Breast Cancer Cell Models Research Articles

An Electrochemical Biosensor Analysis of the Interaction of a Two-Vector Phospholipid Composition of Doxorubicin with dsDNA and Breast Cancer Cell Models In Vitro

Objectives: The main aim of our experiments was to demonstrate the suitability of cell-based biosensors for searching for new anticancer medicinal preparations. Methods: The effect of the substance doxorubicin, doxorubicin embedded in phospholipid nanoparticles, and doxorubicin with phospholipid nanoparticles modified by targeting vectors (cRGD and folic acid) on dsDNA and breast cancer cell lines (MCF-7, MDA-MB-231) was studied. Results: In the obtained doxorubicin nanoforms, the particle size was less than 60 nm. Our study of the percentage of doxorubicin inclusion showed the almost complete embeddability of the substance into nanoparticles for all samples, with an average of 95.4 ± 4.6%. The calculation of the toxicity index of the studied doxorubicin samples showed that all substances were moderately toxic drugs in terms of adenine and guanine. The biosensor analysis using electrodes modified with carbon nanotubes showed an intercalation interaction between doxorubicin and its derivatives and dsDNA, except for the composition of doxorubicin with folic acid with a linker length of 2000 (NPh-Dox-Fol(2.0)). The results of the electroanalysis were normalized to the total cell protein (mg) and cell concentration. The highest intensity of the electrochemical signals was observed in intact control cells of the MCF-7 and MDA-MB-231 cell lines. Conclusions: The proposed electrochemical approach is useful for the analysis of cell line responses to the medicinal preparations.

SRSF6 modulates histone-chaperone HIRA splicing to orchestrate AR and E2F activity in prostate cancer.

Despite novel therapeutic strategies, advanced-stage prostate cancer (PCa) remains highly lethal, pointing out the urgent need for effective therapeutic strategies. While dysregulation of the splicing process is considered a cancer hallmark, the role of certain splicing factors remains unknown in PCa. This study focuses on characterizing the levels and role of SRSF6 in this disease. Comprehensive analyses of SRSF6 alterations (copy number/mRNA/protein) were conducted across eight well-characterized PCa cohorts and the Hi-MYC transgenic model. SRSF6 was up-regulated in PCa samples, correlating with adverse clinical parameters. Functional assays, both in vitro (cell proliferation, migration, colony, and tumorsphere formation) and in vivo (xenograft tumors), demonstrated the impact of SRSF6 modulation on critical cancer hallmarks. Mechanistically, SRSF6 regulates the splicing pattern of the histone-chaperone HIRA, consequently affecting the activity of H3.3 in PCa and breast cancer cell models and disrupting pivotal oncogenic pathways (AR and E2F) in PCa cells. These findings underscore SRSF6 as a promising therapeutic target for PCa/advanced-stage PCa.

Integration of scHi-C and scRNA-seq data defines distinct 3D-regulated and biological-context dependent cell subpopulations

An integration of 3D chromatin structure and gene expression at single-cell resolution has yet been demonstrated. Here, we develop a computational method, a multiomic data integration (MUDI) algorithm, which integrates scHi-C and scRNA-seq data to precisely define the 3D-regulated and biological-context dependent cell subpopulations or topologically integrated subpopulations (TISPs). We demonstrate its algorithmic utility on the publicly available and newly generated scHi-C and scRNA-seq data. We then test and apply MUDI in a breast cancer cell model system to demonstrate its biological-context dependent utility. We find the newly defined topologically conserved associating domain (CAD) is the characteristic single-cell 3D chromatin structure and better characterizes chromatin domains in single-cell resolution. We further identify 20 TISPs uniquely characterizing 3D-regulated breast cancer cellular states. We reveal two of TISPs are remarkably resemble to high cycling breast cancer persister cells and chromatin modifying enzymes might be functional regulators to drive the alteration of the 3D chromatin structures. Our comprehensive integration of scHi-C and scRNA-seq data in cancer cells at single-cell resolution provides mechanistic insights into 3D-regulated heterogeneity of developing drug-tolerant cancer cells.

