoposide (a topoisomerase II inhibitor used to treat relapsed/refractory AML) in human AML cells in vitro. We hypothesized that DSA in combination with doxorubicin or etoposide would exhibit synergistic effects by improving the efficacy of both doxorubicin and etoposide in AML cells. The human AML cell lines (Molm-14 and HL60) were used for all studies. The phenotype of the cells was confirmed by flow cytometry and the IC50 of the drugs were determined using the MTT assay. Our results showed that the AML cells were CD45+, CD33+, CD13+ and CD4+. The IC50 of DSA alone for Molm-14 cells and HL60 cells were 11.12 pM and 114.8 pM, respectively. DSA alone induced DNA double-stranded breaks, significantly decreased the production of colonies and significantly increased apoptosis in AML cells in a dose-dependent manner. The IC50 of doxorubicin alone and etoposide alone was 68.6 nM and 129.7 nM, respectively. DSA in combination with the FDA approved AML drugs showed increased cytotoxic efficacy by observable decreases in AML cell viability. In summary, 1) picomolar concentrations of DSA alone was sufficient to significantly reduce AML cell viability; 2) DSA in combination with doxorubicin or etoposide significantly reduced AML cell viability and reduced the IC50 concentration of doxorubicin and etoposide. Thus, highlighting the therapeutic potential of using DSA in combination with doxorubicin or etoposide as a drug combination strategy to improve AML patient survival outcomes by decreasing the concentration of drugs necessary to treat patients and reducing off-target toxicity. Citation Format: William A. Chen, Diego Aguilar, Leena So, Yasmeen Jawhar, Nhi Nguyen, Natalie Drew, Terry G. Williams, Carlos A. Casiano, Sinisa Dovat, Kristopher Boyle, Olivia L. Francis-Boyle. Investigation of the efficacy of duocarmycin SA in combination with FDA approved drugs in acute myeloid leukemia cells in vitro [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 7224.

Abstract 4762: Tumor treating fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin in vitro and in vivo

Abstract Background: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. While the mechanism of action of TTFields has been associated with an antimitotic effect and impaired DNA damage repair mechanisms, the application of TTFields has also been shown to increase the membrane permeability of glioblastoma cells. The current study examined whether increased cell membrane permeability is relevant in other tumor types and may be leveraged to facilitate uptake of the anticancer agent doxorubicin (DOX). Methods: Breast mammary carcinoma (4T1), breast adenocarcinoma (MCF-7), uterine sarcoma (MES-SA), lung fibroblasts (MRC-5), and brain endothelial (HBMVEC) cells were treated with TTFields using the inovitroTM system. Intracellular 7-aminoactinomycin D (7-AAD) accumulation and cell count were measured using flow cytometry to determine membrane permeability and cytotoxicity, respectively. TTFields (300 kHz) together with DOX were applied to 4T1 cells DOX-sensitive (4T1-S) and DOX-resistant (4T1-R, generated by repeated DOX exposure of the 4T1-S cells), followed by flow cytometry analysis of DOX accumulation (24 h treatment) and cell survival using cell count (72 h treatment). For proof of concept, mice were orthotopically inoculated with 4T1-S cells, followed by TTFields treatment for 72 h and injection of DOX (5 mg/kg) 24 h before ending TTFields treatment. DOX fluorescence was measured by flow cytometry in single-cell tumor suspension and whole tumor in vivo imaging system (IVIS). For the efficacy studies, mice were orthotopically inoculated with 4T1-S or 4T1-R cells. Tumors were allowed to grow, and then the mice were treated with TTFields (300 kHz) or sham-heat continuously for 8 days, with DOX (1 mg/kg for 4T1-S cells; 5 mg/kg for 4T1-R cells) or vehicle 2- and 6-days following treatment initiation. Tumor volumes were measured using caliper. Results: TTFields increased 7-AAD accumulation in cancer but not normal cells. Maximal TTFields-induced cellular permeability was identified at 300 kHz for 4T1 cells, whereas TTFields-induced cytotoxicity was the highest at 150 kHz. The application of TTFields allowed DOX accumulation to the same extent in both DOX-resistant and DOX-sensitive cells and sensitized both cell types to DOX cytotoxicity. A 2- to 3-fold higher DOX accumulation was observed in tumors isolated from mice treated with TTFields relative to control mice. Significantly lower tumor volume was observed in mice treated with TTFields and DOX relative to control or mice treated with DOX alone. Conclusions: TTFields increased cancer cell permeability, resulting in enhanced intracellular accumulation of DOX. Concomitant treatment with TTFields and DOX can improve treatment efficacy in DOX-resistant tumors and sensitize DOX-sensitive tumors to lower DOX concentrations. Citation Format: Bella Koltun, Tali Voloshin, Tal Kan, Cfir David, Lilach Koren, Yaara Porat, Alexandra Volodin, Noa Kaynan, Anat Klein-Goldberg, Rom Paz, Boris Brant, Yiftah Barsheshet, Efrat Zemer-Tov, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin 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 4762.