Positron Emission Tomography‐Assisted Photothermal Therapy with Gold Nanorods

AbstractPhotothermal anticancer therapy based on plasmonic nanoparticles is proposed to enhance treatment efficacy while mitigating unintended side effects. However, most studies blindly rely on the accumulation of nanoparticles at the tumor site, which may result in inefficient treatment. In this study, the aim is to evaluate relevant parameters to improve plasmonic photothermal therapy. Gold nanorods (AuNRs) with an optimized aspect ratio and either amino or carboxylic acid surface functionalization are selected as photothermal agents. AuNR biocompatibility and photothermal activity in 2D and 3D human MDA‐MB‐231 triple‐negative breast cancer cell models, evaluating localized hyperthermal cell death upon irradiation with resonant near‐infrared (NIR) light, are analyzed first. To ensure reliable tracking of biodistribution in vivo, AuNRs are labeled with the positron emitter copper‐64 (64Cu), and their distribution in a murine MDA‐MB‐231 tumor model is studied via positron emission tomography (PET) imaging. PET images reveal enhanced tumor accumulation of carboxylic acid‐functionalized AuNRs compared to amino‐functionalized AuNRs post‐intravenous administration. Relatively low NIR laser power densities (0.5 W cm−2) are used for controlled heating – keeping local temperature below 50 °C – upon irradiation of intravenously and intratumorally administered AuNRs. As a result, tumor growth is significantly decelerated, even 9 days after application of photothermal therapy.

Reversal of the tamoxifen‑resistant breast cancer malignant phenotype by proliferation inhibition with bromosulfonamidine amino‑podophyllotoxin.

One of the lignans isolated from plants within the genus Podophyllum is podophyllotoxin (PPT). PPT and its derivatives are pharmacologically active compounds with potential antiproliferative properties in several kinds of tumors. Although these compounds have been used to treat other malignancies, no PPT derivative-based chemotherapeutic agent has been used to cure tamoxifen (TAM)-resistant breast cancer in clinical trials, to the best of our knowledge. Thus, using TAM-resistant breast cancer as a disease model, the present study assessed the effects of a recently synthesized PPT derivative, bromosulfonamidine amino-PPT (BSAPPT), on TAM-resistant breast cancer. Using the tamoxifen-resistant breast cancer cell model (MCF-7/TAMR) in vitro, Cell Counting Kit-8 and colony formation assays were adopted to evaluate the effect of BSAPPT on cell proliferation. Cell apoptosis and cell cycle assays were used to assess the influence of BSAPPT on cell apoptosis and the cell cycle in MCF-7/TAMR. The targets of the potential mechanism of action were analyzed by RT-qPCR and western blotting. The present study demonstrated that BSAPPT suppressed MCF-7/TAMR cell proliferation in a dose-dependent manner. By modulating the level of expression of genes linked to both apoptosis and the cell cycle, BSAPPT triggered MCF-7/TAMR cells to undergo apoptosis and prevented them from entering the cell cycle. Consequently, BSAPPT blocked these cells from proliferating, thereby halting the malignant advancement of TAM-resistant breast cancer. Therefore, these findings indicate that new therapeutic agents involving BSAPPT may be developed to facilitate the treatment of TAM-resistant breast cancer.

Enhancing Chemotherapy Efficacy: Investigating the Synergistic Impact of Paclitaxel and cd73 Gene Suppression on Breast Cancer Cell Proliferation and Migration.

Background Enhancing chemotherapy efficacy is crucial in breast cancer treatment. This study examines the synergistic effects of paclitaxel, a common chemotherapeutic drug, andCluster of differentiation 73(cd73) gene suppression via siRNA on MDA-MB-231 breast cancer cells. Methods MDA-MB-231 cells were transfected with CD73 siRNA and treated with paclitaxel.Cell viability, apoptosis, and migration wereassessed by using MTT assays, Annexin V-FITC/PI staining, and wound healing assays, respectively, with flow cytometry analyzing cell cycle distribution. Results The combination of CD73 siRNA and paclitaxel significantly reduced cell viability, lowering paclitaxel's IC50 from 14.73 μg/mL to 8.471 μg/mL, indicating enhanced drug sensitivity. Apoptosis rates increased with the combination treatment, while cell migration was significantly inhibited. Flow cytometry revealed cell cycle arrest in the Sub-G1 and G2-M phases. Conclusion These findings suggest that cd73 gene suppression enhances paclitaxel's cytotoxic effects, promoting apoptosis and inhibiting cell migration in MDA-MB-231 breast cancer cell line. This combined strategy shows promise for improving breast cancer treatment outcomes by increasing the efficacy of existing chemotherapeutic regimens, warranting further research to explore its potential clinical applications and effectiveness in other breast cancer cell lines and models.

Modulation of homologous recombination gene activity in breast tumor cells in an <i>in vitro</i> model

Introduction. It has been established that the presence of homologous recombination deficiency in a breast tumor is associated with the effectiveness of treatment. But despite the high chemosensitivity of the tumor to DNA-damaging agents, complete pathological responses to treatment are very rare. And this process may be based on a change in the somatic status of BRCA1, that is, a reversion and return of the wild-type allele occurs and the DNA repair function is restored.Aim. To evaluate changes in the presence of chromosomal aberrations and the expression profile of the main genes of homologous recombination in cell models of breast cancer under the influence of cisplatin and docetaxel.Materials and methods. The study was conducted on breast cancer tumor cell cultures: MCF-7, MDA-MB-231 and MDA-MB-468. A cell model of drug resistance was obtained for two drugs: cisplatin and docetaxel. RNA and DNA were isolated from cell suspension using the RNeasy Plus Mini Kit and QIAamp DNA Mini Kit (Qiagen, Germany), respectively. The expression level of homologous recombination genes was assessed using reverse transcription polymerase chain reaction. To assess the presence of chromosomal aberrations, microarray analysis was performed on DNA chips.Results. Restoration of normal copy number for the BRCA1, CDK12, CHEK1 and RAD51D genes in MCF-7 under the influence of cisplatin was shown. For BRCA2 and PALB2, amplifications were detected. A statistically significant increase in the expression of the BRCA1 (p = 0.04), BRCA2 (p = 0.02), PALB2 (p = 0.01) and RAD51D (p = 0.05) genes was also shown. MDAMB-231 shows that all identified loci with deletions, where the BRCA2, BARD1, CHEK2, PALB2 and RAD54L genes are localized, are restored to normal copy number by cisplatin. The appearance of amplifications was registered for BRCA1, BRIP1, FANCL, RAD51B, PARP1. A similar result was shown for docetaxel. An increase in the expression level is typical for the genes BRCA1 (p = 0.02), BRCA2 (p = 0.02), CHEK2 (p = 0.05), FANCL (p = 0.04), PALB2 (p = 0.05), RAD51C (p = 0.02), PARP1 (p = 0.02), which corresponds to the appearance of amplifications. In the MDA-MB-468 cell culture, an increase in the copy number of only the BRCA1 gene is observed. The effect of docetaxel has no effect on this cell culture. The level of BRCA1 expression increases in direct proportion to the duration of drug action.Conclusion. Thus, the study showed that under the influence of cisplatin, reversion of not only homologous recombination gene mutations, but also other disorders can occur.

Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2µg/ml and 13.2mg/ml after 24h and 48h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Integrated Transcriptome Analysis of Radioresistant Cells Revealed Genes and Pathways Predictive Of Tumor Response to Radiotherapy and Chemotherapy in Breast Cancer

AbstractBreast cancer cells exposed to radiotherapy frequently develop radiation resistance through molecular and phenotypic changes. While there is evidences of pathways controlling radioresistance, the evolution of diverse cell phenotypes and transcriptional changes as mediators of radioresistance in breast cancer are restricted. Moreover, the effectiveness of the chemotherapy on radioresistant cells remains uncertain. In this work, an isogenic model of radioresistant breast cancer cells (RR cells) is used to study this phenotype. RR cells show high survival rates after radiation, moreover, RR cells of the triple negative breast cancer (TNBC) subtype show a significantly advanced invasiveness phenotype. Notably, RR cells are significantly sensitive to chemotherapy by inhibit cell survival and promote apoptosis. Transcriptomics and gene co‐expression network analysis identify differentially expressed genes (DEGs) and hub genes related to survival and apoptosis pathways in the RR cells of luminal subtype, while in TNBC subtype, cell migration, cell differentiation, and immune pathways are enriched. Hub genes predict the failure of radiotherapy in breast cancer patients, but they are also related to pathological complete response after chemotherapy. Transcriptome changes during acquired radioresistance uncover genes and pathways associated to radio and chemotherapy response. These results demonstrate that radioresistant pathways may converge to develop collateral chemo‐sensitivity.