Evaluation of mAb 2C5-modified dendrimer-based micelles for the co-delivery of siRNA and chemotherapeutic drug in xenograft mice model.

Combination therapy with small interfering RNA (siRNA) and chemotherapeutic drug is proven to be effective in downregulating cancer resistance proteins, such as P-glycoprotein (P-gp). These proteins are involved in multidrug resistance (MDR) of tumors. A targeted formulation capable of delivering siRNA and chemotherapeutic drug will not only downregulate P-gp but also increase the concentration of the chemotherapeutic drug at the site of tumor thereby increasing the therapeutic effect and lowering the systemic exposure. In this study, monoclonal antibody 2C5-modified dendrimer-based micelles were used to co-deliver siRNA and doxorubicin (DOX) to the tumor site in both male and female xenograft mouse model. The nucleosome-specific 2C5 antibody recognizes the cancer cells via the cell-surface bound nucleosomes. The ability of ability of the 2C5-modified formulation to affect the metastasis of highly aggressive triple negative breast cancer cell migration in (MDA-MB-231) was assessed by a wound healing. Further, the therapeutic efficacy of the formulation was assessed by measuring the tumor volume progression in which the 2C5-modified nanoparticle group had a similar tumor volume to the free drug group at the end of the study, although a 50% increase in DOX concentrations in blood was observed after the last dose of nanoparticle. The free drug group on the other hand showed body weight reduction as well as the visible irritation around the injection spot. The treatment group with 2C5-modified micelles has shown to be safe at the current dose of DOX and siRNA. Furthermore, the siRNA mediated P-gp downregualtion was studied using western blotting assay. We observed a 29% reduction of P-gp levels in both males and females with respect to the control (BHG). We also conclude that the dose of DOX and siRNA should be further optimized to have a better efficacy in a metastatic tumor model, which will be the subject of our future studies.

Effect of Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells in 3D Culture.

Chemotherapy is one of the most common strategies for cancer treatment, whereas drug resistance reduces the efficiency of chemotherapy and leads to treatment failure. The mechanism of emerging chemoresistance is complex and the effect of extracellular matrix (ECM) surrounding cells may contribute to drug resistance. Although it is well known that ECM plays an important role in orchestrating cell functions, it remains exclusive how ECM stiffness affects drug resistance. In this study, we prepared agarose hydrogels of different stiffnesses to investigate the effect of hydrogel stiffness on the chemoresistance of breast cancer cells to doxorubicin (DOX). Agarose hydrogels with a stiffness range of 1.5 kPa to 112.3 kPa were prepared and used to encapsulate breast cancer cells for a three-dimensional culture with different concentrations of DOX. The viability of the cells cultured in the hydrogels was dependent on both DOX concentration and hydrogel stiffness. Cell viability decreased with DOX concentration when the cells were cultured in the same stiffness hydrogels. When DOX concentration was the same, breast cancer cells showed higher viability in high-stiffness hydrogels than they did in low-stiffness hydrogels. Furthermore, the expression of P-glycoprotein mRNA in high-stiffness hydrogels was higher than that in low-stiffness hydrogels. The results suggested that hydrogel stiffness could affect the resistance of breast cancer cells to DOX by regulating the expression of chemoresistance-related genes.

Effects of pharmacogenetics on pharmacokinetics and toxicity of doxorubicin in Egyptian breast cancer patients

This study investigates the impact of single nucleotide polymorphisms in genes (SLC22A16 and CBR1) involved in the pharmacokinetics and toxicity of doxorubicin (DOX) in Egyptian female patients with breast cancer. Patients administered DOX (60 mg/m2) for 4 cycles every 3 weeks. The peak DOX plasma concentration was measured using a validated chromatographic method. The genotyping for the selected SNPs, SLC22A16 T > C (rs714368), and CBR1 C > T (rs20572), was performed by RT-PCR. Patients were monitored for hematological and cardiac toxicities. The variant carriers of CBR1 C > T (rs20572) exhibited significantly higher DOX concentration, but no significant association to DOX-induced hematological toxicity. On the other hand, SLC22A16 T > C (rs714368) had no significant influence on DOX plasma concentration, but was significantly correlated with lower risk of neutropenia (OR 0.31, 95% CI 0.12–0.75, p = 0.01) and leukopoenia (OR 0.18, 95% CI 0.07–0.5, p = 0.001). DOX-related cardiotoxicity was correlated with the cumulative dose of DOX (R = 0.238, p = 0.017), but not with any of the two examined SNPs. Genetic polymorphisms in SLC22A16 and CBR1 may explain the inter-individual variations in DOX pharmacokinetics and toxicity. Using pharmacogenetic testing is important to customise drug therapy for cancer patients treated with anthracyclines.