Effect of neratinib on HER2 mRNA stability via hsa-miR-23a-5p and efficacy of CDK4/6 inhibitors in HER2-low breast cancer.

e12550 Background: Traditional HER2-negative breast cancer sometimes includes HER2-low components, and CDK4/6 inhibitors are the first- and second-line treatments for advanced HER2-low breast cancer. Nonetheless, little is understood about how the stability of HER2 mRNA in HER2-low breast cancer affects the effectiveness of CDK4/6 inhibitor therapy and the associated intervention techniques. Methods: In order to investigate the impact of HER2-low on the effectiveness of CDK4/6 inhibitor in treating breast cancer, we first used HER2-0 and HER2-low breast cancer cells/mice as the study object and assessed the anti-proliferation effect of CDK4/6 inhibitor using CCK8 technique and monoclonal formation experiment. Second, we increased the treatment of anti-HER2 inhibitor neratinib and assessed its impact on the efficacy of HER2 low breast cancer CDK4/6 inhibitors. By reducing the expression of HER2 in HER2 low breast cancer cells, we analyzed and determined whether HER2 is the primary pathway that influences the poor efficacy of HER2 low breast cancer CDK4/6 inhibitors; At last, actinomycin D was used to detect the mRNA stability of HER2 in the treatment of HER2-low breast cancer by neratinib against CDK4/6 inhibitors, and then microRNA sequencing and luciferase reporter gene detection technology were used. To investigate the specific mechanism of action of neratinib on the mRNA stability of HER2 in the treatment of HER2-low breast cancer by CDK4/6 inhibitors. Results: Our study found that treatment with CDK4/6 inhibitors was significantly less effective against HER2-low breast cancer compared to HER2-0 subtype (P=0.005). Notably, in HER2-low subtype cells, HER2 knockdown significantly inhibited cell proliferation (P<0.001), G1 phase cell cycle arrest (P=0.001), and downregulated cyclin D1 expression and CDK4 protein levels (P=0.007, P=0.003). Second, it was found that in the cell and animal models of HER2-low breast cancer, the addition of the CDK4/6 inhibitor drug neratinib effectively reduced the stability of HER2 mRNA (P=0.001), inhibited the cell cycle of G1 phase (P=0.005). In addition, we observed that lowering the dose of neratinib to 1/2 (20 mg/kg) remained effective and well tolerated (P=0.002). Finally, we found that neratinib can reduce the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3'UTR region of HER2 (P<0.001), thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. Conclusions: We found that low expression of HER2 reduced the efficacy of CDK4/6 inhibitors in HER2-low breast cancer; Neratinib reduces the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3 'UTR region of HER2, thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. These findings provide a tolerable and effective two-drug combination regimen for patients with clinical HER2-low breast cancer.

Parasporins of Bacillus thuringiensis Strain Exhibit Apoptosis-Mediated Selective Cytotoxicity to MDA-MB-231 Cells through Oxidative Stress

Historically, the most important source of both antibiotics and anticancer medications has been microorganisms. Bacillus thuringiensis (Bt) is one of the most prominent bacterial species used as a therapeutic agent targeting cancerous cells in recent worldwide investigations. This study was designed to isolate, molecularly identify, and discover novel Saudi Arabian Bt strains that selectively exhibit cytotoxic properties against MDA-MB-231, a human triple-negative breast cancer (TNBC) cell model. The bacterial strain under investigation was biochemically typed using API 20E and API CH50 and molecularly typed using 16S rDNA sequencing. Flow cytometry and immunoblotting were performed to elucidate the mechanism-of-action (MOA). Molecular typing confirmed the identity of the isolated non-hemolytic strain to be Bt and was named Bt HAU-145. Microscopic examination showed that the strain possessed a parasporal (PS) crystal protein with a spherical morphology. Data of cytotoxicity assay based on MTT revealed that Bt HAU-145 strain exhibited selective and potent cytotoxicity against MDA-MB-231, with a 50 percent inhibition (IC50) of 28 µg/ml. FACS analysis revealed that PS proteins induced both late and early apoptosis in a ROS-dependent manner. Immunoblotting assays showed increased expression of caspase-3 in response to PS treatment, paralleled by a reduction in Bcl-2 expression. This is the first study to investigate the MOA of PS proteins from the Saudi Arabian Bt strain, showing an induction of apoptosis through a ROS-dependent mechanism in TNBC cells. It is hoped that PS-based therapeutic strategies will be investigated at the preclinical scale in non-human primates prior to the clinical scale in randomized clinical trials.

Elevated extracellular vesicular Nm23-H1 subdues the pro-migratory potential of breast cancer cell-derived extracellular vesicles

Metastasis is a key determinant in cancer mortality which is often associated with decreased levels of Nm23-H1, a well-established metastasis suppressor. Despite lacking a secretion signal peptide, Nm23-H1 has been reported to be present in the extracellular space and enclosed within extracellular vesicles (EVs). While the presence of Nm23-H1 proteins in EVs released by cancer cells has been observed through proteomics profiling, the role of vesicular Nm23-H1 remains unclear. Here, we investigated the function of vesicular Nm23-H1 using MDA-MB-231 (highly metastatic, low Nm23-H1) and MCF-7 (low/non-metastatic, high Nm23-H1) breast cancer cell models. Our findings confirm that Nm23-H1 is indeed encapsulated within EVs, and its levels can be manipulated through overexpression and knockdown approaches. Functional assays revealed that EVs derived from MDA-MB-231 cells that contained high levels of Nm23-H1 exhibit impaired pro-migratory properties, suggesting that vesicular Nm23-H1 may act as a metastasis suppressor. Furthermore, EVs with increased levels of Nm23-H1 altered the transcript levels of multiple cancer-related genes in recipient cells and stimulated type I interferon signaling through STAT1 phosphorylation. These results suggest the existence of an unconventional signaling pathway mediated by the uptake of EVs enriched with Nm23-H1, which may contribute to the anti-metastatic effect of Nm23-H1 in the tumor microenvironment. Additionally, our study demonstrates that elevated Nm23-H1 levels can impact the abundance of various other proteins encapsulated within breast cancer cell-derived EVs, such as SUSD2 (Sushi Domain Containing 2) which can also modulate metastasis.

Abstract PO1-24-02: Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer

Abstract Introduction: Claudin-low breast cancer is mostly classified as triple-negative breast cancer and is known to possess cancer stem cell properties with high expression of epithelial-to-mesenchymal transition (EMT) markers. However, specific therapeutic targets for this subtype have not yet been identified. While reports suggest that inhibition of EMT using EMT inhibitors can suppress cell and tumor proliferation, monotherapy may not be sufficient. The expression of CD44, a surface marker of cancer stem cells, has been reported to be involved in multiple signaling pathways, promoting cancer growth and invasion. In this study, we focus on CD44 and the TGF-β receptor, which is involved in EMT, to investigate the effect of inhibiting TGF-β receptor through CD44 inhibition in Claudin-low breast cancer and validate its potential as an effective treatment. Methods: SUM159 and MDA-MB-231 cell lines were used as Claudin-low breast cancer cell models. CD44 knockdown cells were established to examine the effects of TGF-β receptor inhibition and downstream signaling. Results: CD44 knockdown did not affect the expression of TGF-β receptor. Both SUM159 and MDA-MB-231 cells showed comparable cell growth inhibition through CD44 knockdown and TGF-β receptor inhibition, with enhanced effects observed in CD44 knockdown cells in response to TGF-β receptor inhibition. Furthermore, CD44 knockdown combined with TGF-β receptor inhibition significantly reduced the expression of Smad2/3. Conclusion: In Claudin-low breast cancer cells, CD44 knockdown enhances the effectiveness of TGF-β receptor inhibition and significantly influences the expression level of Smad2/3. Citation Format: Ryoichi Matsunuma, Sae Imada, Shoko Sato, Ryosuke Hayami, Michiko Tsuneizumi. Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer [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 PO1-24-02.

A Scaffold-Assisted 3D Cancer Cell Model for Surface-Enhanced Raman Scattering-Based Real-Time Sensing and Imaging.

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situ sensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold's adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

Abstract 4167: Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes

Abstract Tumors consist of a complex mixture of resident and infiltrating cells that can support their survival and evasion of treatment. Selective detection of genetically stable cells in the tumor microenvironment (TME) offers insight into tumor biology beyond the malignant cells, which are the focus of most molecular imaging approaches to cancer detection. Motivated by the goal of non-invasively understanding the primary breast TME, we have developed fluorescent molecular probes that target cancer-associated fibroblasts (CAFs), a major component of the stromal compartment of many tumors, and emit light in the near-infrared (NIR) spectral window, providing access to deeper tissues than visible probes. We synthesized and characterized a series of CAF-targeted antibody- and small molecule-based fluorescent imaging agents utilizing a highly hydrophilic NIR dye and ligands that target Fibroblast Activation Protein (FAP). FAP is a key cell-surface marker of breast cancer CAFs with an S1 phenotype, which are modulators of immunosuppression, metastatic spread, and are associated with worse outcomes, especially in HER2+ and triple-negative breast cancer. Modifications to the chemical structure of the linker and targeting ligands allowed us to alter the specificity and brightness of the CAF-probes in cellulo. Imaging of a co-implanted tumor model of SKBR3 human breast cancer cells and FAP expressing fibroblasts after administering our lead small-molecule CAF imaging agent revealed selective tumor targeting of CAF-rich tumors compared to cancer cell predominant tumors with favorable pharmacokinetics and tumor-to-background ratio (TBR) greater than 2. Similar imaging studies with MMTV-PyMT transgenic mice, an established murine model of breast cancer, showed analogous enhanced uptake in mammary tumors (TBR > 2) and clearance over 24 hours. NIR fluorescence also correlated with CAFs isolated from PyMT tumor tissue via flow cytometry, consistent with our hypothesis that this agent can specifically detect endogenous CAFs. This study identified a novel, bright, and water-soluble FAP-specific NIR agent for in vivo imaging, setting the stage for simultaneous optical imaging of malignant cells and activated fibroblasts in the TME to elucidate cancer-host interactions. Citation Format: Megan S. Michie, Junwei Du, Jiayu (Jennifer) Ye, Kathleen Duncan, Mikhail Y. Berezin, Samuel Achilefu. Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes [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 4167.

Development of L-phenylalanine-grafted poly (vinyl alcohol) scaffold for selective capturing of cancer cells

Breast cancer is the most common cancer and the leading cause of cancer death in women. The majority of deaths from breast cancer do not result from the primary tumor itself but from metastasis to other organs in the body. Here, we report the development of a novel polymeric material that selectively traps breast cancer cells. The cancer cell-capturing polymer was synthesized by grafting L-phenylalanine onto a biocompatible poly(vinyl alcohol) (PVA-Phe). L-phenylalanine exhibits high affinity to L-type amino acid transporter 1 (LAT1), which is overexpressed on various cancer cells. We evaluated cancer cell-selective adhesion ability of PVA-Phe using MCF-7, a human hormone receptor-positive breast cancer model cell expressing LAT1. Our results suggest that LAT1 on cancer cells recognizes the L-phenylalanine and that MCF-7 selectively adheres to the synthesized PVA-Phe. For patients at risk of cancer recurrence, we expect that PVA-Phe synthesized in this study could serve to capture the remaining cancer cells when implanted in the body. Together with an appropriate scaffold that functions to attract cancer cells, PVA-Phe may have the potential to completely prevent cancer recurrence.

Intracellular Delivery of Therapeutic Protein via Ultrathin Layered Double Hydroxide Nanosheets.

The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable protein, gelonin, towards efficient and specific cancer treatment. The LDH nanosheet was synthesized via a facile method without using exfoliation agents and showed a high loading capacity of proteins (up to 182%). Using 2D and 3D 4T1 breast cancer cell models, LDH-gelonin demonstrated significantly higher cellular uptake efficiency, favorable endosome escape ability, and deep tumor penetration performance, leading to a higher anticancer efficiency, in comparison to free gelonin. This work provides a promising strategy and a generalized nanoplatform to efficiently deliver biofunctional proteins to unlock their therapeutic potential for cancer treatment.

Autophagy and senescence facilitate the development of antiestrogen resistance in ER positive breast cancer.

Estrogen receptor positive (ER+) breast cancer is the most common breast cancer diagnosed annually in the US with endocrine-based therapy as standard-of-care for this breast cancer subtype. Endocrine therapy includes treatment with antiestrogens, such as selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). Despite the appreciable remission achievable with these treatments, a substantial cohort of women will experience primary tumor recurrence, subsequent metastasis, and eventual death due to their disease. In these cases, the breast cancer cells have become resistant to endocrine therapy, with endocrine resistance identified as the major obstacle to the medical oncologist and patient. To combat the development of endocrine resistance, the treatment options for ER+, HER2 negative breast cancer now include CDK4/6 inhibitors used as adjuvants to antiestrogen treatment. In addition to the dysregulated activity of CDK4/6, a plethora of genetic and biochemical mechanisms have been identified that contribute to endocrine resistance. These mechanisms, which have been identified by lab-based studies utilizing appropriate cell and animal models of breast cancer, and by clinical studies in which gene expression profiles identify candidate endocrine resistance genes, are the subject of this review. In addition, we will discuss molecular targeting strategies now utilized in conjunction with endocrine therapy to combat the development of resistance or target resistant breast cancer cells. Of approaches currently being explored to improve endocrine treatment efficacy and patient outcome, two adaptive cell survival mechanisms, autophagy, and "reversible" senescence, are considered molecular targets. Autophagy and/or senescence induction have been identified in response to most antiestrogen treatments currently being used for the treatment of ER+ breast cancer and are often induced in response to CDK4/6 inhibitors. Unfortunately, effective strategies to target these cell survival pathways have not yet been successfully developed. Thus, there is an urgent need for the continued interrogation of autophagy and "reversible" senescence in clinically relevant breast cancer models with the long-term goal of identifying new molecular targets for improved treatment of ER+ breast cancer.

Efficacy and delivery strategies of the dual Rac/Cdc42 inhibitor MBQ-167 in HER2 overexpressing breast cancer

Trastuzumab and trastuzumab-based treatments are the standard of care for breast cancer patients who overexpress the human epidermal growth factor receptor 2 (HER2). However, patients often develop resistance to trastuzumab via signaling from alternative growth factor receptors that converge to activate guanine nucleotide exchange factors (GEFs) that in turn activate the Rho GTPases Rac and Cdc42. Since Rac and Cdc42 have been implicated in high tumor grade and therapy resistance, inhibiting the activity of Rac and Cdc42 is a rational strategy to overcome HER2-targeted therapy resistance. Therefore, our group developed MBQ-167, a dual Rac/Cdc42 inhibitor with IC50s of 103 nM and 78 nM for Rac and Cdc42, respectively, which is highly effective in reducing cell and tumor growth and metastasis in breast cancer cell and mouse models. Herein, we created a trastuzumab resistant variant of the SKBR3 HER2 positive breast cancer cell line and show that Rac activation is a central mechanism in trastuzumab resistance. Next, we tested the potential of targeting MBQ-167 to HER2 overexpressing trastuzumab-resistant cell lines in vitro, and show that MBQ-167, but not trastuzumab, reduces cell viability and induces apoptosis. When MBQ-167 was targeted to mammary fatpad tumors established from HER2 overexpressing cells via immunoliposomes functionalized with trastuzumab, MBQ-167 and MBQ-167-loaded liposomes show equal efficacy in reducing the viability of trastuzumab-resistant cells, inhibiting tumor growth in mouse xenografts, and reducing metastasis to lungs and liver. This study demonstrates the efficacy of MBQ-167 as an alternative therapeutic in HER2 overexpressing cancers, delivered either in free form or in liposomes.

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.